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* Department of Pharmacology, University of Illinois College of Medicine, Chicago, IL 60612;
Department of Medicine, Division of Pulmonary and Critical Care Medicine, University of Michigan Medical School, Ann Arbor, MI 48109;
Laboratory of Host Defenses, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Bethesda, MD 20892; and
Department of Pediatrics, University of Indiana School of Medicine, Indianapolis, IN 46202
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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2 generated by the NADPH oxidase
complex in the mechanism of polymorphonuclear leukocyte (PMN)
accumulation and transalveolar migration and lung microvascular injury.
Studies were made in mice lacking the p47phox
and gp91phox subunits of NADPH oxidase
(p47phox-/- and
gp91phox-/-) in which PMN are incapable
of the respiratory burst. The mice were challenged i.p. with live
Escherichia coli to induce sepsis. We observed
time-dependent increases in PMN sequestration and migration from 1 to
6 h after challenge with 2 x 108 E.
coli. The responses in knockout mice were greater
post-E. coli challenge compared with control mice; i.e.,
transalveolar PMN migration post-E. coli challenge
increased by 
50% in the null mice above values in wild type. The
increased PMN infiltration was associated with decreased lung bacterial
clearance. The generation of the chemoattractant
macrophage-inflammatory protein-2 in lung tissue was greater in NADPH
oxidase-defective mice after E. coli challenge than
control mice; moreover, macrophage-inflammatory protein-2 Ab
pretreatment prevented the PMN infiltration. We also observed that
E. coli failed to increase lung microvascular
permeability in p47phox-/- and
gp91phox-/- mice despite the greater
lung PMN sequestration. Thus, O2 production is required for
the induction of sepsis-induced lung microvascular injury. We conclude
that NADPH oxidase-derived O2 generation has an important
bactericidal role, such that an impairment in bacterial clearance in
NADPH oxidase-defective mice results in increased chemokine generation
and lung tissue PMN infiltration. |
Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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2) by the NADPH oxidase complex of PMN is
indispensable to the host defense response, as it is essential for
killing of invading microorganisms (5, 6, 7, 8). NADPH oxidase
is a multisubunit complex that generates O2 in one-electron
reduction of O2 using electrons supplied by NADPH
(9). NADPH oxidase subunits,
gp91phox and p47phox, are
essential components of this complex (10, 11, 12, 13, 14, 15). Mutations
in these subunits in chronic granulomatous disease (CGD) patients
(8, 16) or targeted deletions of these genes in mice
(10, 11) resulted in PMN incapable of the respiratory
burst and failure of PMN bactericidal function.
gp91phox knockout mice also had increased the
susceptibility to Staphylococcus aureus and
Aspergillus fumigatus, two important causes of infection in
patients with CGD, and a high mortality compared with wild-type mice
(11, 17).
Although it is known that the acute lung injury associated with
Gram-negative sepsis is dependent on PMN infiltration and activation
(1, 18), it is unclear the extent to which oxidant
generation itself is a determinant of PMN infiltration in lung tissue.
