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The Journal of Immunology, 2002, 169: 2093-2101.
Copyright © 2002 by The American Association of Immunologists

Nitric Oxide Stimulates Macrophage Inflammatory Protein-2 Expression in Sepsis1

Randal A. Skidgel2,*, Xiao-pei Gao*, Viktor Brovkovych*, Arshad Rahman*, David Jho*, Sanda Predescu*, Thedodore J. Standiford{dagger} and Asrar B. Malik*

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


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
NO is a crucial mediator of the inflammatory response, but its in vivo role as a determinant of lung inflammation remains unclear. We addressed the in vivo role of NO in regulating the activation of NF-{kappa}B and expression of inflammatory proteins using an in vivo mouse model of sepsis induced by i.p. injection of Escherichia coli. We observed time-dependent degradation of I{kappa}B and activation of NF-{kappa}B accompanied by increases in inducible NOS, macrophage inflammatory protein-2 (MIP-2), and ICAM-1 expression after E. coli challenge, which paralleled the ability of lung tissue to produce high-output NO. To determine the role of NO in this process, mice were pretreated with the NO synthase (NOS) inhibitor NG-methyl-L-arginine. Despite having relatively modest effects on NF-{kappa}B activation and ICAM-1 or inducible NOS expression, the NOS inhibitor almost completely inhibited expression of MIP-2 in response to E. coli challenge. These responses were associated with the inhibition of migration of neutrophils in lung tissue and increased permeability induced by E. coli. In mice pretreated with NG-methyl-L-arginine, coadministration of E. coli with the NO donor (Z)-1-[N-(2-aminoethyl)-N-(2-ammonioethyl)amino]diazen-1-ium-1,2-diolate substantially restored MIP-2 expression but decreased ICAM-1 expression. The results suggest that NO generated after administration of E. coli serves as an important proinflammatory signal to up-regulate MIP-2 expression in vivo. Thus, NO production in high quantities may be important in the mechanism of amplification of the lung inflammatory response associated with sepsis.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
Acute lung injury associated with Gram-negative sepsis is characterized by the accumulation of large numbers of neutrophils in the lungs and a pulmonary inflammatory response (1, 2, 3, 4). The recruitment of polymorphonuclear leukocytes (PMN)3 to the endothelial cell surface of the microvessels plays an important role in the initiating event that leads to multiple organ inflammation and dysfunction during sepsis (2, 5, 6, 7, 8, 9). This complex process involves the well-coordinated expression of adhesion molecules (3, 10) and increased production of chemoattractant cytokines (chemokines) (11).

Chemokines have been implicated as important mediators in the pathogenesis of endotoxin-induced lung injury by controlling the nature and magnitude of inflammatory cell infiltration (1, 12, 13). The C-X-C chemokines promote neutrophil adherence to endothelial cells and transendothelial migration into lung tissue. Macrophage inflammatory protein (MIP)-2, a rodent C-X-C chemokine generated by macrophages in response to LPS, is involved in the pathogenesis of acute lung injury (1, 13). Some studies indicate that MIP-2 plays a significant role in the LPS-induced inflammatory response (14). For example, anti-MIP-2 Abs significantly reduced neutrophil infiltration into the lungs and alveolar PMN accumulation in animal models of acute lung injury (15, 16). In addition, MIP-2 may be affected by NO generation as hydroxylamine, an NO donor, dose dependently up-regulated release of MIP-2 after allergen challenge in mice (17). However, the interplay between NO and chemokines in regulating lung leukocyte activation and lung injury during sepsis in vivo remains to be determined.

NO, synthesized from the amino acid L-arginine by the action of NO synthase (NOS), is a highly reactive radical gas that regulates cellular functions in both physiologic and pathologic conditions (18, 19, 20, 21). NO has a variety of activities on the cardiovascular and nervous systems and, at high concentrations, is bacteriostatic/bactericidal and fungistatic. In physiologic states, NO can serve a protective function, but under conditions of high output NO may contribute to tissue damage by reacting with superoxide to form peroxynitrite, a strong oxidant (22). LPS induces the production of large amounts of NO and superoxide in alveolar macrophages and lung epithelial, endothelial, and interstitial cells (22, 23, 24, 25). The primary mechanism by which this occurs is induction of inducible NOS (iNOS) mRNA expression in various organs, including lung (26, 27), via activation of the transcription factor NF-{kappa}B (28, 29, 30). NF-{kappa}B also regulates the transcription of numerous other genes involved in varied inflammatory and immune responses, including TNF-{alpha}, ICAM-1, VCAM-1, and MIP-2 (31).

