Division of Pulmonary Sciences and Critical Care Medicine, University of Colorado Health Sciences Center, Denver, Colorado 80262
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ABSTRACT |
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Acute lung injury is
characterized by accumulation of neutrophils in the lungs, accompanied
by the development of interstitial edema and an intense inflammatory
response. To assess the role of neutrophils as early immune effectors
in hemorrhage- or endotoxemia-induced lung injury, mice were made
neutropenic with cyclophosphamide or anti-neutrophil antibodies.
Endotoxemia- or hemorrhage-induced lung edema was significantly reduced
in neutropenic animals. Activation of the transcriptional regulatory
factor nuclear factor-B after hemorrhage or endotoxemia was
diminished in the lungs of neutropenic mice compared with
nonneutropenic controls. Hemorrhage or endotoxemia was followed by
increases in pulmonary mRNA and protein levels for interleukin-1
(IL-1
), macrophage inflammatory protein-2 (MIP-2), and tumor
necrosis factor-
(TNF-
). Endotoxin-induced increases in
proinflammatory cytokine expression were greater than those found after
hemorrhage. The amounts of mRNA or protein for IL-1
, MIP-2, and
TNF-
were significantly lower after hemorrhage in the lungs of
neutropenic versus nonneutropenic mice. Neutropenia was associated with
significant reductions in IL-1
and MIP-2 but not in TNF-
expression in the lungs after endotoxemia. These experiments show that
neutrophils play a centrol role in initiating acute inflammatory
responses and causing injury in the lungs after hemorrhage or endotoxemia.
cytokines; nuclear factor-B; interleukin-1
; tumor necrosis
factor-
; macrophage inflammatory protein-2
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INTRODUCTION |
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ACUTE LUNG INJURY is
characterized by the accumulation of large numbers of neutrophils into
the lungs and a pulmonary inflammatory response in which there is
increased production of immunoregulatory cytokines (13, 18, 26,
27). Macrophages, neutrophils, endothelial cells, and other
pulmonary cell populations have all been demonstrated to express
proinflammatory cytokines, but the relative importance of each of these
cell populations in contributing to the development of acute lung
injury has not been well defined. Interleukin (IL)-1 appears to be
the most important proinflammatory cytokine in bronchoalveolar lavage
specimens from patients with acute respiratory distress syndrome
(ARDS), the most severe form of acute lung injury (25).
Tumor necrosis factor-
(TNF-
) and IL-8 also appear to have
central roles in the initiation and potentiation of acute lung injury
(8, 11, 19, 24).
Blood loss and sepsis are major risk factors for the development of acute lung injury (5, 15). Experimental models of hemorrhage or endotoxin administration demonstrate that both of these pathophysiological insults produce acute inflammatory lung injury (9, 17, 31). Neutrophils appear to be important in the genesis of acute lung injury since induced neutropenia followed by endotoxin challenge or complement activation attenuates increases in vascular permeability and other indexes of pulmonary damage (14, 32). However, the in vivo mechanisms by which neutrophils mediate lung injury after hemorrhage or endotoxemia remain incompletely understood.
Neutrophils can express proinflammatory cytokines, including IL-1,
IL-1
, IL-8, and TNF-
(2, 7, 23, 30. 34). After hemorrhage or
endotoxemia, neutrophils are major contributors to lung IL-1
production (23). Expression of TNF-
and macrophage
inflammatory protein-2 (MIP-2), a murine homologue of IL-8, is also
increased in lung neutrophils after blood loss or endotoxin
administration (2, 30, 33). Binding sites for the
transcriptional regulatory factor nuclear factor-
B (NF-
B) are
present in the promoters of IL-1
, TNF-
, and MIP-2, and activation
of NF-
B is important in modulating the expression of these as well
as other immunoregulatory mediators (3, 4, 28). After
hemorrhage or endotoxemia, NF-
B activation is increased in lung but
not in blood neutrophils, providing an explanation for the enhanced
expression of proinflammatory cytokines in pulmonary neutrophil
populations (30).
Although neutrophils are an important component of the inflammatory
response that characterizes acute lung injury, limited information is
available concerning their role as early immune effectors in this
process. To examine this issue, we performed a series of experiments in
which neutrophils were depleted and then pulmonary cytokine expression,
NF-B activation, and parameters of lung injury were examined after
either hemorrhage or endotoxemia. These studies demonstrate that
activated neutrophils have an important role in initiating the
inflammatory processes involved in the development of hemorrhage- or
endotoxemia-induced acute lung injury.