Some reports indicate that impaired O2 production can promote
increased leukocyte migration (17, 19, 20). For example,
NADPH oxidase knockout mice (p47phox-/-
and gp91phox-/-) exhibited increased
peritoneal leukocytosis in response to thioglycolate (10, 11). In the present study, we used
p47phox-/- and
gp91phox-/- mice to address the role of
the PMN respiratory burst in regulating PMN sequestration in lung
tissue and migration into airspaces and the contribution of oxidant
generation in the mechanism of lung microvascular injury. We observed
that there is greater lung tissue PMN sequestration and transalveolar
PMN migration in p47phox-/- and
gp91phox-/- mice compared with wild-type
mice after live Escherichia coli challenge. However, the
lack of PMNO2 generation in these mice prevented lung
microvascular injury. Lung tissue PMN sequestration and transalveolar
migration were associated with increased bacterial load and dependent
on the generation of ELR+ (glutamic
acid-leucine-arginine motif-positive) CXC chemokine,
macrophage-inflammatory protein (MIP)-2, the functional murine homolog
of IL-8. Thus, PMN infiltration in lung tissue can occur in the absence
of overt lung microvascular injury. Moreover, increased bacterial load
in NADPH oxidase deficiency is a critical factor in activating the
release of chemokines and in thereby augmenting PMN sequestration and
migration into lung tissue.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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p47phox-/- and gp91phox-/- mice, generated as described (10, 11), lack the cytosolic p47phox and membrane gp91phox subunits, respectively. Both subunits are required for assembly of the active NADPH oxidase complex. Wild type of C57BL/6 mice (obtained from The Jackson Laboratory, Bar Harbor, ME) were used as controls. Mice (n = 425) weighing 22–26 g, 9–12 wk old, were used in all experiments. Mice were housed in pathogen-free conditions with free access to food and water at the University of Illinois Animal Care Facility (Chicago, IL). The knockout mice were backcrossed to the C57BL/6 background. All studies were made in accordance with institutional and National Institutes of Health guidelines, and after approval from the Institutional Review Board.
Reagents
Murine recombinant MIP-2 and keratinocyte-derived cytokine (KC), used for generation of Abs and standards for ELISA, were purchased from R&D Systems (Minneapolis, MN). Polyclonal anti-murine MIP-2 or anti-KC antisera used in the ELISA were produced by immunization of rabbits with recombinant murine MIP-2 or KC following multiple intradermal sites with CFA. MIP-2 and KC antisera and control rabbit antiserum were made as described (21, 22). We showed that the polyclonal anti-murine MIP-2 Ab was specific, as it did not react with other members of the murine ELR+ CXC chemokine family or with ELR- CXC, C, or CC chemokines (23, 24).
Isolation of murine AM
Mice maintained under pathogen-free conditions were anesthetized
and exsanguinated, and the exposed trachea was intubated using a 1.7-mm
OD polyethylene catheter. Bronchoalveolar lavage (BAL) was performed by
instilling 0.5-ml aliquots of PBS containing 5 mM EDTA to obtain
alveolar macrophages (AM). Approximately 10 ml of lavage fluid was
retrieved per mouse, resulting in isolation of 2.5–5 x
105 AM per animal. BAL from 15 mice was pooled
for analysis. The alveolar cells were washed using complete medium,
followed by cell counting and differential cell analysis. Under basal
conditions >95% cells present in BAL were AM in the both wild-type
and p47phox-/- mice. Cell viability (as
determined by trypan blue exclusion) was >90% in the AM isolated
from both control C57BL/6 and NADPH oxidase knockout mice; thus,
differences in cell viability is unlikely to explain the differential
release of chemokines between the groups. AM were
adherence-purified, then cultured at a concentration of 5 x
105 AM per ml RPMI 1640 in a 24-well culture
plate for protein analysis.
Morphometric analysis of lung tissue PMN sequestration
We used a computer-based stereological method to quantify the
number of PMN in lung tissue. This assessment was made in a blinded
fashion without knowledge of tissue sections, which were prepared by
inflating the mouse lungs with 10% formalin and embedding in paraffin.
Tissue blocks were sectioned to 5 µm thick and mounted onto glass
slides. The H&E-stained tissue sections were visualized using a high
magnification objective with a high numerical aperture. The
computerized optical counting system consisted of a microscope,
computer-controlled x-y-z motorized stage, high-sensitivity video
camera, computer-assisted image capture, and stereological software
program from MicroBrightField (Colchester, VT). The instrumentation was
calibrated before each measurement. The middle region (30
mm2) of the upper lobe of the left lung was
outlined at low magnification (x1.25). At least 5% of the outlined
region was measured with a systematic random design of counting frame
using a x100 oil immersion objective with a 1.4 numerical aperture
(18, 25). The total number of PMN in the outlined region
of lung was determined using the following formula: n =
Q- x SSF/ASF, where
Q- is total number of PMN counted by optical
evaluation using a random design procedure for all measurements, ASF is
the area sampling fraction, and SSF is the section sampling fraction.
