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The Journal of Immunology, 2002, 168: 3974-3982.
Copyright © 2002 by The American Association of Immunologists

Role of NADPH Oxidase in the Mechanism of Lung Neutrophil Sequestration and Microvessel Injury Induced by Gram-Negative Sepsis: Studies in p47phox-/- and gp91phox-/- Mice1

Xiao-pei Gao*, Thedodore J. Standiford{dagger}, Arshad Rahman*, Michael Newstead{dagger}, Steven M. Holland{ddagger}, Mary C. Dinauer§, Qing-hui Liu* and Asrar B. Malik2,*

* Department of Pharmacology, University of Illinois College of Medicine, Chicago, IL 60612; {dagger} Department of Medicine, Division of Pulmonary and Critical Care Medicine, University of Michigan Medical School, Ann Arbor, MI 48109; {ddagger} 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


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
We addressed the role of O2 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
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
Accumulation of polymorphonuclear leukocytes (PMN)3 in lungs is a central event in the pathogenesis of inflammatory lung injury associated with Gram-negative sepsis (1, 2, 3, 4). Generation of superoxide anions (O2) 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.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Mice

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{phi}

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{phi}). Approximately 10 ml of lavage fluid was retrieved per mouse, resulting in isolation of 2.5–5 x 105 AM{phi} 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{phi} in the both wild-type and p47phox-/- mice. Cell viability (as determined by trypan blue exclusion) was >90% in the AM{phi} 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{phi} were adherence-purified, then cultured at a concentration of 5 x 105 AM{phi} 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 = {Sigma}Q- x SSF/ASF, where {Sigma}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-{alpha}, IL-2, IL-4, IL-6, IFN-{gamma}, or with other members of murine chemokine family, murine JE/monocyte chemoattractant protein-1, MIP-{alpha}, growth-related oncogene-{alpha}, 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-{kappa}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-{kappa}B 5'-AGTTGAGGGGACTTTCCCAGGC-3', contains the consensus NF-{kappa}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-{kappa}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{phi} 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.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
PMN sequestration in lungs after E. coli challenge

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. 1GoA; 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 IGo).



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  		FIGURE 1. A, PMN sequestration in lungs of wild-type (C57BL/6) and NADPH oxidase-deficient (p47phox-/- and gp91phox-/-) mice. All mice were challenged for 1 or 6 h with 2 x 108 E. coli (i.p.) and lungs were harvested for morphometric assay of PMN in interstitial space (see Materials and Methods). Asterisks indicate increase (p < 0.05) in PMN sequestration compared with basal (*) or 1-h wild-type value (**). #, Increase (p < 0.05) in PMN sequestration compared with 6-h wild-type value. B, Inhibitory effect of MIP-2 Ab on PMN sequestration after 6-h E. coli challenge. {dagger}, Decrease (p < 0.05) in PMN sequestration due to MIP-2 Ab. **, Increase (p < 0.05) in PMN sequestration relative to wild type. Results are mean values of six experiments; bars show mean ± SEM.

 

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  		Table I. PMN sequestration and migration induced by E. coli and heat-inactivated E. coli (6-h values)

 
Because chemoattractants such as MIP-2 may mediate the PMN uptake response in NADPH oxidase-defective mice, we studied the effects of MIP-2 Ab. Pretreatment with MIP-2 Ab prevented lung tissue PMN sequestration after E. coli challenge in both wild-type and NADPH oxidase knockout mice (Fig. 1GoB; 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. 2GoA). The increased PMN migration at 6 h was prevented by heat-inactivated E. coli challenge of p47phox-/- and gp91phox-/- mice (Table IGo). Pretreatment with MIP-2 Ab prevented the augmentation of PMN migration in both wild-type and NADPH oxidase-deficient mice (Fig. 2GoB). Control rabbit serum had no effect on transalveolar PMN migration.



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  		FIGURE 2. A, PMN migration into airspace of wild-type (C57BL/6) or NADPH oxidase-deficient (p47phox-/- and gp91phox-/-) mice. All mice were challenged for 1 (left panel) or 6 (right panel) h with 2 x 108 E. coli (i.p.). PMN counts in BAL fluid were determined post-E. coli (see Materials and Methods). Asterisks indicate increase (p < 0.05) in PMN migration compared with basal (*) or 6-h wild-type (**) value. B, Inhibitory effect of anti-MIP-2 Ab on PMN migration at 6 h after E. coli challenge. {dagger}, Decrease (p < 0.05) in PMN migration due to MIP-2 Ab. **, Increase (p < 0.05) in PMN migration relative to wild-type. PMN counts in BAL fluid are based on lavage of a comparable number of leukocytes from lungs of C57BL/6 or NADPH oxidase-deficient mice. Results are mean values of six experiments; bars show mean ± SEM.

