(Circulation. 2000;102:3015.)
© 2000 American Heart Association, Inc.
Basic Science Reports |
Endotoxin-Induced Mortality Is Related to Increased Oxidative Stress and End-Organ Dysfunction, Not Refractory Hypotension, in Heme Oxygenase-1–Deficient Mice
From the Program of Developmental Cardiovascular Biology, the Cardiovascular Division (P.W., A.P.P., A.P., K. Maemura., S.-F.Y., M.-E.L., M.A.P.) and the Pulmonary and Critical Care Division (M.A.P.), Brigham and Women’s Hospital; the Department of Medicine (A.P., S.-F.Y., M.-E.L., M.A.P.) Harvard Medical School; and the Cardiovascular Biology Laboratory (P.W., A.P.P., N.D., P.B.M., C.U.S., A.P., K. Maemura, S.-F.Y., M.-E.L., M.A.P.) and the Physiology Program (B.W.L., K. Marino, C.M.D.), Harvard School of Public Health, Boston, Mass. Dr Doerschuk is now at the Department of Pediatrics, Case Western Reserve University, Cleveland, Ohio.
Correspondence to Mark A. Perrella, MD, Program of Developmental Cardiovascular Biology, Brigham and Women’s Hospital, 75 Francis St, Boston, MA 02115. E-mail mperrella{at}rics.bwh.harvard.edu
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Abstract |
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Background—Heme oxygenase (HO)-1 is an enzyme that degrades heme to generate CO (a vasodilatory gas), iron, and the potent antioxidant bilirubin. A disease process characterized by decreases in vascular tone and increases in oxidative stress is endotoxic shock. Moreover, HO-1 is markedly induced in multiple organs after the administration of endotoxin (lipopolysaccharide [LPS]) to mice.
Methods and Results—To determine the role of HO-1 in endotoxemia, we administered LPS to mice that were wild-type (+/+), heterozygous (±), or homozygous null (-/-) for targeted disruption of HO-1. LPS produced a similar induction of HO-1 mRNA and protein in HO-1+/+ and HO-1+/- mice, whereas HO-1-/- mice showed no HO-1 expression. Four hours after LPS, systolic blood pressure (SBP) decreased in all the groups. However, SBP was significantly higher in HO-1-/- mice (121±5 mm Hg) after 24 hours, compared with HO-1+/+ (96±7 mm Hg) and HO-1+/- (89±13 mm Hg) mice. A sustained increase in endothelin-1 contributed to this SBP response. Even though SBP was higher, mortality was increased in HO-1-/- mice, and they exhibited hepatic and renal dysfunction that was not present in HO-1+/+ and HO-1+/- mice. The end-organ damage and death in HO-1-/- mice was related to increased oxidative stress.
Conclusions—These data suggest that the increased mortality during endotoxemia in HO-1-/- mice is related to increased oxidative stress and end-organ (renal and hepatic) damage, not to refractory hypotension.
Key Words: endotoxin • shock • vasoconstriction • perfusion
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Introduction |
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Sepsis is a disease process caused by a severe underlying infection.1 2 If left unchecked, this process may progress to refractory hypotension, multiple organ system failure, and death. Beyond refractory hypotension, oxygen-derived free radicals are believed to contribute to the cellular and tissue injury associated with endotoxin-induced inflammation. Investigators have suggested that this oxidative damage may be a major cause of organ failure and mortality associated with sepsis3 and that the administration of antioxidants may be an adjuvant to conventional therapy (such as vasoconstrictors) in the management of sepsis.4 Other than the cardiovascular response and its associated hypotension, abnormalities of the renal, hepatic, pulmonary, and hematologic systems are common during endotoxemia. In the course of sepsis, the development of multiple organ failure and of pulmonary nosocomial infection5 contributes significantly to morbidity and mortality.