The area sampling fraction (ASF) is the counting frame (6400
µm2) and section sampling fraction (SSF) is the
fraction of section sampled in the region of lung. This morphometric
approach enabled the quantification of lung tissue PMN uptake
(18).
PMN counts in BAL fluid
BAL was performed by cannulating the trachea with a blunt-ended 21-gauge needle, instilling 0.5 ml of sterile PBS containing 1 mM EDTA, and collecting the fluid by gentle aspiration. The total fluid 0.4 ml was centrifuged for 5 min at 300 rpm using a Cytospin3 (Thermo Shandon, Pittsburgh, PA), and BAL cells were stained with HEMA3 (Fisher, Chicago, IL). PMN count was determined by counting 300 cells per slide (1, 26).
Measurement of lung tissue chemokines
Mice were sacrificed by i.p. anesthetic at designated time points. Whole lungs were then obtained for measurement of MIP-2 and KC concentrations. Before removal of lungs, the pulmonary vasculature was perfused with 1 ml of PBS containing 5 mM EDTA via the right ventricle. After removal, lungs were homogenized in 3 ml of lysis buffer containing 0.5% Triton X-100, 150 mM NaCl, 15 mM Tris, 1 mM CaCl2, and 1 mM MgCl2 (pH 7.4) using a tissue homogenizer (Dremel, Racine, WI). Homogenates were incubated on ice for 30 min and centrifuged at 1500 x g for 10 min. Supernatants were collected, passed through a 0.45-µm filter (Gelman Sciences, Ann Arbor, MI), and stored at -20°C for measurement of chemokine concentrations (27).
Chemokine ELISA
Murine MIP-2 and KC concentrations were quantitated by a
modification of a double ligand method as described (27).
In brief, flat-bottom 96-well microtiter plates (Immuno-Plate I 96-F;
Nunc, Roskilde, Denmark) were coated with 50 µl/well rabbit
anti-MIP-2 Ab or anti-KC Ab (1 µg/ml in 0.6 M NaCl, 0.26
MH3BO4, and 0.08 N NaOH, pH
9.6) for 16 h at 4°C, and then washed with PBS (pH 7.5) with
0.05% Tween 20 (wash buffer). Nonspecific binding sites on microtiter
plates were blocked with 2% BSA in PBS. After incubation for 90 min at
37°C, the plates were rinsed four times with wash buffer and diluted
(neat and 1/10). Cell-free supernatants (50 µl) in duplicate were
added, and this was followed by incubation for 1 h at 37°C.
Plates were washed four times, followed by the addition of 50 µl/well
biotinylated rabbit anti-MIP-2 or anti-KC Ab (3.5 µg/ml in
PBS (pH 7.5) with 0.05% Tween 20 and 2% FCS), and plates were
incubated for 30 min at 37°C. Plates were washed four times,
streptavidin-peroxidase conjugate (Bio-Rad, Richmond, CA) was added,
and the plates were incubated for 30 min at 37°C. Plates were washed
again four times, and chromogen substrate (Bio-Rad) was added. Plates
were incubated at room temperature to the desired extinction, and the
reaction was terminated with 50 µl/well 3 M
H2SO4 solution. Plates were
read at 490 nm in an ELISA reader. Standards were 0.5-log dilutions of
recombinant murine MIP-2 or KC from 1 pg/ml to 100 ng/ml. The ELISA
method was able to detect murine MIP-2 and KC concentrations
above 25 pg/ml. The ELISA did not cross-react with TNF-
, IL-2, IL-4,
IL-6, IFN-
, or with other members of murine chemokine family, murine
JE/monocyte chemoattractant protein-1, MIP-, growth-related
oncogene-, neutrophil-activating peptide-2, epithelial
neutrophil-activating peptide-78, or granulocyte chemotactic
protein-2 (27). The anti-MIP-2 and anti-KC Abs
also did not interfere with measurements of MIP-2 and KC by ELISA
(21, 24).