 
Generation of MIP-2 and KC in lung tissue

Lung tissue m MIP-2 and KC concentrations increased after E. coli challenge at 1 and 6 h in wild-type mice (Fig. 3GoA). 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. 3Go, B and C) that paralleled the augmented tissue PMN sequestration (Fig. 1Go) and PMN migration (Fig. 2Go) responses in the null mice.



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  		FIGURE 3. A, Lung tissue chemokines measured by ELISA at 1 or 6 h after 2 x 108 E. coli challenge in wild-type mice (C57BL/6). *, Increases (p < 0.05) in lung tissue chemokines compared with control values (without E. coli challenge); **, changes (p < 0.05) compared with E. coli challenge at 1 h. B, Lung tissue mMIP-2 level after E. coli challenge without or with MIP-2 Ab pretreatment or control Ab at 6 h in wild-type and in NADPH oxidase-deficient mice (p47phox-/- and gp91phox-/-). C, Lung tissue mKC level after E. coli challenge without or with MIP-2 Ab pretreatment or control Ab at 6 h in wild-type mice and in NADPH oxidase-deficient mice (p47phox-/- and gp91phox-/-). Asterisks indicate increase (p < 0.05) in lung tissue chemokines. *, Increase (p < 0.05) in lung tissue chemokines compared with control values (without E. coli challenge); **, increase (p < 0.05) in lung tissue chemokines compared with 6-h wild-type value; {dagger}, decrease (p < 0.05) in concentration of lung tissue chemokines due to MIP-2 Ab. Results are mean of six experiments; bars indicate mean ± SEM.

 
Both mMIP-2 and mKC concentrations in lungs were reduced significantly in the p47phox-/- and gp91phox-/- mice after challenge with heat-inactivated E. coli compared with E. coli challenge, whereas there was no significant reduction in wild-type mice (Table IIGo). Pretreatment with MIP-2 Ab prevented the induction of lung tissue mMIP-2 in wild-type mice and both NADPH oxidase-deficient groups (Fig. 3Go, B and C). Control antiserum had no effect on the increases in chemokine concentrations.


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  		Table II. MIP-2 and KC concentrations in lungs induced by E. coli and heated-inactivated E. coli (6-h values)

 
Generation of MIP-2 and KC in AM{phi}

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{phi} obtained from E. coli-challenged mice. In contrast to data from whole lungs (above), mMIP-2 and mKC concentrations in AM{phi} obtained at 6 h after E. coli challenge in p47phox-/- mice were reduced compared with wild-type mice (Fig. 4Go; p < 0.05, n = 6 mice in each group). The AM{phi} 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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  		FIGURE 4. mMIP-2 and mKC concentrations in AM{phi} in wild-type and p47phox-/- mice challenged with E. coli. A, mMIP-2 concentration in AM{phi} obtained at 6 h after E. coli challenge. B, mKC concentration in AM{phi} obtained at 6 h after E. coli challenge. Asterisks indicate increase (p < 0.05) in AM{phi} chemokine concentrations compared with basal level. **, Decrease (p < 0.05) in concentration of AM{phi} chemokine concentrations in p47phox-/- mice compared with wild-type mice. Results are mean of six experiments; bars indicate mean ± SEM.

 
NF-{kappa}B activation and ICAM-1 expression

We observed increases in lung tissue NF-{kappa}B activation peaking at 1 h after E. coli challenge (Fig. 5GoA) and ICAM-1 expression peaking at 6 h (Fig. 5GoB). However, both NF-{kappa}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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  		FIGURE 5. E. coli-induced NF-{kappa}B activation (A) and ICAM-1 expression (B) in mouse lungs. Lungs were isolated at the indicated times after 2 x 108 E. coli challenge of wild-type, p47phox-/-, or gp91phox-/- mice. A, Nuclear extracts were assayed for NF-{kappa}B binding activity by EMSA (see Materials and Methods). B, Total cell lysates were separated by SDS-PAGE and immunoblotted with an Ab against ICAM-1 (see Materials and Methods). Basal expression of ICAM-1 is similar in NADPH oxidase knockout mice as compared with wild-type controls. These studies were conducted four times with similar results.