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METHODS |
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Mice. Male BALB/c mice, 8-12 wk of age, were purchased from Harlan Sprague Dawley (Indianapolis, IN). The mice were kept on a 12:12-h light-dark cycle with free access to food and water. All experiments were conducted in accordance with institutional review board-approved protocols.
Models of hemorrhage and endotoxemia. The murine hemorrhage model used in these experiments was developed in our laboratory and reported on previously (1, 2, 23, 29-31). With this model, 30% of the calculated blood volume (~0.55 ml for a 20-g mouse) is withdrawn over a 60-s period by cardiac puncture from a methoxyflurane-anesthetized mouse. The period of methoxyflurane anesthesia is <2 min in all cases. The mortality rate with this hemorrhage protocol is ~12%.
The model of endotoxemia used was reported previously (2, 23, 30). Mice received an intraperitoneal injection of lipopolysaccharide at dose of 1 mg/kg in 200 µl of PBS. This dose produces neutrophil infiltration into the lungs and acute neutrophilic alveolitis in mice, histologically consistent with acute lung injury.Generation of neutropenia. In designated experiments, mice were given 150 mg/kg cyclophosphamide intraperitoneally in 0.2 ml of PBS as previously reported (23). Control mice were given 0.2 ml of PBS intraperitoneally at the same time points. The effects of cyclophosphamide treatment on neutrophil numbers were determined by preparing Wright's stains using blood withdrawn from the tail of each mouse. In mice treated with this dose of cyclophosphamide 4 days and 1 day before hemorrhage or administration of endotoxin, there was >95% reduction in neutrophil numbers compared with that in control mice. Mice given a single dose of cyclophosphamide 1 day before endotoxin administration or hemorrhage had neutrophil counts that were not different from those in controls.
Neutropenia also was produced by treating mice with absorbed rabbit anti-neutrophil antibodies (Accurate Chemicals, Westbury, NY) (12). In these experiments, mice were treated intraperitoneally with anti-neutrophil antibodies diluted 1:15 in 0.2 ml of PBS on days 3 and 4 before endotoxin administration or hemorrhage, followed by 2 days of treatment with 0.2 ml of undiluted antibodies. Wright's staining of tail blood was used in each mouse to determine the degree of neutropenia achieved with this regimen. In each case, >95% of neutrophils, compared with that in PBS-treated controls, were eliminated with such anti-neutrophil antibody treatment.Myeloperoxidase assay.
Myeloperoxidase activity was assayed as reported previously (2,
23). Excised lungs from three to four mice per treatment group
were frozen in liquid nitrogen, weighed, and stored at 86°C. Lungs
were homogenized for 30 s in 1.5 ml of 20 mM potassium phosphate, pH 7.4, and centrifuged at 4°C for 30 min at 40,000 g. The
pellet was resuspended in 1.5 ml of 50 mM potassium phosphate, pH 6.0, containing 0.5% hexadecyltrimethylammonium bromide, sonicated for
90 s, incubated at 60°C for 2 h, and centrifuged. The
supernatent was assayed for peroxidase activity corrected to lung weight.
Wet-to-dry lung weight ratios. All mice used for lung wet-to-dry weight ratios were of identical ages. Lungs were excised, rinsed briefly in PBS, blotted, and then weighed to obtain the "wet" weight. Lungs were then dried in an oven at 80°C for 7 days to obtain the "dry" weight.
Cytokine ELISA.
After the lung vascular bed had been flushed by injecting 5 ml of
chilled (4°C) PBS into the right ventricle, the lungs were homogenized for 30 s in lysis buffer containing 10 mM HEPES, 150 mM NaCl, 1 mM EDTA, 0.6% ipegal, 5 mM phenylmethylsulfonyl fluoride, 1 µg/ml leupeptin, 1 µg/ml aprotinin, 10 µg/ml soybean trypsin inhibitor, and 1 µg/ml pepstatin. The homogenates were
centrifuged at 10,000 rpm at 4°C for 10 min, and the supernatants
were collected. Protein content of the supernatants was determined
using the bicinchoninic acid protein assay kit from Pierce Chemical
(Pittsburgh, PA). Immunoreactive IL-1, TNF-
, and MIP-2
were quantitated with commercially available ELISA kits (R&D Systems,
Minneapolis, MN). With these assays, the threshold of sensitivity for
IL-1
and MIP-2 is 3 pg/ml, and for TNF-
, it is 10 pg/ml.