Nuclear protein isolation
Nuclear proteins were isolated as described (28). Briefly, lungs were minced and incubated on ice for 30 min in 0.5 ml of ice-cold buffer A (10 mM HEPES (pH 7.9), 1.5 mM KCl, 10 mM MgCl2, 0.5 mM DTT, 0.1% IGEPAL CA-630, and 0.5 mM PMSF. The minced tissue was homogenized using a Dounce homogenizer followed by centrifugation at 5000 x g at 4°C for 10 min. The crude nuclear pellet was suspended in 200 µl of buffer B (20 mM HEPES (pH 7.9), 25% glycerol, 1.5 mM KCl, 420 mM NaCl, 0.5 mM DTT, 0.2 mM EDTA, 0.5 mM PMSF, and 4 µM leupeptidin) and incubated on ice for 30 min. The suspension was centrifuged at 16,000 x g at 4°C for 30 min. The supernatant (nuclear protein) was collected and kept at -70°C until use.
EMSA
EMSA was performed as described (29, 30). Briefly,
nuclear extract (10–15 µg) was incubated with 1 µg of poly(dI-dC)
in a binding buffer (10 mM Tris-HCl (pH 7.5), 50 mM NaCl, 0.5 mM DTT,
10% glycerol (20 µl final volume)) for 15 min at room temperature.
Then end-labeled double-stranded oligonucleotides containing the
NF-
B site (30,000 cpm each) were added and the reaction mixtures
were incubated for 15 min at room temperature. The DNA-protein
complexes were resolved by native PAGE in low ionic strength buffer
(0.25x Tris-borate-EDTA). The oligonucleotide used for the gel shift
analysis, NF-B 5'-AGTTGAGGGGACTTTCCCAGGC-3',
contains the consensus NF-B binding site. The sequence motifs within
the oligonucleotide are underlined.
Determination of plasma and lung CFU
Plasma was collected at the time of sacrifice, and the lungs were removed aseptically and placed in 3 ml of sterile saline. Tissue was homogenized with a tissue homogenizer under a vented hood. Plasma and lung homogenates were placed on ice and serial 1/10 dilutions were made. Ten microliters of each dilution was plated on soy-based blood agar plates (Difco, Detroit, MI) and incubated for 18 h at 37°C, after which the formed colonies were counted (22, 27). Bacterial colonies were identified using substrate-specific use criteria (BBL Enterotube II; BD Biosciences, Sparks, MD).
ICAM-1 Western blotting
ICAM-1 expression in lung tissue was determined by Western blotting (18). Briefly, lungs were homogenized in PBS containing protease inhibitor mixture (Sigma-Aldrich, St. Louis, MO). Protein concentration was measured in an aliquot of the tissue homogenate. Homogenates (containing equal amounts of protein) were then electrophoresed on 8% SDS-PAGE gels, transferred to Immobilon-P (Millipore, Bedford, MA), blocked with 5% nonfat milk, and analyzed by Western blotting using the anti-ICAM-1 Ab (Santa Cruz Biotechnology, Santa Cruz, CA).