NO participates in the inflammatory response not only via its physiological effects but also by its ability to regulate the expression of inflammatory proteins (28, 32). In general, NO has an inhibitory effect on the transcription of inflammatory genes thought to be mediated by the inhibition of NF-{kappa}B activation by increasing the transcription and stability of the NF-{kappa}B inhibitory protein I{kappa}B (33, 34). For example, NF-{kappa}B activation was inhibited by treatment of cytokine-stimulated endothelial or vascular smooth muscle cells with S-nitrosothiols (33, 35), and NF-{kappa}B activity in endothelial cells and LPS-stimulated macrophages was augmented by NOS inhibitors (33). However, these effects are not straightforward, as some studies have suggested that NO can increase the expression of some inflammatory response proteins such as cyclooxygenase-2 (COX-2), TNF-{alpha}, and iNOS (36, 37). One explanation for these varied effects is that NO may regulate NF-{kappa}B in a biphasic manner, as shown in a mouse monocyte/macrophage cell line where NO activated NF-{kappa}B at early time points after LPS administration or at lower levels of NO and inhibited its activation at later times or at higher doses of NO (38). Thus, it has been suggested that NO may exert both deleterious and protective effects in sepsis by regulating NF-{kappa}B either positively or negatively, depending on species, timing, the cell type, inflammatory stimulus, the NO concentration, and NO-related metabolites generated (25).

The present study was undertaken to investigate the role of NO in regulating the expression of inflammatory proteins using an in vivo Gram-negative bacteremia model. The major aims of the present study were 1) to determine the effect of NO on the regulation of NF-{kappa}B-associated iNOS and adhesion molecule expression and 2) to determine the ability of NO to regulate the expression of chemokine MIP-2 and its role in the subsequent recruitment of neutrophils and development of lung permeability changes.


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

NG-methyl-L-arginine (L-NMMA) and NG-methyl-D-arginine (D-NMMA) were purchased from Sigma-Aldrich (St. Louis, MO). (Z)-1-[N-(2-aminoethyl)-N-(2-ammonioethyl)amino]diazen-1-ium-1,2-diolate (DETA NONOate) was purchased from Cayman Chemical (Ann Arbor, MI). Murine recombinant MIP-2 used for the generation of Abs and standards for ELISA were purchased from R&D Systems (Minneapolis, MN). Polyclonal anti-murine MIP-2 antisera used in the ELISA were produced by immunization of rabbits with recombinant murine MIP-2 in multiple intradermal sites with CFA. MIP-2 antisera and control rabbit antiserum were made as described (16). The polyclonal anti-murine MIP-2 Ab is very specific, as it did not react with other members of the murine ELR+ XC chemokine family or with ELR-CXC, C, or CC chemokines (16, 39).

Escherichia coli infection

Pathogen-free C57BL/6 male mice (n = 340), 8–10 wk old (obtained from The Jackson Laboratory, Bar Harbor, ME), were used in all experiments and were housed in specific pathogen-free conditions with free access to food and water in the University of Illinois Animal Care Facility. Studies were done in accordance with institutional and National Institutes of Health guidelines, and after review and approval by the Animal Care Committee. 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 25992; American Type Culture Collection, Manassas, VA) (12). 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. The lungs obtained at different time points after E. coli challenge (1 and 6 h) were used to assess NO production, I{kappa}B degradation, NF-{kappa}B activation, endothelial NOS (eNOS), iNOS, ICAM-1, and MIP-2 expression, concentration of murine chemokine (murine MIP (mMIP)-2), leukocyte migration, and lung edema formation. To determine the effects of NO, mice were first injected i.p. with L-NMMA (100 mg/kg, 100 µl). In another group, D-NMMA was used as negative control of L-NMMA effects. At 1 h after injection, mice were challenged i.p. with 2 x 108 live E. coli/100 µl as above. In another group, mice pretreated with L-NMMA for 1 h were given E. coli combined with NO donor DETA NONOate (20 mg/kg i.v. in 100 µl). The lungs obtained at different time points after E. coli challenge (0.5, 1, 3 and 6 h) were used to assess the changes as described above.

Effects of anti-MIP-2 Ab

Mice were first injected i.p. with 0.5 ml MIP-2 antiserum, in which the Ab titer is between 105 and 106. The Ab titer of 105–106 indicates that the antiserum is capable of detecting murine MIP-2 out to dilutions of between 1/1 x 105 and 1/106. At 2 h after injection, mice were challenged i.p. with 2 x 108 live E. coli/100 µl. In the control group, mice were injected i.p. with the same dosage of control rabbit serum.