 
Bacterial clearance

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. 6Go; p < 0.05, n = 6 mice in each group).



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  		FIGURE 6. Lungs and plasma bacterial clearances at 6 h following i.p. administration of E. coli (2 x 108 CFU) in wild-type and in NADPH oxidase-deficient mice. A, Lung bacterial burden after E. coli challenge at 6 h in wild-type and in NADPH oxidase-deficient mice (p47phox-/- and gp91phox-/-). B, Plasma bacterial burden after E. coli challenge at 6 h in wild-type mice and in NADPH oxidase-deficient mice (p47phox-/- and gp91phox-/-). *, Increase (p < 0.05) in bacterial burden in NADPH oxidase-deficient mice compared with wild-type (C57BL/6) mice; {dagger}, increase (p < 0.05) in bacterial burden of gp91phox-/- compared with p47phox-/- value. Results are mean of six experiments; bars indicate mean ± SEM.

 
Lung microvessel permeability and isogravimetric edema formation

E. coli infection resulted in increases in pulmonary microvessel Kf,c (vessel wall liquid permeability) (Fig. 7GoA) and isogravimetric lung wet weight (Fig. 7GoB). 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. 7GoC) and lung wet weight (Fig. 7GoD) did not increase significantly in either knockout group challenged with E. coli. Increases in Kf,c (Fig. 8GoA) 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. 8Go, B and C). Control Ab had no effect in modifying the increased pulmonary microvessel Kf,c and isogravimetric weight change.



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  		FIGURE 7. A, Pulmonary Kf,c of wild-type mice (C57BL/6) challenged in vivo with E. coli for the indicated periods (1, 3, or 6 h). *, Increases (p < 0.05) in Kf,c from basal value (without E. coli challenge). B, Time course of changes in wet weight of perfused lung preparations challenged in vivo with E. coli. The control group received sham injection of PBS 6 h before lung isolation. *, Increase (p < 0.05) in lung wet weight compared with control. C, Pulmonary Kf,c of wild-type mice or mice with NADPH oxidase deficiency; lungs were isolated and studied 6 h after E. coli challenge or sham injection (Control). *, Increase (p < 0.05) in Kf,c compared with control value; **, decrease (p < 0.05) in Kf,c value than in wild-type mice challenged with E. coli for 6 h (filled bar). D, Time course of change in wet weight of perfused lung preparations. Control preparations received sham injection. Wild-type (C57BL/6) and NADPH oxidase-deficient (p47phox-/- or gp91phox-/-) mice were challenged with E. coli for 6 h. Changes in lung isogravimetric water content were obtained at different times after E. coli challenge of wild-type mice and NADPH oxidase-deficient mice. *, Increase (p < 0.05) of lung wet weight compared with control. Results are mean of six experiments; bars indicate mean ± SEM.

 