Pulmonary microvascular permeability and isogravimetric lung water determinations
Mice were anesthetized with an i.p. injection of ketamine (60 mg/kg), xylazine (2.5 mg/kg), and acepromazine (2.5 mg/kg) in PBS. The trachea was cannulated with a polyethylene tube (PE 60; BD Biosciences, Parsippany, NJ) for constant positive pressure ventilation (rate of 186 breaths per minute). Heparin (50 U) was injected into the jugular vein as an anticoagulant. The abdominal cavity was opened to expose the diaphragm, which was ventrally punctured and cut free from the rib cage. A thoracotomy was then performed and the rib cage was retracted to expose the heart and lungs. The heart was caudally retracted with a silk suture (6-0; Ethicon, Somerville, NJ) to make the pulmonary artery accessible for cannulation. An incision was made in the right ventricle at the base of the pulmonary artery for introducing an arterial cannula and another incision was made in the left atrium for drainage of venous effluent. In some preparations, a left atrial catheter was inserted. A polyethylene cannula (PE 60) was advanced into the pulmonary artery through the pulmonic valve and secured by means of a suture around the pulmonary artery that included the aorta. The lungs were perfused in situ using a peristaltic pump and the ventilation was continued with room air. The heart and exsanguinated lungs were rapidly excised and transferred en bloc to the perfusion apparatus, where lung preparations were suspended from a 6-cm Perspex lever arm anchored to the sensor element of a force-displacement transducer (FT03; Astro-Med, West Warwick, RI). The isolated lungs were ventilated (186 per minute) and perfused at constant flow (2 ml/min), temperature (37°C), and venous pressure (0 cm H2O) with modified Krebs-Henseleit solution (31, 32), supplemented with 5 g/100 ml of BSA (Fraction V, 99% pure and endotoxin-free; Sigma-Aldrich). Pulmonary arterial pressure was monitored throughout the experiment using a Gould pressure transducer (model P23ID; Gould, Dayton, OH). Lung wet weight was electronically nulled when the tissue was mounted and subsequent weight changes due to gain or loss of fluid from the lung were recorded. Lung weight and arterial pressure recordings were continuously displayed on a video monitor with aid of amplifiers (Astro-Med), an analog-to-digital converter (Scientific Solution, Solon, OH), and commercial software for acquisition of data (Notebook Pro for Windows; Labtech, Andover, MA). All lung preparations underwent a 20-min equilibration period.
Capillary filtration coefficient (Kf,c) was measured to determine pulmonary microvascular permeability to liquid as described (33, 34). Briefly, after the standard 20-min equilibration perfusion, a step increase in the outflow pressure to 10 cm H2O was applied for 2 min. The lung wet weight changed in a ramp-like fashion, reflecting the net transvacular fluid extravasation. At the end of each experiment, lungs were dissected free of nonpulmonary tissue, and lung dry weight was determined. Kf,c (milliliter per minute per centimeter of H2O per dry gram) was calculated from the slope of the recorded weight change normalized to the pressure change and lung dry weight.
As another approach for the assessment of leakiness of pulmonary microvessels, we determined the rate of pulmonary edema formation by continuously monitoring the lung wet weight change. Lung weight change of lungs obtained from different groups was followed for 90 min after beginning of the perfusion (described above). As the perfusate albumin concentration was constant at the onset of perfusion and pulmonary arterial pressure did not change during the 90-min monitoring period, the rate and magnitude of the increase in lung wet weight (i.e., attainment of a new isogravimetric state) provided another index of permeability of pulmonary microvessels.
Experimental protocols
E. coli infection.
Mice from different groups were challenged i.p. with a defined number
of CFU per milliliter (2 x 108 live
E. coli/100 µl; ATCC 25922; American Type Culture
Collection, Manassas, VA) (35). This E.
coli dosage did not result in death within the 6-h experimental
period after challenge. Control mice were injected i.p. with an equal
volume of PBS. Lungs obtained at 1 and 6 h after challenge were
used to assess PMN sequestration, PMN migration into airspace,
concentrations of murine (m)KC and mMIP-2, NF-B activation, ICAM-1
expression, Kf,c, and rate of lung
edema formation (until attainment of isogravimetric state). We also
determined bacterial clearance in lungs and plasma after E.
coli infection. AM were harvested from E.
coli-challenged mice to determine the generation of chemokines.