NO measurements

For NO measurements, lungs were removed and cut into slices that were 1 mm thick. The slices were placed in HBSS at 37°C. NO measurements were performed in this bath using a three-electrode system that consisted of a porphyrinic microsensor (NOP-1, Quanteon; University of Colorado Health Sciences Center) a platinum counter electrode, and a silver-silver chloride reference electrode. The system was coupled with a FAS1 femtostat and an IBM-compatible computer with electrochemical software (Gamry Instruments, Warminster, PA). The microsensor had a response time of 10 ms at the detection limit of 10 nmol/L. With the aid of a micromanipulator, the NO sensor was carefully placed on the surface of the lung slice and the baseline was recorded. To determine potential constitutive NOS (cNOS) activity, 1 mM L-arginine was added to the medium, NO generation was stimulated by application of 10 µM calcium ionophore A23187, and NO release was recorded for 20 s. To measure the lung iNOS activity, slices of lung were incubated in HBSS without L-arginine, and NO generation was initiated by the application of 1 mM L-arginine. NOS activity was expressed as the area under the curve of the recorded signal for 20 s and 20 min for cNOS and iNOS, respectively.

Nuclear protein isolation

Nuclear proteins were isolated as described (30, 40). 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 detergent (Sigma-Aldrich), 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 mM leupeptin) 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 (40). 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) in a 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 was NF-{kappa}B 5'-AGTTGAGGGGACTTTCCCAGGC-3' containing the consensus NF-{kappa}B binding site present in the Ig {kappa} L chain enhancer.

Western blotting

We determined eNOS, iNOS, ICAM-1, and MIP-2 expression in lung tissue by Western blotting. Briefly, lungs were homogenized in 0.05 M Tris, 0.138 M NaCl, 0.0027 M KCl (pH 8) containing protease inhibitor mixture (Sigma-Aldrich). Protein concentration was then measured in an aliquot of the tissue homogenate. Homogenates containing equal amounts of protein were then electrophoresed on gradient gels (5–20%), transferred to 0.22-µm nitrocellulose membranes (Osmonics, Minnetonka, MN), blocked with 5% nonfat milk, and analyzed by Western blotting using Abs specific for eNOS or iNOS (BD Transduction Laboratories, Lexington, KY), ICAM-1 (Santa Cruz Biotechnology, Santa Cruz, CA), or MIP-2 (R&D Systems).

Measurement of lung tissue chemokines

Mice were sacrificed by i.p. anesthetic at designated time points. Whole lungs were then harvested for measurement of MIP-2 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, the whole 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, then 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 levels (41).

Chemokine ELISA

Murine MIP-2 concentrations were quantitated by a modification of a double ligand method as described (41). In brief, flat-bottom 96-well microtiter plates (ImmunoPlate I 96-F; Nunc, Roskilde, Denmark) were coated with 50 µl/well rabbit anti-MIP-2 Ab (each at a concentration of 1 µg/ml in 0.6 M NaCl, 0.26 M H3BO4, 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). Microtiter plate nonspecific binding sites were blocked with 2% BSA in PBS and incubated for 90 min at 37°C. Plates were rinsed four times with wash buffer and diluted (neat and 1/10). Cell-free supernatants (50 µl) in duplicate were added, 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 Abs (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 from 1 pg/ml to 100 ng/ml. The ELISA method was able to detect murine MIP-2 concentrations >25 pg/ml. The ELISA did not cross-react with TNF-{alpha}, IL-2, IL-4, IL-6, or IFN-{gamma}, or with other members of the murine chemokine family, including murine JE/monocyte chemoattractant protein-1, MIP-1{alpha}, growth-related oncogene {alpha}, or ENA-78 (41). The anti-MIP-2 Abs also did not interfere with measurements of MIP-2 by ELISA.

Lung tissue MPO activity

Lungs were homogenized in 1 ml of 50 mM PBS (pH 6) with 5% of hexadecyltrimethylammonium bromide and 5 mM EDTA for quantification of PMN sequestration by myeloperoxidase (MPO) activity as described by us (2). The homogenates were sonicated, centrifuged at 40,000 x g for 20 min, and frozen and thawed two times followed by homogenization and centrifugation as before. The supernatant was mixed 1/30 (v/v) with assay buffer (0.2 mg/ml of o-dianisidine hydrochloride and 0.0005% H2O2), and the absorbance change was measured at 460 nm for 3 min. MPO activity based on lung weight was calculated as change in absorbance over time.