Heme oxygenase (HO)-1 is a stress response enzyme that is induced by stimuli associated with oxidative stress.6 HO-1 catalyzes the degradation of heme to generate biliverdin, CO, and iron.7 Biliverdin is subsequently converted to bilirubin, a potent endogenous antioxidant.8 CO shares many similarities with NO, such as its ability to increase cGMP levels and promote vasodilation, thereby modulating tissue perfusion.9 10 We have previously demonstrated that interleukin-1ß and lipopolysaccharide (LPS) markedly induce HO-1 expression in cultured vascular smooth muscle cells and several organs of endotoxemic rats, respectively,11 12 suggesting that HO-1 may be involved in the pathogenesis of endotoxic shock. These data are supported by human studies showing an elevation in carboxyhemoglobin levels in septic trauma patients.13 We have also demonstrated that Zn-protoporphyrin IX, an inhibitor of HO activity, abrogates endotoxin-induced hypotension in rats.11 These results imply that the marked induction of HO-1 during endotoxemia contributes to the decrease in systemic blood pressure. Conversely, other investigators have shown that the administration of LPS to rats receiving high doses of HO inhibitors14 or mice lacking HO-115 leads to increased mortality. Using high doses of LPS (25 mg/kg), investigators have shown that the increased mortality in HO-1 null (HO-1-/-) mice is associated with hepatic necrosis in vivo.15 Taken together, these results suggest that although an exaggerated induction of HO-1 may participate in the hypotensive response to LPS, basal HO-1 expression is needed to resist oxidative stress.
To better understand the role of HO-1 in the pathophysiology of endotoxemia, we evaluated LPS-induced hypotension and end-organ dysfunction in HO-1-/- mice.16 The goal of the present study was to define the role of HO-1 in LPS-induced hypotension and to determine whether refractory hypotension and/or exaggerated oxidative stress was responsible for the mortality in HO-1-/- mice.
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Methods |
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Mouse Model of Endotoxemia
Mice that were wild-type (+/+), heterozygous (±), or homozygous null (-/-) for targeted disruption of HO-116 were studied. These mice were maintained on a 129SvxBALB/c genetic background, and littermates were used for the studies. Salmonella typhosa LPS (Sigma Chemical Co) was administered intraperitoneally (5 mg/kg) to 20- to 30-g female mice that were 16 weeks of age. Pilot experiments revealed that 5 mg/kg LPS produced a 15% to 20% decrease in systolic blood pressure (SBP). Mice were injected with 0.9% saline to act as controls for animals receiving LPS. HO-1-/- mice receiving LPS were also treated with the endothelin (ET) type A and B (ETA/ETB) receptor antagonist PD142893 (3 µmol/kg IP,17 Sigma) or the antioxidant N-acetylcysteine (NAC, 150 mg/kg). The mice were killed 24 hours after LPS administration. Tissue and plasma samples were collected and stored at -80°C until further processing. The Harvard Medical Area Standing Committee on Animals approved the protocol.
Northern Blot Analysis
Total RNA was obtained from mouse tissue by guanidinium
isothiocyanate extraction and silica-gel-membrane spin technology
(RNeasy midi kit, Qiagen). Northern blot analysis was performed
as described,11 12 and filters were hybridized with
32P-labeled rat HO-1, rat HO-2, or mouse ET-1
probes. To correct for differences in RNA loading, the filters were
also hybridized with a 32P-labeled
oligonucleotide probe complementary to 28S ribosomal
RNA. Images were displayed, and radioactivity was measured on a
PhosphorImager running ImageQuant software (Molecular Dynamics).
Western Blot Analysis
Tissue samples were homogenized in 13.2 mmol/L
Tris-HCl, 5.5% glycerol, 0.44% SDS, and 10% ß-mercaptoethanol. An
equal amount of soluble protein (50 µg) was fractionated by
Tris-glycine-SDS–polyacrylamide gel (12%) electrophoresis,
and Western blotting was performed as described12 with use
of an antibody to recombinant rat HO-1 or HO-2 protein (1:1000,
Stressgen).
Blood Pressure Measurements
Baseline SBP was measured 12 hours before the administration of
LPS or saline vehicle and then 4 and 24 hours after LPS or saline
vehicle. A tail-cuff method was used to measure SBP. Mice were trained
by placing them in restraints, 1 hour daily, for 10 days before the
experiments. Once they were fully trained, conscious mice were
restrained and gently warmed with use of a heating lamp. An occlusion
cuff and a piezoelectric pulse sensor were placed around the tail (Kent
Scientific), and SBP was measured after a 15-minute acclimatization
period. A minimum of 8 serial measurements were made, and the average
value was calculated (Mac Laboratory software, version 3.5, AD
Instruments). Both training and blood pressure measurements were
performed the same time each day (afternoon).