Heat-inactivated E. coli. Live E. coli bacteria were heated at 70°C for 10 min, which was sufficient to prevent growth of bacteria in culture plates for >48 h. The purpose of challenging mice with inactivated bacteria was to address the contribution of E. coli replication in the mechanisms of lung tissue PMN sequestration and microvessel injury. Lung PMN accumulation and transalveolar PMN migration, concentration of mKC and mMIP-2, Kf,c, and lung edema formation were determined as described above.
Effects of anti-MIP-2 Ab. Mice were injected i.p. with 0.5 ml MIP-2 antiserum (with Ab titer of 105–106), which is capable of detecting murine MIP-2 out to dilutions of 1/1 x 105 to 1/106. We administer 5 mg/mouse MIP-2 antiserum. Anti-MIP-2 antiserum and control rabbit antiserum were prepared as described (22). At 2 h after injection of antiserum, mice were challenged i.p. with 2 x 108 live E. coli/100 µl.
Statistical analysis
Data are expressed as mean ± SEM. Statistical analysis was performed using the two-way analysis of variance and Newman-Keuls test for multiple comparisons. The numbers of experiments in the different groups are given in the figures. The value of p < 0.05 was used to define significance.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Lung tissue PMN sequestration increased from basal value of
524 ± 79 cells/30 mm2 lung tissue to
3131 ± 114 cells at 1 h and 3437 ± 104 at 6 h
after live E. coli challenge in wild-type mice. A
significantly greater number of PMN was seen at 6 h in
p47phox-/- and
gp91phox-/- mice compared with wild-type
mice (Fig. 1
A;
n = 6 mice per group). The increase in PMN
sequestration observed at 6 h did not occur following challenge of
p47phox-/- and
gp91phox-/- mice with heat-inactivated
E. coli challenge (Table I).
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B; p <
0.05). Control Ab had no effect in altering lung PMN sequestration. Transalveolar PMN migration after E. coli challenge
Migration of PMN into the alveolar space showed the same pattern
as lung tissue infiltration of PMN. PMN migration increased with peak
response observed at 6 h after E. coli challenge. There
was 40–50% greater transalveolar PMN migration at 6 h in
p47phox-/- and
gp91phox-/- mice compared with wild-type
mice (Fig. 2A). The increased
PMN migration at 6 h was prevented by heat-inactivated E.
coli challenge of p47phox-/- and
gp91phox-/- mice (Table I). Pretreatment
with MIP-2 Ab prevented the augmentation of PMN migration in both
wild-type and NADPH oxidase-deficient mice (Fig. 2B).
Control rabbit serum had no effect on transalveolar PMN migration.
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Lung tissue m MIP-2 and KC concentrations increased after E.
coli challenge at 1 and 6 h in wild-type mice (Fig. 3A). There was a greater
induction of mMIP-2 at 1 h than 6 h after E. coli
challenge, whereas the mKC concentration continued to increase from 1
to 6 h. There were greater increases in mMIP-2 and mKC
concentrations at 6 h in p47phox-/-
and gp91phox-/- mice compared with
wild-type (Fig. 3, B and C) that paralleled the
augmented tissue PMN sequestration (Fig. 1) and PMN migration (Fig. 2)
responses in the null mice.
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). Pretreatment with MIP-2 Ab
prevented the induction of lung tissue mMIP-2 in wild-type mice and
both NADPH oxidase-deficient groups (Fig. 3, B and
C). Control antiserum had no effect on the increases in
chemokine concentrations.
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To address whether increased production of chemokines could
account for their augmented generation in lungs of null mice, we
determined the generation of MIP-2 and KC in cultured AM obtained
from E. coli-challenged mice. In contrast to data from whole
lungs (above), mMIP-2 and mKC concentrations in AM obtained at
6 h after E. coli challenge in
p47phox-/- mice were reduced compared
with wild-type mice (Fig. 4;
p < 0.05, n = 6 mice in each group).
The AM from wild-type and null mice demonstrated similar viability
(as determined by trypan blue exclusion) at the end of culture
period.