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  		FIGURE 8. A, Pulmonary Kf,c in wild-type mice (C57BL/6) after 3 or 6 h of E. coli challenge without or with pretreatment of MIP-2 Ab. *, Increase (p < 0.05) in Kf,c from control value (without E. coli challenge). {dagger}, Decrease (p < 0.05) in Kf,c secondary to MIP-2 Ab. Results are mean of six experiments; bars indicate mean ± SEM. B, Time course of changes in wet weight of lungs of wild-type mice (C57BL/6) after a 3-h E. coli challenge without or with pretreatment of MIP-2 Ab. The control group received sham injection of PBS before lung isolation. *, Increases (p < 0.05) in lung wet weight compared with control. {dagger}, Decrease (p < 0.05) in lung wet weight secondary to MIP-2 Ab. C, Time course of changes in wet weight of perfused lung preparations of wild-type mice after a 6-h E. coli challenge without or with pretreatment of MIP-2 Ab. The control group received sham injection of PBS before lung isolation. *, Increase (p < 0.05) in lung wet weight compared with control. {dagger}, Decrease (p < 0.05) in wet weight of lungs secondary to MIP-2 Ab. Results are mean of six experiments; bars indicate mean ± SEM.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
The superoxide anions generated by the NADPH oxidase complex of phagocytic cells serve the primary function of killing invading microorganisms (6, 8, 36). This is evident by the finding of greater susceptibility to bacterial infections such as S. aureus and A. fumigatus in p47phox-/- and gp91phox-/- mice (11, 17). Acute lung injury associated with Gram-negative sepsis is characterized by marked lung tissue infiltration of PMN (1, 18). As the mechanisms underlying lung tissue PMN influx remain unclear, we addressed the possibility that superoxide generated by NADPH oxidase has an additional role in contributing to the sequestration of PMN in lungs. In addition, we addressed whether the sequestered PMN may be involved in the mechanism of lung microvascular injury, an important feature of sepsis-induced acute lung injury. To study the in vivo role of NADPH oxidase-derived superoxide, we used p47phox-/- and gp91phox-/- mice in which the phagocytic cells are incapable of the respiratory burst (11, 37). We observed that E. coli challenge of control mice resulted in increases in lung tissue PMN sequestration and migration from 1 to 6 h after the challenge, whereas these responses in the null mice were augmented; for example, transalveolar PMN migration was 40–50% greater in the NADPH oxidase-defective mice after E. coli challenge than in control mice. An explanation of this finding is evident from the observation that PMN sequestration was prevented by challenging the mice with heat-inactivated E. coli; thus, it appears that the augmented response is secondary to impaired microbial killing in the null mice. We also observed that bacterial clearance from lungs and blood of NADPH oxidase-deficient mice was impaired consistent with the disrupted bactericidal function in these animals. In previous studies, p47phox-/- and gp91phox-/- mice also showed increased peritoneal leukocytosis in response to thioglycolate (10, 11). Also, intratracheal inoculation of heat-killed A. fumigatus in a X-linked mouse model of CGD (gp91phox-/-) was accompanied by enhanced PMN accumulation (11). Thus, the present studies demonstrate a role of superoxide-mediated bacterial killing as an important factor that limits lung tissue PMN infiltration.

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{phi} isolated from E. coli-challenged mice was also reduced in the macrophages from null mice. NF-{kappa}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-{kappa}B, and activation of NF-{kappa}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-{kappa}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-{kappa}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{phi} 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 IIGo). 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{phi}.

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.


   Acknowledgments
 
We gratefully acknowledge Dr. Shuan Y. Ma for his expertise in analyzing morphometric data of lung tissue PMN and Dr. Khandakar Anwar for helpful technical assistance.


   Footnotes
 
1 This work was supported by National Institutes of Health Grants HL60678, HL27601, and HL45638 (to A.B.M.), and HL57243 and P50HL60289 (to T.J.S.). Back

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 Back

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{phi}, alveolar macrophage. Back

Received for publication October 22, 2001. Accepted for publication February 6, 2002.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