Biochemical Measurements
Alanine aminotransferase (ALT), aspartate aminotransferase
(AST), and creatinine (Cr) levels were measured in plasma
by use of commercial kits (Sigma), according to the manufacturer’s
recommendations. Lipid peroxidation products were measured in liver
tissue by use of the lipid peroxidation assay kit from Calbiochem. This
colorimetric assay is specific for malondialdehyde
(MDA) and 4-hydroxy-2-nonenal (4-HNE). Values are expressed as the sum
of MDA+4-HNE levels (micromoles MDA+4-HNE per gram wet tissue).
Characterization of Lung Neutrophils, Edema, and Alveolar
Destruction
Twenty-four hours after intratracheal administration of LPS (5
mg/kg), the percentage of distal lung tissue occupied by neutrophils,
edema, or destroyed alveolar walls was quantified on paraffin-embedded
histological sections by using point-counting
techniques.18 19 Total circulating white blood cell (WBC)
and neutrophil counts were measured with use of a hemocytometer and
blood smears stained with Leukostat (Fisher Scientific).
Statistics
Where indicated, comparisons between groups were made by
factorial ANOVA followed by the Fisher least significant difference
test when appropriate. Comparisons of mortality between groups were
made by the 
2 goodness of fit test.
Statistical significance was accepted at P<0.05. Data are
expressed as mean±SE.
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Results |
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LPS Induction of HO-1 Expression in Multiple Organs
We initially evaluated HO-1 expression in the livers, kidneys, and lungs of HO-1 wild-type mice at baseline and then 4 and 24 hours after LPS administration. HO-1 mRNA was markedly induced by LPS in all organs after 4 and 24 hours (Figure 1
). When
comparing HO-1 expression 24 hours after LPS, we found that HO-1 mRNA
was markedly induced in both HO-1+/+ and
HO-1+/- mice (Figure 2a). As expected, HO-1 mRNA was not
detected in HO-1-/- mice.
A constitutive isoform of HO (HO-2) was not affected by LPS in these
organs. Moreover, HO-2 was not upregulated in
HO-1-/- mice compared
with HO-1+/+ and HO-1+/-
mice (in the presence or absence of LPS); thus, HO-2 did not compensate
for the absence of HO-1 (Figure 2a). To determine whether these
changes in HO-1 mRNA levels translated into similar changes in HO-1
protein levels, we subjected liver protein extracts to Western blot
analysis. Again, the induction of HO-1 was similar in
HO-1+/+ and HO-1+/- mice,
whereas no HO-1 protein was detected in the
HO-1-/- mice (Figure 2b). HO-2 protein levels were not increased by LPS, and they
were not upregulated in
HO-1-/- mice compared
with HO-1+/+ and HO-1+/-
mice (Figure 2b).
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Recovery of LPS-Induced Hypotension in
HO-1-/- but Not HO-1+/+ and
HO-1+/- Mice
HO-1–derived CO is a vasoactive gas that may participate in the
regulation of vascular tone. Therefore, we measured SBP in
HO-1+/+, HO-1+/-, and
HO-1-/- mice challenged
with LPS. SBP was not different between the groups at baseline. Four
hours after LPS, the decrease in SBP was similar in all the groups;
however, after 24 hours, SBP was significantly higher in
HO-1-/- mice (121±5
mm Hg) compared with HO-1+/+ (96±7 mm Hg)
and HO-1+/- (89±13 mm Hg) mice (Figure 3). These data suggest that HO-1
contributes to the sustained, but not the acute, hypotension associated
with endotoxemia.
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Twenty-four hours after LPS administration, we assessed mortality in the HO-1+/+, HO-1+/-, and HO-1-/- mice. Despite the lack of sustained hypotension in HO-1-/- mice, mortality was increased (60%, P<0.05) compared with HO-1+/+ (10%) and HO-1+/- (10%) mice.
Sustained Induction of ET-1 mRNA in
HO-1-/- Mice After LPS
Administration
In addition to its direct vasodilatory effect, CO is also known to
inhibit the expression of ET-1, a potent
vasoconstrictor.20 Thus, we hypothesized that in
HO-1-/- mice, unabated
ET-1 gene expression may contribute to the restoration of blood
pressure. We performed Northern blot analysis to evaluate ET-1
mRNA levels in the kidney, an organ that robustly expresses ET-1 and
plays a pivotal role in the regulation of blood
pressure.21 Four hours after LPS administration, the
induction of ET-1 mRNA was similar in HO-1+/+ and
HO-1-/- mice (Figure 4a). In HO-1+/+
mice, ET-1 mRNA had returned to baseline levels by 24 hours. However,
at this time point, ET-1 mRNA levels were still markedly increased in
HO-1-/- mice (Figure 4a). Twenty-four hours after LPS, the increase in ET-1 mRNA was
also noted in liver and lung tissue from
HO-1-/- mice (Figure 4b). These data suggest that the lack of HO-1 allowed a
sustained induction of ET-1 message in multiple organs.