PMN counts in BAL fluid

The trachea was cannulated and bronchoalveolar lavage (BAL) was performed using 1 ml of PBS. BAL fluid was centrifuged for 5 min at 300 RPM using a cytospin (Thermo Shandon, Pittsburgh, PA) and BAL cells were stained with HEMA3 (catalog no. 122-911ABC; Fisher, Hanover Park, IL). PMN counts were determined by counting 500 cells per slide (2).

Determination of lung edema

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 for constant positive pressure ventilation (186 breaths/min). 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 performed to expose the heart and lung. 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 via 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 a 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/min) and perfused at constant flow (2 ml/min), temperature (37°C), and venous pressure (0 cm H2O) with a modified Krebs-Henseleit solution, 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 Instruments, 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 computer 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).

We determined the rate of pulmonary edema formation by monitoring the lung wet weight changes as described before (2). Weight change of lungs obtained from different groups of animals 6 h after treatment was followed for 90 min after beginning of the perfusion (described above). Because 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 magnitude of the increase in lung wet weight at 90 min was used as an index of permeability of pulmonary microvessels.

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 value of p < 0.05 was used as the criterion for significance. Blots shown are representative results from experiments done two to three times.


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

To assess the protein levels of NOS in mouse lungs after E. coli challenge, Western blots were performed with Abs specific for either eNOS or iNOS (Fig. 1Go). eNOS immunoreactivity in lung extracts remained constant for up to 6 h after E. coli treatment, indicating that this cNOS isoform is not regulated by inflammatory mediators. eNOS protein levels were also not affected by E. coli challenge in animals pretreated with L-NMMA in the absence or presence of the NO donor, DETA NONOate (Fig. 1Go). In contrast, iNOS protein levels were substantially increased at 1 h and especially 6 h after injection of E. coli, consistent with the known ability of iNOS to be strongly induced during sepsis (30, 42). Pretreatment of animals with L-NMMA reduced iNOS induction at 1 and 6 h, and in the presence of L-NMMA the NO donor, DETA NONOate, actually increased iNOS expression at 0.5 h over E. coli alone and restored iNOS expression at 1 h, but not after 6 h (Fig. 1Go).



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  		FIGURE 1. E. coli-induced eNOS and iNOS expression in mouse lungs. Lungs were isolated at the indicated times after E. coli challenge without or with pretreatment with L-NMMA or L-NMMA plus the NO donor (NOD) DETA NONOate. Aliquots of tissue lysates were separated by SDS-PAGE (adjusted for equal protein loading) and immunoblotted with Abs against eNOS or iNOS, as described in Materials and Methods.

 
We wanted to determine whether the lung NOS protein levels corresponded to the ability of lung tissue to produce NO, because it is possible for the protein to be present but inactive. Thus, constitutive Ca2+-activated NOS activity (cNOS) and iNOS activity were measured in lung slices using a sensitive porphyrinic microelectrode that is specific for NO and allows the determination of NO production in real time. To distinguish cNOS activity from iNOS, two separate slices from each lung were used and treated as follows. To one, the calcium ionophore A23187 was added in medium containing 1 mM Arg, which evoked a rapid, maximal generation of NO from cNOS that returned to baseline within 20 s (Fig. 2Go). The peak area of NO over 20 s was integrated as a measure of maximal cNOS activity (i.e., the potential of the tissue to generate NO via this pathway). Although it is likely that a majority of the activity was contributed by eNOS due to the high endothelial content of lung tissue, some generation of NO by nNOS cannot be ruled out, because this isoform is also present in lung, primarily in the epithelium (43). Because iNOS activity is substrate controlled (it is not agonist or Ca2+ dependent), the other slice was preincubated in HBSS without Arg and then 1 mM Arg was added to initiate NO generation. When conducted with slices from control lungs, addition of 1 mM Arg caused a small, slow rise in NO production that returned to baseline within 20 min. However, after a 6-h E. coli challenge, Arg addition caused a continuous, increasing production of a high level of NO that was still increasing at 20 min (Fig. 2Go). Integration of the area under the curve for 20 min was used as a standard measure of potential iNOS activity.



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  		FIGURE 2. Representative recordings of real-time measurement of NO release from tissue slices of lungs from control animals or animals challenged with E. coli for 1 or 6 h. cNOS (A–C)-dependent NO release in lungs was stimulated by application of 10 µM calcium ionophore A23187, and iNOS (D–F)-dependent NO release was stimulated by application of 1 mM L-Arg. NO release was measured with a porphyrinic microelectrode.

 
cNOS activity in control lungs and lungs after a 1-h E. coli challenge was essentially identical (Fig. 2Go, A and B). However, after 6 h of E. coli challenge, the maximum response to ionophore was diminished (Fig. 2GoC) and the total amount of NO produced in 20 s was somewhat reduced (Fig. 3GoA).