Quantitative PCR.
Groups of five mice, with results obtained from individual mice, were
used for each experimental condition. PCR was used in these studies
because the amount of RNA obtained from each mouse was insufficient to
prepare Northern blots for cytokine analysis. The animals were
anesthetized with methoxyfluorane and then killed by cervical
dislocation. The thorax was opened with two lateral incisions along the
rib cage. The right heart was injected with cold, sterile PBS (3-5
ml) until the lungs had been flushed thoroughly. The lungs were excised
with care to avoid the peritracheal lymph nodes and rinsed in PBS. The
lungs were briefly blotted. The lungs were homogenized for 30 s on
ice in 300 µl of buffer RLT (QIAGEN, Valenica, CA) with 3 µl of
-mercaptoethanol (Sigma, St. Louis, MO). RNA was isolated
using the RNAeasy kit (QIAGEN) following the manufacturer's protocol.
Briefly, proteinase K was added to each sample, incubated at 55°C for
10 min, and then centrifuged at 12,000 rpm for 3 min. Ethanol (100%)
was added to clear the lysate, and the samples were centrifuged at
12,000 rpm for 15 s. After washes, the samples were treated with
DNase for 15 min at room temperature and the membrane was dried in
buffer RPE. RNA was eluted from the membrane in 30 µl
RNase-free water, and the quantity of RNA was determined at 260-nm absorbance.
Preparation of nuclear extracts.
Nuclear extracts were prepared as previously described (17,
18). In brief, lungs were snap-frozen in liquid nitrogen and then homogenized in buffer A. After cytoplasm was removed
from the nuclei by 15 passages through a 25-gauge needle, nuclei were centrifuged at 4°C for 6 min at 600 g. After the nuclear
pellet was incubated on ice for 15 min in buffer C, the
extract was centrifuged at 4°C for 10 min at 12,000 g. The
supernatant was collected, divided into aliquots, and stored at
86°C. Protein concentration was determined using Coomassie Plus
protein assay reagent (Pierce Chemical) standardized to bovine serum
albumin according to the manufacturer's protocol.
Electrophoretic mobility shift assay analysis.
Activation of the transcriptional factor NF-B was determined by
electrophoretic mobility shift assay (EMSA) analysis, as described
previously in our laboratory (22, 29, 30). The
B DNA sequence of the immunoglobulin gene was used. Synthetic DNA
sequences (with enhancer motifs underlined) were annealed, forming
double-strand DNA probes with single-strand ends consisting of
sequences of four thymidines, allowing the ends to be labeled by base
pairing with [
-32P]dATP using Sequenase DNA
polymerase:
B, 5'-TTTTCGAGCTCGGGACTTTCCGAGC-3' and
3'-GCTCGAGCCCTGAAAGGCTCGTTTT-5'.
In situ immunolocalization of TNF-.
Immunohistochemistry was performed as described previously
(24). Briefly, control mice or endotoxin-treated mice were
prepared as previously described. After the right ventricle was
perfused with 5 ml of PBS (4°C), the lungs were gently infiltrated
through the trachea with 1% low-melting-point agarose (Seakem,
Rockland, ME) at 42°C. Lungs were then removed en bloc and fixed in
4% paraformaldehyde-0.23 M sucrose solution overnight. Tissue was then
embedded and treated with 0.2 M glucose and 1.5 U/ml glucose oxidase in
PBS for 30 min followed by 10% hydrogen peroxide in PBS for 15 min,
after which 5-µm sections were prepared. Immunohistochemistry was
conducted using either rabbit polyclonal anti-mouse TNF-
antibodies
or control rabbit serum (PharMingen, San Diego, CA) at a dilution of
1:1,000 using the Vectastain immunohistochemistry kit following the
manufacturer's protocol (Vector, Burlingame, CA).
Statistical analysis. For each experimental condition, the entire group of animals was prepared and studied at the same time. Mice in all groups had the same birth date and had been housed together. Separate groups of mice were used for myeloperoxidase assays, PCR, and EMSA. For PCR, each animal was analyzed individually before group data. Data are presented as means ± SD for each experimental group were calculated. One-way ANOVA and the Tukey-Kramer multiple comparisons test were used when more than two experimental groups were compared. Student's t-test was used for comparisons between two data groups. P < 0.05 was considered significant.
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RESULTS |
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Elimination of neutrophils reduces hemorrhage- or
endotoxemia-induced lung injury.