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To determine whether this sustained induction of ET-1 contributed to
the higher SBP in HO-1-/-
mice after 24 hours of LPS, we administered an
ETA/ETB receptor
antagonist (PD142893, 3 µmol/kg IP) or vehicle to
HO-1-/- mice. Before LPS
administration, the ETA/ETB
receptor antagonist caused no change in SBP (data not
shown). However, in the presence of LPS,
HO-1-/- mice receiving
the ETA/ETB receptor
antagonist had a significantly lower SBP (90±7
mm Hg) compared with SBP in null mice receiving vehicle (121±5
mm Hg) (Figure 4c
). SBP in
HO-1-/- mice receiving
the ETA/ETB receptor
antagonist was comparable to SBP in
HO-1+/+ and HO-1+/- mice
after 24 hours of LPS (Figure 3).
End-Organ Dysfunction in Endotoxemic
HO-1-/- Mice
LPS at a dose of 5 mg/kg caused no hepatocellular destruction by
histological analysis; however, iron deposition
was present in the liver (data not shown). To further determine
whether end-organ damage occurred in
HO-1-/- mice in the
absence of prolonged hypotension, we evaluated markers of hepatic and
renal insult 24 hours after LPS administration. Plasma levels of AST
and ALT were measured to evaluate the occurrence of hepatocellular
injury. Levels of both transaminases were similar in
HO-1+/+, HO-1+/-, and
HO-1-/- mice at baseline
(Figure 5a). However, AST and ALT levels
were markedly induced in
HO-1-/- mice after LPS
but not in HO-1+/+ and
HO-1+/- mice. Similarly, there was no difference
in plasma Cr levels between the groups at baseline (Figure 5b).
After LPS administration, there was a significant increase in plasma Cr
in HO-1-/-mice but not in
HO-1+/+ and HO-1+/- mice.
These data show a significant LPS-induced deterioration in hepatic and
renal function in the absence of HO-1.
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Increased Susceptibility to LPS-Induced Oxidative Damage in the
Absence of HO-1
Oxidative stress induces HO-1 in a variety of cell types and
pathophysiological conditions, and HO-1 constitutes
an important antioxidant defense mechanism. To determine whether the
absence of HO-1 may lead to increased oxidative damage in target organs
of endotoxemic mice, we measured 2 aldehydes (MDA and 4-HNE) that are
generated during lipid peroxidation (Figure 6a). A specific
colorimetric method was used to detect both aldehydes
(see Methods) from liver homogenates. MDA+4-HNE levels were
similar at baseline in HO-1+/+ and
HO-1-/- mice (Figure 6a). Twenty-four hours after LPS administration, a 3-fold
increase in aldehyde levels was evident in
HO-1+/+ mice. However, MDA+4-HNE levels were
induced >7-fold in
HO-1-/- mice receiving
LPS. These data demonstrate increased oxidative liver injury in the
absence of HO-1.
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P<0.05
vs all other groups. b, HO-1-/- mice received
the antioxidant NAC (150 mg/kg IP, n=6) or vehicle (n=10) 1 hour before
and 12 hours after LPS administration (5 mg/kg IP). Death rate was
assessed after 24 hours. *P<0.05 vs no (-) NAC
treatment.
To determine whether increased oxidative stress and damage may have
contributed to increased organ dysfunction and mortality,
HO-1-/- mice receiving
LPS were treated with vehicle or the antioxidant NAC (150 mg/kg).
Mortality was assessed after 24 hours. In contrast to vehicle-treated
mice that had a death rate of 60%, mice receiving NAC had a death rate
of 0% after receiving LPS (Figure 6b). Moreover, end-organ
dysfunction improved in the presence of NAC. Plasma Cr was similar to
baseline in HO-1-/- mice
receiving NAC before LPS (26±4 µmol/L); however, plasma Cr was
significantly increased in the absence (56±2 µmol/L) of
NAC.