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  		FIGURE 3. NO release from tissue slices of lungs from control animals or animals challenged with E. coli for 1 or 6 h with or without pretreatment with NOS inhibitor L-NMMA or L-NMMA plus NO donor (NOD) DETA NONOate. cNOS-dependent NO release (A) and iNOS-dependent NO release (B) were measured with a porphyrinic NO-specific microelectrode as described in Materials and Methods and Fig. 2Go. Data are represented as the mean ± SEM for the area under the NO curve for 20 s and 20 min for cNOS and iNOS, respectively (n = 4–6). *, Increase (p < 0.05) vs control (basal); {dagger}, decrease (p < 0.05) vs the same time E. coli challenge; **, increase (p < 0.05) vs the same time L-NMMA plus E. coli challenge.

 
Treatment of control mice with L-NMMA alone did not change the cNOS activity subsequently measured in their lung tissue slices (Fig. 3GoA). This was expected, because L-NMMA is a reversible inhibitor that was injected into animals 1 h before challenge. Then, after removal of the lungs, tissue slices were washed and bathed in medium, which would dilute and remove any residual L-NMMA. The purpose of the inhibitor treatment was to inhibit NOS activity in vivo during the E. coli challenge while still allowing the measurement of total NOS activity in lung tissue slices after treatment. The lack of effect of L-NMMA on measured cNOS activity in control lung slices supports the validity of this approach. Thus, under these conditions, any change in NOS activity by L-NMMA would be due to changes in active protein levels mediated by NOS inhibition in vivo during E. coli challenge.

In animals challenged with E. coli for 1 or 6 h, L-NMMA did not significantly change the level of cNOS activity compared with animals treated with E. coli alone (Fig. 3GoA). In animals treated with L-NMMA, the NO donor, DETA NONOate, had little effect on cNOS activity except after 6 h of E. coli challenge, where the activity was modestly but significantly elevated over animals treated with E. coli alone (Fig. 3GoA).

In contrast to the cNOS activity, E. coli challenge significantly increased iNOS activity, especially at 6 h (Fig. 3GoB), which correlated with the increase in iNOS protein measured by Western blot (Fig. 1Go). L-NMMA treatment of the animals resulted in a reduction in the ability of E. coli to stimulate an increase in iNOS levels. To verify that the effects of L-NMMA were specific for inhibition of NOS activity in vivo, the inactive isomer D-NMMA was used in experiments on control lung slices and lung slices from mice 6 h after E. coli challenge. In both cases, there was no effect of D-NMMA pretreatment on subsequent cNOS or iNOS activity measured in lung slices (data not shown). Addition of DETA NONOate to the L-NMMA treatment restored iNOS activity to the same level as that in animals treated with E. coli alone at 1 h but was unable to reverse the decrease in iNOS activity caused by L-NMMA at 6 h (Fig. 3GoB). In general, the measurements of NOS activity paralleled the protein levels measured by Western blot.

NF-{kappa}B activation

We observed a time-dependent increase in lung tissue NF-{kappa}B activation with a peak response at 1 h after E. coli challenge, which paralleled but was delayed with respect to I{kappa}B degradation (Fig. 4Go). L-NMMA inhibited NF-{kappa}B activation at 3 h after E. coli infection but not at 0.5 or 1 h, whereas I{kappa}B degradation was inhibited 1 h after E. coli challenge but not at 0.5 h (Fig. 4Go). The resynthesis of I{kappa}B, evident at the 3-h time point, was not affected by L-NMMA treatment. When DETA NONOate was used to restore NO production in animals treated with E. coli plus L-NMMA, it restored NF-{kappa}B activation at 1 h and I{kappa}B degradation at 1 h and also blunted the resynthesis of I{kappa}B at 3 h (Fig. 4Go).



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  		FIGURE 4. E. coli-induced I{kappa}B{alpha} degradation and NF-{kappa}B activation in mouse lungs. Lungs were isolated from animals at the indicated times after E. coli challenge with or without pretreatment with NOS inhibitor L-NMMA or L-NMMA plus NO donor (NOD) DETA NONOate. Nuclear extracts were assayed for NF-{kappa}B binding activity by EMSA. Total cell lysates were separated by SDS-PAGE and immunoblotted with an Ab against I{kappa}B{alpha} (for further details see Materials and Methods).