Hemorrhage or endotoxemia results in accumulation of neutrophils in the
lungs (Fig. 1) and increased wet-to-dry
weight ratios (Fig. 2). Endotoxemia- or
hemorrhage-induced lung edema was significantly reduced in mice made
neutropenic by treatment with cyclophosphamide (Fig. 2A).
The ameliorative effects of cyclophosphamide treatment on hemorrhage-
or endotoxemia-associated increases in lung wet-to-dry weight ratios
appeared to be due to neutropenia, but not to other effects of
cyclophosphamide, since there was no effect on lung edema in
nonneutropenic cyclophosphamide-treated animals (i.e., given
cyclophosphamide 1 day before endotoxin administration or hemorrhage).
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Effects of neutrophils on NF-B activation in the lungs after
hemorrhage or endotoxemia.
The transcriptional regulatory factor NF-
B was activated in the
lungs after hemorrhage or endotoxemia but to a greater extent by
endotoxemia than by hemorrhage (Fig. 3).
To determine the relative importance of neutrophils in affecting
pulmonary NF-
B activation, we treated mice with either
cyclophosphamide or anti-neutrophil antibodies and then examined
nuclear translocation of NF-
B in whole lung extracts obtained after
endotoxin administration or hemorrhage (Fig. 3). The increases in lung
NF-
B activation produced by hemorrhage or endotoxemia were reduced
in mice made neutropenic by administration of either cyclophosphamide
or anti-neutrophil antibodies. Mice treated with a single dose of
cyclophosphamide 1 or 4 days before hemorrhage or endotoxin
administration or with anti-neutrophil antibodies for 2 days before
hemorrhage or endotoxin administration were not neutropenic and showed
levels of NF-
B activation similar to those found in control mice not
given either cyclophosphamide or anti-neutrophil antibodies.
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Role of neutrophils in hemorrhage- or endotoxemia-induced increases
in lung cytokine expression.
Hemorrhage or endotoxemia resulted in increased pulmonary
mRNA levels for IL-1, MIP-2, and TNF-
(Fig.
4). Although the amounts of mRNA
for these proinflammatory cytokines were significantly elevated
compared with control level after hemorrhage, the increases produced by
endotoxin injection were ~10- to 100-fold greater than those
occurring after hemorrhage.
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DISCUSSION |
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In these experiments, neutrophils were demonstrated to have a
central role in initiating an acute inflammatory response and in
causing injury to the lungs after hemorrhage or endotoxemia. Elimination of neutrophils using two techniques with different mechanisms for producing neutropenia, cyclophosphamide or
anti-neutrophil antibody therapy, diminished hemorrhage- or
endotoxin-induced lung edema, activation of NF-B, and expression of
IL-1
and MIP-2. TNF-
generation in the lungs after hemorrhage but
not after endotoxin administration also appeared to be neutrophil dependent.
Previous experiments have shown that neutrophil depletion prevented
endotoxin-induced lung edema (14). However, the role that
neutrophils have in producing lung injury after hemorrhage has not been
well explored previously. In the present studies, both hemorrhage and
endotoxemia induced NF-B activation and proinflammatory cytokine
expression in the lungs. However, the magnitude of NF-
B activation
and enhancement of cytokine expression was greater after endotoxemia
than after hemorrhage. For example, in the case of pulmonary IL-1
mRNA or protein, the increase from baseline, control conditions
produced by hemorrhage was less than fivefold, whereas levels rose more
than 20-fold after endotoxemia. Similar differences between hemorrhage-
and endotoxemia-induced effects were found for MIP-2 and TNF-
. These
results indicate that the magnitude of induction of signaling pathways
initiated by endotoxemia and leading to NF-
B activation and
proinflammatory cytokine expression is substantially greater than that
produced by hemorrhage.
In a previous study (30), we found that the enhanced
expression of IL-1, TNF-
, and MIP-2 in lung neutrophils is
dependent on activation of xanthine oxidase after hemorrhage but not
after endotoxemia. Additionally, activation of the transcription factor cAMP-responsive element binding protein was regulated by xanthine oxidase-dependent pathways after hemorrhage but not after
endotoxemia. Our previous study (30), coupled with
the present results in which both the magnitude and patterns of
endotoxin-induced increases in pulmonary cytokine expression differed
from those present after hemorrhage, indicated that the intracellular
signaling pathways initiated by endotoxemia and leading to increased
expression of proinflammatory cytokines in lung neutrophils are
distinct from those associated with hemorrhage.