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B activation and ICAM-1 expression
We observed increases in lung tissue NF-B activation peaking at
1 h after E. coli challenge (Fig. 5A) and ICAM-1 expression
peaking at 6 h (Fig. 5B). However, both NF-B
activation and ICAM-1 expression were lower after E. coli
challenge of p47phox-/- and
gp91phox-/- mice as compared with values
in wild-type mice.
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We assessed bacterial clearance in mice infected with 2 x
108 live E. coli i.p. The bacterial
burden in blood and lungs at 6 h after E. coli
challenge was greater in p47phox-/- and
gp91phox-/- groups compared with
wild-type mice (Fig. 6; p
< 0.05, n = 6 mice in each group).
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E. coli infection resulted in increases in pulmonary
microvessel Kf,c (vessel wall liquid
permeability) (Fig. 7A) and
isogravimetric lung wet weight (Fig. 7B). Increases in
Kf,c and lung wet weight were time
dependent (p < 0.05 and n = 6
in each group) in the wild-type mice.
Kf,c (Fig. 7C) and lung wet
weight (Fig. 7D) did not increase significantly in either
knockout group challenged with E. coli. Increases in
Kf,c (Fig. 8A) and isogravimetric lung
wet weight observed at both 1 and 6 h after E. coli
challenge were prevented by i.p. injection of MIP-2 Ab 2 h before
the bacterial challenge (Figs. 8, B and C).
Control Ab had no effect in modifying the increased pulmonary
microvessel Kf,c and isogravimetric
weight change.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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We addressed the possibility that release of the ELR+ CXC chemokines, MIP-2 and KC, induced by E. coli (38, 39, 40, 41, 42, 43, 44, 45) could be responsible for attracting PMN into the airspace. We observed that lung tissue MIP-2 concentration increased 16-fold within 1 h and decreased by 6 h, and that KC concentration also increased but with a delayed time course. The increases in MIP-2 and KC concentrations were greater in NADPH oxidase-deficient mice. Pretreatment with the anti-MIP-2 Ab prevented both MIP-2 and KC release consistent with role of chemokines such as MIP-2 as autocrine mediators capable to activating production of other chemokines (46). Pretreatment with anti-MIP-2 Ab prevented the E. coli-induced PMN infiltration in control mice and also the augmented PMN infiltration response observed in p47phox-/- and gp91phox-/- mice. A possible explanation of this finding is that impairment of bactericidal function in the null mice induced the greater generation of the potent PMN chemoattractants, MIP-2 and KC, that in turn mediated the increased PMN influx.
We observed that ICAM-1 expression did not increase in NADPH oxidase
knockout mice in response to E. coli in contrast to the
marked ICAM-1 expression observed in wild-type mice. The
production of chemokines MIP-2 and KC in AM isolated from
E. coli-challenged mice was also reduced in the macrophages
from null mice. NF-B activation was impaired in the null mice in
response to E. coli challenge. Thus, NADPH
oxidase-derived superoxide generation may have a role in signaling the
activation of NF-B, and activation of NF-B-dependent genes such
as ICAM-1 and MIP-2 (47), in a redox-sensitive manner. The
reduced generation of chemokines by the isolated macrophages contrasts
with the increased concentration of these chemokines present in lung
tissue of the NADPH oxidase-null mice challenged with E.
coli. Although the basis of the latter observation is not clear,
the increased PMN migrating into lung tissue in the null mice could be
a source of the higher chemokine concentrations observed in these
animals. Another possibility is that the augmented PMN uptake in the
NADPH oxidase-null mice occurred by a NF-B-independent mechanism
(48). The present results are consistent with the recent
observations of Koay et al. (48). These investigators
showed that i.p. LPS induced the activation of NF-B in lung tissue,
which was reduced in p47phox-/- mice. As
in the present study, PMN migration into the airspace was augmented in
association with increased MIP-2 generation in the NADPH
oxidase-defective mice.