 
ICAM-1 expression

As expected, ICAM-1 expression was increased after 30 min of E. coli challenge and remained elevated at 6 h (Fig. 5Go). Pretreatment of animals with L-NMMA did not substantially affect the expression of ICAM-1 in response to E. coli (Fig. 5Go). However, the combination of L-NMMA and DETA NONOate noticeably reduced ICAM-1 expression in response to E. coli at 1 and 6 h (Fig. 5Go).



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  		FIGURE 5. E. coli-induced ICAM-1 and MIP-2 expression in mouse lungs. Lungs were isolated at the indicated times after E. coli challenge with or without pretreatment with NOS inhibitor L-NMMA or L-NMMA plus NO donor (NOD) DETA NONOate. Total cell lysates were separated by SDS-PAGE and immunoblotted with Abs against ICAM-1 or MIP-2.

 
MIP-2 expression

E. coli challenge strongly induced lung tissue MIP-2 expression, beginning at 30 min, becoming maximal at 1 h, and persisting to 6 h as determined by Western blotting (Fig. 5Go) or by a more quantitative ELISA (Fig. 6Go). The increased expression was almost abolished in lungs from the L-NMMA-pretreated group at all time points (Figs. 5Go and 6Go), indicating a significant role for NOS activity in the induction of MIP-2. Indeed, when animals pretreated with L-NMMA were challenged with E. coli combined with DETA NONOate to generate NO, MIP-2 expression was substantially restored (~60–80%), with the most prominent effects at early time points. To verify the specificity of L-NMMA, the similar, but inactive, isomer D-NMMA was used and found to have no effect on MIP-2 expression after E. coli challenge (data not shown). These data indicate that NO plays a major role in regulating chemokine production.



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  		FIGURE 6. Lung tissue chemokines measured by ELISA at 1 or 6 h after E. coli challenge, with or without pretreatment with NOS inhibitor L-NMMA or L-NMMA plus NO donor (NOD) DETA NONOate. *, Increases (p < 0.05) in lung tissue mMIP-2 level compared with control values (without E. coli challenge); **, increases (p < 0.05) in lung tissue mMIP-2 level compared with L-NMMA plus E. coli challenge; {dagger}, decreases (p < 0.05) in lung tissue mMIP-2 level compared with E. coli challenge alone. Results are mean of six experiments; bars indicate ± 1 SEM.

 
PMN sequestration in lungs after E. coli challenge

Infiltration of PMN in lung, as determined by either MPO activity or the percentage of PMN in BAL, increased after E. coli challenge, with a peak response observed at 6 h (Fig. 7Go). The more impressive increase in MPO activity in lung homogenates, compared with the smaller increase in the percentage of PMN in BAL, is consistent with intravascular or interstitial trapping of PMN at this time point. The increase in lung PMN was inhibited by pretreatment with L-NMMA at 6 h but not at 1 h (Fig. 7GoA; p < 0.05). A combination of E. coli with DETA NONOate in animals pretreated with L-NMMA significantly potentiated the E. coli alone-induced increase in PMN ~50% at 1 h, but not at 6 h, as determined by lung MPO activity (Fig. 7GoA). There were no significant changes in lung PMN compared with E. coli treatment alone after pretreatment with inactive D-NMMA isomer (Fig. 7GoA). Consistent with the effects of L-NMMA on lung MPO activity, L-NMMA also reduced the PMN recovered in lung BAL at 6 h but not at 1 h (Fig. 7GoB).



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  		FIGURE 7. A, PMN sequestration in lungs of mice. Control mice (Basal) or mice challenged for 1 or 6 h with E. coli with or without pretreatment with NOS inhibitor L-NMMA or L-NMMA plus NO donor (NOD) DETA NONOate were euthanized and lungs were harvested for measurement of MPO activity (see Materials and Methods). *, Increases (p < 0.05) in PMN sequestration compared with basal (without E. coli challenge); **, increases (p < 0.05) in PMN sequestration compared with L-NMMA plus E. coli challenge; {dagger}, decreases (p < 0.05) in lung PMN sequestration compared with E. coli alone. B, PMN migration into airspace. All mice were challenged for 1 or 6 h with 2 x 108 E. coli (i.p.) without or with pretreatment with L-NMMA. PMN counts in BAL fluid were determined (see Materials and Methods). *, Increases (p < 0.05) in PMN migration compared with basal (without E. coli challenge); {dagger}, Decreases (p < 0.05) in PMN migration compared with E. coli challenge alone. Results are mean of six experiments; bars indicate ± 1 SEM.