  1. Zhou, M. Y., S. K. Lo, M. Bergenfeldt, C. Tiruppathi, A. Jaffe, N. Xu, A. B. Malik. 1998. In vivo expression of neutrophil inhibitory factor via gene transfer prevents lipopolysaccharide-induced lung neutrophil infiltration and injury by a {beta}2 integrin-dependent mechanism. J. Clin. Invest. 101:2427.[Medline]
  2. Kaslovsky, R. A., K. Parker, A. Siflinger-Birnboim, A. B. Malik. 1995. Increased endothelial permeability after neutrophil activation occurs by a diffusion-dependent mechanism. Microvasc. Res. 49:227.[Medline]
  3. Malik, A. B., S. K. Lo. 1996. Vascular endothelial adhesion molecules and tissue inflammation. Pharmacol. Rev. 48:213.[Medline]
  4. Doerschuk, C. M., J. P. Mizgerd, H. Kubo, L. Qin, T. Kumasaka. 1999. Adhesion molecules and cellular biomechanical changes in acute lung injury: Giles F. Filley lecture. Chest 116:37S.[Free Full Text]
  5. Chang, Y. C., B. H. Segal, S. M. Holland, G. F. Miller, K. J. Kwon-Chung. 1998. Virulence of catalase-deficient Aspergillus nidulans in p47phox-/- mice: implications for fungal pathogenicity and host defense in chronic granulomatous disease. J. Clin. Invest. 101:1843.[Medline]
  6. Hiraoka, W., N. Vazquez, W. Nieves-Neira, S. J. Chanock, Y. Pommier. 1998. Role of oxygen radicals generated by NADPH oxidase in apoptosis induced in human leukemia cells. J. Clin. Invest. 102:1961.[Medline]
  7. Leusen, J. H. W., A. J. Verhoeven, D. Roos. 1996. Interactions between the components of the human NADPH oxidase: a review about the intrigues in the phox family. Front. Biosci. 1:d72.[Medline]
  8. Gallin, J. I., E. S. Buescher, B. E. Seligmann, J. Nath, T. Gaither, P. Katz. 1983. National Institutes of Health conference: recent advances in chronic granulomatous disease. Ann. Intern. Med. 99:657.
  9. Dodd, O. J., D. B. Pearse. 2000. Effect of the NADPH oxidase inhibitor apocynin on ischemia-reperfusion lung injury. Am. J. Physiol. 279:H303.[Abstract/Free Full Text]
  10. Jackson, S. H., J. I. Gallin, S. M. Holland. 1995. The p47phox mouse knock-out model of chronic granulomatous disease. J. Exp. Med. 182:751.[Abstract/Free Full Text]
  11. Pollock, J. D., D. A. Williams, M. A. Gifford, L. L. Li, X. Du, J. Fisherman, S. H. Orkin, C. M. Doerschuk, M. C. Dinauer. 1995. Mouse model of X-linked chronic granulomatous disease, an inherited defect in phagocyte superoxide production. Nat. Genet. 9:202.[Medline]
  12. Pagano, P. J.. 2000. Vascular gp91phox: beyond the endothelium. Circ. Res. 87:1.[Free Full Text]
  13. Yu, L., M. T. Quinn, A. R. Cross, M. C. Dinauer. 1998. gp91phox is the heme binding subunit of the superoxide-generating NADPH oxidase. Proc. Natl. Acad. Sci. USA 95:7993.[Abstract/Free Full Text]
  14. Finkel, T.. 2000. Redox-dependent signal transduction. FEBS Lett. 476:52.[Medline]
  15. Hsich, E., B. H. Segal, P. J. Pagano, F. E. Rey, B. Paigen, J. Deleonardis, R. F. Hoyt, S. M. Holland, T. Finkel. 2000. Vascular effects following homozygous disruption of p47phox: an essential component of NADPH oxidase. Circulation 101:1234.[Abstract/Free Full Text]
  16. Segal, B. H., T. M. Doherty, T. A. Wynn, A. W. Cheever, A. Sher, S. M. Holland. 1999. The p47phox-/- mouse model of chronic granulomatous disease has normal granuloma formation and cytokine responses to Mycobacterium avium and Schistosoma mansoni eggs. Infect. Immun. 67:1659.[Abstract/Free Full Text]
  17. Morgenstern, D. E., M. A. Gifford, L. L. Li, C. M. Doerschuk, M. C. Dinauer. 1997. Absence of respiratory burst in X-linked chronic granulomatous disease mice leads to abnormalities in both host defense and inflammatory response to Aspergillus fumigatus. J. Exp. Med. 185:207.[Abstract/Free Full Text]