Despite differences between endotoxemia or hemorrhage on NF-B
activation and cytokine expression, the magnitude of increase in lung
edema was similar with the two conditions. Although these results are
consistent with there being a threshold effect for pulmonary damage,
with no additional injury occurring even if there are further increases
in NF-
B activation and proinflammatory cytokine release, it is
possible that histopathological alterations, other than the measured
increase in lung edema, may differ between hemorrhage and endotoxemia,
reflecting the more pronounced effect of endotoxemia on NF-
B
activation and cytokine expression. An alternate explanation for the
present findings is that additional potent neutrophil-derived
proinflammatory mediators not measured in these experiments were
released after hemorrhage in amounts similar to those after endotoxemia
and that such mediators were more important than IL-1
or MIP-2 in
causing lung injury.
Neutrophil depletion prevented lung edema but did not decrease
pulmonary TNF- mRNA or protein levels after endotoxemia. These findings, like those of our previous experiments (30),
indicate that transcriptional regulatory pathways activated in the
lungs by hemorrhage and leading to enhanced proinflammatory cytokine expression are distinct from those associated with endotoxemia. These
results also suggest that TNF-
does not have a central role in
producing acute lung injury after endotoxemia. Such findings are
consistent with those in humans with ARDS, where IL-1
was demonstrated to be the most potent proinflammatory mediator in bronchoalveolar lavage, with TNF-
being much less significant (25).
Activation of the transcriptional factor NF-B is important in
modulating acute inflammatory responses (3, 4, 28). Association of NF-
B heterodimers with
B binding sites in the promoters of cytokines such as IL-1
, TNF-
, and MIP-2, as well as
of other proinflammatory mediators, including intercellular adhesion
molecule-1 and tissue factor, enhances expression of these proteins.
Increased activation of NF-
B occurs among lung cell populations in
models of acute lung injury due to hemorrhage or endotoxemia (6,
20, 21, 29, 30). NF-
B activation is also enhanced in
alveolar macrophages from patients with ARDS (22).
Previous experiments showed that inhibition of NF-
B activation prevented hemorrhage- or endotoxin-induced proinflammatory cytokine expression and neutrophil accumulation in the lungs (6, 20, 30).
In these experiments, hemorrhage- or endotoxemia-induced NF-B
activation was diminished in the lungs of neutropenic mice. We
previously demonstrated that NF-
B is activated in lung neutrophils after hemorrhage or endotoxemia (30). One explanation for
the present results is that most of the activation of NF-
B occurring in the lungs after hemorrhage or endotoxemia is in infiltrating neutrophils. In that case, neutrophil depletion decreases NF-
B activation in whole lung extracts simply by eliminating the major cell
population in which such activation of NF-
B occurs. However, an
alternate explanation is that neutrophils, by generating reactive oxygen intermediates, cytokines, or other proinflammatory mediators, initiate an inflammatory response in the lungs through which NF-
B becomes activated in other pulmonary cell populations. In this scenario, neutrophils are the initiators of an inflammatory response but are not the major cell population in which NF-
B is activated. Further in situ studies will resolve this issue.
Inhibition of neutrophil accumulation in the lungs, such as through the
use of anti-adhesion molecule therapies, decreases the severity of lung
injury after endotoxin administration, ischemia-reperfusion, or other
pathophysiological insults (10, 16). The benefit of such
interventions generally has been ascribed to inhibiting the effector
functions of neutrophils, primarily the release of reactive oxygen
intermediates and proteolytic mediators by which the lungs are directly
damaged. However, neutrophils may have an alternate role in which they
initiate inflammatory responses. Neutrophils are transcriptionally
active and can produce a range of immunoregulatory molecules, such as
cytokines, that are capable of potentiating inflammatory responses
(2, 7, 30, 33). The present experiments are consistent
with this early immunomodulatory role for neutrophils because they show
that neutrophils are responsible for the initial activation of NF-B,
as well as of the increases in proinflammatory cytokines that occur in
the lungs after hemorrhage or endotoxemia.
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ACKNOWLEDGEMENTS |
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This work was supported by the National Heart, Lung, and Blood Institute Grants HL-50284 and HL-62221.
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FOOTNOTES |
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Address for reprint requests and other correspondence: E. Abraham, Div. of Pulmonary Sciences and Critical Care Medicine, Box C272, Univ. of Colorado Health Sciences Center, 4200 E. Ninth Ave., Denver, Colorado 80262 (E-mail: edward.abraham{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 9 March 2000; accepted in final form 10 June 2000.
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