 
Pretreatment of animals with MIP-2 Ab significantly inhibited lung tissue PMN sequestration after E. coli challenge at both 1 and 6 h, whereas control Ab had no effect (Fig. 8Go).



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  		FIGURE 8. Inhibitory effect of anti-MIP-2 Ab on PMN sequestration at 1 or 6 h after E. coli challenge. Mice were challenged for 1 or 6 h with 2 x 108 E. coli (i.p.) without or with anti-MIP-2 Ab pretreatment, and lungs were harvested for measurement of MPO activity (see Materials and Methods). *, Increases (p < 0.05) in PMN sequestration compared with basal (without E. coli challenge); {dagger}, decreases (p < 0.05) in PMN sequestration compared with E. coli challenge alone. Results are mean of six experiments; bars indicate ± 1 SEM.

 
Lung edema formation

E. coli infection for 6 h, followed by a 90-min lung perfusion in vitro (see Materials and Methods), resulted in significant increases in lung wet weight compared with perfused lungs from control animals (Fig. 9Go; p < 0.05). The weight changes are relatively large, as the typical basal wet weight of a mouse lung is ~0.15 g. Increases in lung wet weight caused by E. coli infection were significantly reduced in lungs from animals treated with L-NMMA (Fig. 9GoA, p < 0.05). In L-NMMA-treated animals, DETA NONOate administration partially restored the lung weight gain in response to E. coli challenge (Fig. 9GoA). MIP-2 Ab administration before bacterial challenge also substantially prevented edema formation (Fig. 9GoB). These data suggest that NO plays a role in the recruitment of neutrophils, likely via enhancement of MIP-2 expression, and development of lung permeability changes.



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  		FIGURE 9. A, Changes in wet weight of perfused lung preparations challenged in vivo with E. coli for 6 h with or without pretreatment with NOS inhibitor L-NMMA or L-NMMA plus NO donor (NOD) DETA NONOate. The control group (Basal) received sham injection of PBS 6 h before lung isolation. *, Increases (p < 0.05) in lung wet weight compared with control; {dagger}, decreases (p < 0.05) in lung wet weight compared with E. coli challenge alone. B, Changes in wet weight of lungs challenged with E. coli for 6 h without or with pretreatment with MIP-2 Ab or control Ab. The control group (Basal) received sham injection of PBS before lung isolation. *, Increases (p < 0.05) in lung wet weight compared with control (without E. coli challenge); {dagger}, reduction (p < 0.05) in wet weight gain of lungs compared with E. coli alone or E. coli plus control Ab. Results are mean of six experiments; bars indicate ± 1 SEM.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
Acute lung injury is an important feature of sepsis. Increased iNOS expression and subsequent NO production are important contributors to endotoxin-induced lung injury (23). Although many organs can express iNOS and contribute to total NO generated during sepsis, the major site of NO production that results in pulmonary edema and injury is in the lung itself (44). Alveolar macrophages and airway epithelial cells were demonstrated to be major cells in which cytokines induce iNOS expression and NO formation, although endothelial cells can also be induced to express iNOS (45, 46).

Toxic products released in sepsis activate systemic host defenses involving neutrophils, macrophages, monocytes, and endothelial cells, which produce potentially harmful mediators such as superoxide, cytokines, kinins, eicosanoids, and NO. Activation of the transcription factor NF-{kappa}B is an early obligatory event in the initiation of acute inflammation in E. coli septicemia. For example, pyrrolidine dithiocarbamate, an inhibitor of I{kappa}B degradation and NF-{kappa}B activation, inhibited in vivo expression of proinflammatory genes such as iNOS, ICAM-1, COX-2, cytokine-induced neutrophil chemoattractant, and TNF-{alpha} (30, 31) and reduced LPS-induced microvascular injury in multiple organs (47).