  18. Gao, X., N. Xu, M. Sekosan, D. Mehta, S. Y. Ma, A. Rahman, A. B. Malik. 2001. Differential role of CD18 integrins in mediating lung neutrophil sequestration and increased microvascular permeability induced by Escherichia coli in mice. J. Immunol. 167:2895.[Abstract/Free Full Text]
  19. Gallin, J. I., E. S. Buescher. 1983. Abnormal regulation of inflammatory skin responses in male patients with chronic granulomatous disease. Inflammation 7:227.[Medline]
  20. Cooper, A. M., B. H. Segal, A. A. Frank, S. M. Holland, I. M. Orme. 2000. Transient loss of resistance to pulmonary tuberculosis in p47phox-/- mice. Infect. Immun. 68:1231.[Abstract/Free Full Text]
  21. Standiford, T. J., R. M. Strieter, N. W. Lukacs, S. L. Kunkel. 1995. Neutralization of IL-10 increases lethality in endotoxemia: cooperative effects of macrophage inflammatory protein-2 and TNF. J. Immunol. 155:2222.[Abstract]
  22. Moore, T. A., M. W. Newstead, R. M. Strieter, B. Mehrad, B. L. Beaman, T. J. Standiford. 2000. Bacterial clearance and survival are dependent on CXC chemokine receptor-2 ligands in a murine model of pulmonary Nocardia asteroides infection. J. Immunol. 164:908.[Abstract/Free Full Text]
  23. Greenberger, M. J., R. M. Strieter, S. L. Kunkel, J. M. Danforth, L. L. Laichalk, D. C. McGillicuddy, T. J. Standiford. 1996. Neutralization of macrophage inflammatory protein-2 attenuates neutrophil recruitment and bacterial clearance in murine Klebsiella pneumonia. J. Infect. Dis. 173:159.[Medline]
  24. Tateda, K., T. A. Moore, M. W. Newstead, W. C. Tsai, X. Zeng, J. C. Deng, G. Chen, R. Reddy, K. Yamaguchi, T. J. Standiford. 2001. Chemokine-dependent neutrophil recruitment in a murine model of Legionella pneumonia: potential role of neutrophils as immunoregulatory cells. Infect. Immun. 69:2017.[Abstract/Free Full Text]
  25. Ma, S. Y., B. J. Ciliax, G. Stebbins, S. Jaffar, J. N. Joyce, E. J. Cochran, J. H. Kordower, D. C. Mash, A. I. Levey, E. J. Mufson. 1999. Dopamine transporter-immunoreactive neurons decrease with age in the human substantia nigra. J. Comp. Neurol. 409:25.[Medline]
  26. Saltini, C., A. J. Hance, V. J. Ferrans, F. Basset, P. B. Bitterman, R. G. Crystal. 1984. Accurate quantification of cells recovered by bronchoalveolar lavage. Am. Rev. Respir. Dis. 130:650.[Medline]
  27. Greenberger, M. J., R. M. Strieter, S. L. Kunkel, J. M. Danforth, R. E. Goodman, T. J. Standiford. 1995. Neutralization of IL-10 increases survival in a murine model of Klebsiella pneumonia. J. Immunol. 155:722.[Abstract]
  28. Liu, S. F., X. Ye, A. B. Malik. 1997. In vivo inhibition of NF-{kappa}B activation prevents inducible nitric oxide synthase expression and systemic hypotension in a rat model of septic shock. J. Immunol. 159:3976.[Abstract]
  29. Rahman, A., K. N. Anwar, A. B. Malik. 2000. Protein kinase C-{zeta} mediates TNF-{alpha}-induced ICAM-1 gene transcription in endothelial cells. Am. J. Physiol. 279:C906.
  30. Hou, J., V. Baichwal, Z. Cao. 1994. Regulatory elements and transcription factors controlling basal and cytokine-induced expression of the gene encoding intercellular adhesion molecule 1. Proc. Natl. Acad. Sci. USA 91:11641.[Abstract/Free Full Text]
  31. Gao, X., P. Kouklis, N. Xu, R. D. Minshall, R. Sandoval, S. M. Vogel, A. B. Malik. 2000. Reversibility of increased microvessel permeability in response to VE-cadherin disassembly. Am. J. Physiol. 279:L1218.[Abstract/Free Full Text]
  32. Vogel, S. M., X. Gao, D. Mehta, R. D. Ye, T. A. John, P. Andrade-Gordon, C. Tiruppathi, A. B. Malik. 2000. Abrogation of thrombin-induced increase in pulmonary microvascular permeability in PAR-1 knockout mice. Physiol. Genomics 4:137.[Abstract/Free Full Text]
  33. Lo, S. K., J. Everitt, J. Gu, A. B. Malik. 1992. Tumor necrosis factor mediates experimental pulmonary edema by ICAM-1 and CD18-dependent mechanisms. J. Clin. Invest. 89:981.