Another possible explanation for the observed reduction in MIP-2 and KC
generation in the AM isolated from NADPH oxidase knockout mice could
be that these cells become refractory to the E. coli;
however, this is unlikely, because MIP-2 and KC generation in the NADPH
oxidase-null mice was significantly reduced as compared with control
mice in response to an equivalent amount of heat-inactivated E.
coli (Table II). This would not occur had the macrophages become
refractory to the exposure to E. coli. Thus, the data
suggest a role of oxidant signaling in the regulation of chemokine
production in AM.
An important aspect of the present study involves the mechanism of lung microvascular injury induced by E. coli. Generation of oxidants by PMN and other phagocytic cells not only is essential for the host defense function directed toward killing invading microorganisms, as shown above, but also may injure the endothelial cell barrier. As LPS and inflammatory mediators failed to induce superoxide production in cells from p47phox-/- mice (49), the NADPH oxidase-null mice enabled us to address the in vivo role of NADPH oxidase-derived superoxide in mediating lung microvessel injury. We observed that pulmonary Kf,c (a measure of vessel wall liquid permeability) and isogravimetric lung wet weight failed to increase significantly in the knockout mice after challenge with E. coli. This was evident even in the face of marked lung PMN infiltration. Thus, the results demonstrate that NADPH oxidase-derived superoxide generation is required for the induction of lung microvascular injury in this murine model of Gram-negative sepsis.
The measurement of lung microvascular permeability in the present study involves determining the Kf,c, which provides a quantitative index of microvessel permeability to liquid (33, 34). An increase in lung microvascular permeability is an indication of endothelial barrier injury, although it does not define the site of injury. The ex vivo preparation is stable for a 3-h period, as evidenced by reproducible measurements of Kf,c during this time. Unlike methods relying on the accumulation of a tracer, in which the tracer uptake can be explained by increased vascular surface area and convective flux, the present studies show that the Kf,cis increased in control mouse lungs in response to E. coli challenge and that this response is abrogated in NADPH oxidase knockout mice.
The increase in lung microvascular permeability following
E. coli-induced septicemia was evident in the presence of a
relatively small increase in the number of PMN in the airspace (i.e.,
with only 4% PMN in BAL). In other studies, we showed that
intratracheal instillation of the same concentration and strain of
E. coli (such that the bacterial infection is
"compartmentalized" in the airspace) resulted in 80% PMN in BAL
but failed to induce lung microvessel injury (N. Xu, X. Gao, and Q.
Liu, unpublished observation). Thus, a small number of PMN activated by
bacteremia are capable of inducing lung microvessel injury. We also
observed that pretreatment of mice with anti-MIP-2 Ab prevented the
PMN infiltration into lung tissue as well as increased lung
microvascular permeability and increased isogravimetric wet weight
gain. These data point to an important role of MIP-2-mediated lung
tissue PMN infiltration in the mechanism of lung microvascular injury
induced by Gram-negative sepsis. Taken together, these data suggest
that the basis of lung microvessel injury involves 1) MIP-2-directed
PMN sequestration in lungs and 2) the secondary generation of NADPH
oxidase-derived superoxide anions.
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Acknowledgments |
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Footnotes |
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2 Address correspondence and reprint requests to Dr. Asrar B. Malik, Department of Pharmacology, University of Illinois College of Medicine, 835 South Wolcott Avenue (M/C 868), Chicago, IL 60612. E-mail address: abmalik{at}uic.edu
3 Abbreviations used in this paper: PMN, polymorphonuclear leukocyte; CGD, chronic granulomatous disease; BAL, bronchoalveolar lavage; MIP, macrophage-inflammatory protein; Kf,c, capillary filtration coefficient; KC, keratinocyte-derived cytokine; m, murine; AM, alveolar macrophage.
Received for publication October 22, 2001. Accepted for publication February 6, 2002.
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