No Increased Lung Damage in HO-1-/- Mice
Exposed to LPS but Increased Circulating Inflammatory Cells
Similar to the histological analysis of
the liver, there were no abnormalities in the lung tissue of
HO-1-/- mice after
intraperitoneal administration of LPS (data not
shown). In addition, there was no gross increase in lung iron
deposition in these mice. Because pulmonary nosocomial
infections and acute lung injury are associated with increased
morbidity and mortality in septic patients, the inflammatory response
during LPS-induced pneumonia was evaluated in
HO-1-/- and
HO-1+/+ mice.18 19 There was no
difference in distal airway neutrophil accumulation, pulmonary
edema, or alveolar destruction in
HO-1-/- mice compared
with HO-1+/+ mice (Figure 7a). Baseline numbers of circulating WBCs
were not different between the groups. However, WBCs in
HO-1-/- mice increased
after LPS administration compared with WBCs in
HO-1+/+ mice (Figure 7b). We next examined
the circulating level of neutrophils, a critical source of inflammatory
mediators during endotoxemia.22 There was a marked
increase in circulating neutrophils in
HO-1-/- mice, but not in
HO-1+/+ mice, after LPS administration (Figure 7b).
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Discussion |
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TopAbstractIntroductionMethodsResultsDiscussionReferences |
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Sepsis is a systemic response to a severe underlying infection. Even with intense efforts to improve therapeutic strategies, mortality rates remain high, and the incidence of sepsis continues to rise.23 In some patients, intractable hypotension leads to death, whereas in others, the failure of one or more organs is the cause of demise. Beyond antibiotics, supportive care remains the mainstay of therapy at present.24 25 If the blood pressure remains low, vasoconstrictor agents are administered. Although these agents increase blood pressure, they also have the potential to decrease end-organ perfusion and promote organ damage.24 Thus, a delicate balance exists between vasoconstrictors and vasodilators during septic shock. Systemic blood pressure needs to be increased to reverse hypotension, yet this cannot occur at the expense of peripheral organ perfusion.
In the present study, SBP was measured before and after (4 and 24
hours) the administration of LPS to HO-1+/+,
HO-1+/-, and
HO-1-/- mice. Four hours
after LPS, SBP was reduced in mice from all 3 groups. However, SBP was
significantly increased after 24 hours in
HO-1-/- mice compared
with HO-1+/+ and HO-1+/-
mice (Figure 3). SBP was not different between
HO-1+/+ and HO-1+/- mice,
and HO-1 expression was similar (Figure 2). Even though SBP was
higher in HO-1-/- mice
after LPS, we found increased mortality. These data demonstrate for the
first time that HO-1 contributes to the sustained hypotension
associated with endotoxemia and that a lack of HO-1 results in
increased mortality that is not associated with intractable
hypotension.
An endogenous vasoconstrictor that plays an important role
in the pathophysiology of septic shock is ET-1.26
Investigators have suggested that the release of endogenous
ET-1 during endotoxemia may help to counteract severe
hypotension,27 especially in the setting of NO synthase
inhibition.28 However, the increased levels of ET-1 may
lead to excessive vasoconstriction in peripheral vascular
beds, and ET-1 contributes to the dysfunction of multiple organs during
endotoxemia, including liver,29 kidney,30 and
lung.30 Because HO-1–derived CO is known to suppress
ET-1,20 we investigated whether ET-1 mRNA levels would be
altered in HO-1-/- mice.
ET-1 was induced by LPS in the kidneys of HO-1+/+
and HO-1-/- mice after 4
hours; however, ET-1 mRNA levels remained elevated only in
HO-1-/- mice after 24
hours (Figure 4a). The sustained increase in ET-1 mRNA was also
noted in the liver and lungs of
HO-1-/- mice (Figure 4b). Administration of an
ETA/ETB receptor
antagonist to
HO-1-/- mice receiving
LPS resulted in a significantly lower SBP compared with SBP in
HO-1-/- mice receiving
vehicle (Figure 4c). Moreover, none of the null mice receiving
the ETA/ETB receptor
antagonist died after 24 hours of LPS stimulation (data not
shown). These data suggest that increased levels of ET-1 in multiple
organs contributed not only to higher SBP but also to increased
mortality in HO-1-/- mice
receiving LPS.
The development of multiple organ failure is a key predictor of outcome
in septic patients.31 Thus, we examined the mice for
LPS-induced end-organ damage.