  34. Selig, W. M., T. C. Noonan, D. F. Kern, A. B. Malik. 1986. Pulmonary microvascular responses to arachidonic acid in isolated perfused guinea pig lung. J. Appl. Physiol. 60:1972.[Abstract/Free Full Text]
  35. Xu, N., A. Rahman, R. D. Minshall, C. Tiruppathi, A. B. Malik. 2000. {beta}2-Integrin blockade driven by E-selectin promoter prevents neutrophil sequestration and lung injury in mice. Circ. Res. 87:254.[Abstract/Free Full Text]
  36. Ward, P. A., G. O. Till, R. Kunkel, C. Beauchamp. 1983. Evidence for role of hydroxyl radical in complement and neutrophil-dependent tissue injury. J. Clin. Invest. 72:789.
  37. Segal, B. H., N. Sakamoto, M. Patel, K. Maemura, A. S. Klein, S. M. Holland, G. B. Bulkley. 2000. Xanthine oxidase contributes to host defense against Burkholderia cepacia in the p47phox-/- mouse model of chronic granulomatous disease. Infect. Immun. 68:2374.[Abstract/Free Full Text]
  38. Kopydlowski, K. M., C. A. Salkowski, M. J. Cody, N. van Rooijen, J. Major, T. A. Hamilton, S. N. Vogel. 1999. Regulation of macrophage chemokine expression by lipopolysaccharide in vitro and in vivo. J. Immunol. 163:1537.[Abstract/Free Full Text]
  39. Tsai, W. C., R. M. Strieter, B. Mehrad, M. W. Newstead, X. Zeng, T. J. Standiford. 2000. CXC chemokine receptor CXCR2 is essential for protective innate host response in murine Pseudomonas aeruginosa pneumonia. Infect. Immun. 68:4289.[Abstract/Free Full Text]
  40. Wagner, J. G., K. E. Driscoll, R. A. Roth. 1999. Inhibition of pulmonary neutrophil trafficking during endotoxemia is dependent on the stimulus for migration. Am. J. Respir. Cell Mol. Biol. 20:769.[Abstract/Free Full Text]
  41. Duffy, A. J., B. Nolan, K. Sheth, H. Collette, M. De, P. E. Bankey. 2000. Inhibition of alveolar neutrophil immigration in endotoxemia is macrophage inflammatory protein 2 independent. J. Surg. Res. 90:51.[Medline]
  42. Mercer-Jones, M. A., M. S. Shrotri, M. Heinzelmann, J. C. Peyton, W. G. Cheadle. 1999. Regulation of early peritoneal neutrophil migration by macrophage inflammatory protein-2 and mast cells in experimental peritonitis. J. Leukocyte Biol. 65:249.[Abstract]
  43. Zhang, P., G. J. Bagby, J. K. Kolls, D. A. Welsh, W. R. Summer, J. Andresen, S. Nelson. 2001. The effects of granulocyte colony-stimulating factor and neutrophil recruitment on the pulmonary chemokine response to intratracheal endotoxin. J. Immunol. 166:458.[Abstract/Free Full Text]
  44. Kernacki, K. A., R. P. Barrett, J. A. Hobden, L. D. Hazlett. 2000. Macrophage inflammatory protein-2 is a mediator of polymorphonuclear neutrophil influx in ocular bacterial infection. J. Immunol. 164:1037.[Abstract/Free Full Text]
  45. Matsubara, T., M. Ziff. 1986. Increased superoxide anion release from human endothelial cells in response to cytokines. J. Immunol. 137:3295.[Abstract]
  46. Czermak, B. J., V. Sarma, N. M. Bless, H. Schmal, H. P. Friedl, P. A. Ward. 1999. In vitro and in vivo dependency of chemokine generation on C5a and TNF-{alpha}. J. Immunol. 162:2321.[Abstract/Free Full Text]
  47. Schmal, H., B. J. Czermak, A. B. Lentsch, N. M. Bless, B. Beck-Schimmer, H. P. Friedl, P. A. Ward. 1998. Soluble ICAM-1 activates lung macrophages and enhances lung injury. J. Immunol. 161:3685.[Abstract/Free Full Text]
  48. Koay, M. A., J. W. Christman, B. H. Segal, A. Venkatakrishnan, T. R. Blackwell, S. M. Holland, T. S. Blackwell. 2001. Impaired pulmonary NF-{kappa}B activation in response to lipopolysaccharide in NADPH oxidase-deficient mice. Infect. Immun. 69:5991.[Abstract/Free Full Text]
  49. Kono, H., I. Rusyn, M. Yin, E. Gabele, S. Yamashina, A. Dikalova, M. B. Kadiiska, H. D. Connor, R. P. Mason, B. H. Segal, et al 2000. NADPH oxidase-derived free radicals are key oxidants in alcohol-induced liver disease. J. Clin. Invest. 106:867.[Medline]



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