HO-1-/- mice at this age
showed no evidence of increased organ dysfunction (Figures 5a and 5b), oxidative damage (Figure 6a), or systemic
inflammation (Figure 7b) compared with
HO-1+/+ mice at baseline. However, after LPS
stimulation, plasma ALT and AST levels dramatically increased in
HO-1-/- mice (Figure 5a) in the absence of intractable hypotension. Furthermore,
plasma Cr levels were significantly increased in
HO-1-/- mice after LPS
administration (Figure 5b). The end-organ insults were not
present in HO-1+/+ or
HO-1+/- mice exposed to LPS. These data show
LPS-induced hepatocellular damage and renal dysfunction only in mice
lacking HO-1.
The absence of HO-1–induced CO and the sustained induction of ET-1 may
have contributed to end-organ damage due to oxidative damage, resulting
from decreased tissue perfusion. Furthermore, decreased generation of
bilirubin (an important antioxidant8 ) and iron deposition
provide an environment susceptible to oxidative stress and damage in
the absence of HO-1. The administration of LPS to wild-type mice caused
a 3-fold increase in lipid peroxidation products in liver tissue
(MDA+4-HNE, Figure 6a), which is consistent with
increased oxidative stress associated with endotoxemia. In
HO-1-/- mice, however,
lipid peroxidation products were increased 7-fold after the
administration of LPS. This level of lipid peroxidation products in
HO-1-/- mice was
significantly higher than that in wild-type mice (Figure 6a).
Previously, investigators have shown that HO-1–deficient cells from
mice15 and humans32 in culture are more
sensitive to injury by pro-oxidants (such as hemin, hydrogen peroxide,
paraquat, and cadmium chloride). In the present study, we show that
the administration of a pathophysiological
stimulus, LPS, can enhance tissue oxidative damage in
HO-1-/- mice in vivo. The
importance of increased oxidative stress and damage in these mice was
underscored by experiments demonstrating that the administration of an
antioxidant, NAC, improved end-organ dysfunction and prevented
LPS-induced death in
HO-1-/- mice (Figure 6b).
Studies have suggested that HO helps to protect the lung from
oxidant-induced injury. For example, Dennery et al33
showed that mice lacking HO-2 were more susceptible to
hyperoxia-induced injury, whereas Otterbein et al34
demonstrated that overexpression of HO-1 can protect the lung from
hyperoxia. This latter effect of HO-1 appears to be mediated, in part,
by the attenuation by CO of neutrophil infiltration into the
airways.35 In patients with sepsis, nosocomial infections
and acute lung injury are complications associated with increased
morbidity and mortality.5 Thus, we evaluated LPS-induced
pneumonia18 in
HO-1-/- mice to determine
whether the lack of HO-1 would enhance lung injury. Unexpectedly, in
this model, we found no increase in distal airway neutrophil
accumulation, pulmonary edema, or alveolar destruction in
HO-1-/- mice compared
with HO-1+/+ mice (Figure 7a). However,
circulating WBCs were increased in
HO-1-/-, but not
HO-1+/+, mice exposed to LPS (Figure 7b).
This increase was due primarily to a marked increase in circulating
neutrophils (a critical source of inflammatory mediators) in
HO-1-/- mice. These data
suggest that the release of neutrophils from the bone marrow may be
enhanced. This response may represent a globally exaggerated
systemic inflammatory response.
Taken together, we show that endotoxin-induced mortality in mice lacking HO-1 is not the result of intractable hypotension but is associated with end-organ damage (liver and renal) and increased oxidative stress. These data suggest that in conjunction with supportive care and the use of vasoconstrictor agents to treat hypotension, strategies aimed at reducing inflammation and oxidative injury in target organs during sepsis may have therapeutic benefit.
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Acknowledgments |
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This study was supported in part by National Institutes of Health grants HL-03194 and HL-60788 (to Dr Perrella), GM-53249 (to Dr Lee), and HL-48160 (to Dr Doerschuk); an American Heart Association Grant-in-Aid (to Dr Perrella); grants from Novartis and the SICPA Foundation (to Dr Wiesel); and a grant from the Bristol-Myers Squibb Pharmaceutical Research Institute. We dedicate this study to the memory of Dr Mu-En Lee, who was a constant source of inspiration and support for our work. We also thank Dorothy Zhang for her technical assistance.
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Footnotes |
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Drs Wiesel and Patel contributed equally to this study.
Received April 24, 2000; revision received June 20, 2000; accepted June 30, 2000.
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