The precise roles of NO in sepsis and lung inflammation are still under debate, because it is beneficial in certain conditions but it also can have toxic effects (48, 49, 50). This is reflected in the conflicting reports regarding the effects of iNOS inhibition or knockout on overall survival in sepsis (49). Part of the difficulty in assigning a precise role to NO is the dual nature of this important mediator. Thus, NO can be cytotoxic at high levels, especially when combined with superoxide to form the potent oxidant ONOO- (51). In contrast, NO can also blunt the inflammatory response via its ability to inhibit NF-{kappa}B activation by increasing the expression, nuclear translocation, and stabilization of its inhibitory protein I{kappa}B (33, 34). For example, NO blocks the NF-{kappa}B-mediated up-regulation of inflammatory proteins such as iNOS itself (33, 52), VCAM-1 (53), ICAM-1, E-selectin (34), and COX-2 (54). However, most of these studies have been conducted in cell cultures using well-defined stimuli (e.g., TNF-{alpha}, LPS, IL), not in vivo, where the response to sepsis is much more complex. In the present study, we used an in vivo Gram-negative bacteremia model to address the role of NO in regulating chemokine-associated lung PMN activation and lung edema. The large magnitude of the wet weight change (~3- to 4-fold increase) argues for alveolar edema and thus breakdown of the alveolar epithelial barrier. A strictly interstitial edema would register a barely detectable weight increase because of the small volume of the interstitial space. To support this conclusion, evidence was recently obtained that albumin permeability of the alveolar-capillary barrier is markedly increased in this model system (S. M. Vogel and X.-p. Gao, unpublished data).

As indicators of lung inflammation, we measured NF-{kappa}B activation and three proteins known to be regulated by NF-{kappa}B: iNOS, MIP-2, and ICAM-1. Interestingly, the effects of L-NMMA and the NO donor DETA NONOate on the resulting levels of iNOS, MIP-2, and ICAM-1 differed and did not directly correlate with the effects of the treatments on NF-{kappa}B activation, which were relatively modest. Thus, our results indicate that the changes in inflammatory proteins caused by iNOS inhibitor or NO donor administration cannot be due solely to the effects of NO on NF-{kappa}B activation.

A complicating factor in interpreting the effects of L-NMMA on the regulation of inflammatory proteins, especially in vivo, is that NO is not the only relevant product generated by iNOS. Recent studies have made it clear that NOS can generate significant amounts of superoxide under conditions of low substrate (arginine) or tetrahydrobiopterin (55), as can occur during sepsis (56, 57). Superoxide and its metabolites are oxidants, which can be potent stimuli of NF-{kappa}B activation (58). For example, iNOS, by producing both superoxide and NO, can generate ONOO-, which was recently found to stimulate cytokine production by human monocytes (59) or IL-8 gene expression and production by human leukocytes (60) in an NF-{kappa}B-dependent manner. Whether L-NMMA would inhibit the production of superoxide by iNOS in vivo is not clear. Although it is generally accepted that L-NMMA inhibits only NO production by NOS whereas N-nitro-L-arginine methyl ester inhibits both NO and superoxide production (61), most of the studies have been conducted on eNOS or nNOS, and the mechanism of inhibition is not identical for the three isoforms (62). One study that directly investigated the production of superoxide by iNOS showed almost complete L-NMMA inhibition of superoxide production in mouse macrophages (63). In addition, detailed mechanistic studies showed that inactivation of iNOS by L-NMMA was accompanied by significant loss of heme from the enzyme (64), which would abolish the ability of iNOS to produce superoxide. Our approach to address the dual nature of iNOS activity was to use an NO donor in the presence of L-NMMA to restore NO levels while inhibiting endogenous NOS activity. Thus, if L-NMMA inhibited superoxide production in our studies, the restoration of the responses by an NO donor in the presence of L-NMMA would indicate the direct involvement of NO. However, if superoxide generation were not inhibited by L-NMMA, the restoration of the response could be interpreted as indicating a role for ONOO- in the response.

We observed that E. coli induced iNOS expression and activity in a time-dependent manner in lungs. Pretreatment with the NOS inhibitor L-NMMA reduced iNOS expression, whereas the NO donor, although it reversed the small L-NMMA inhibition at early time points (0.5–1 h), did not have any effect at 6 h, when the iNOS induction was maximal. The reversal of L-NMMA effects by DETA NONOate treatment at earlier time points, but not at 6 h, was also noticed with MIP-2 expression and lung MPO activity (Figs. 5Go, 6Go, and 7GoA). One possible explanation of these results is that the half-life of DETA NONOate in vivo is short and therefore does not continue generating NO over the 6-h time of the studies. However, although the clearance of DETA NONOate in vivo has not been carefully studied, it has the longest half-life in vitro of any NO donor (20 h at pH 7.4) and is generally considered to generate constant levels of NO over prolonged periods of time (65). Another possible explanation is that NO generation early in sepsis has a positive effect on expression of iNOS and MIP-2, but that the primary effect of L-NMMA at later time points (i.e., 6 h) is due to inhibition of oxidant generation by iNOS, which is not restored by use of an NO donor. This is consistent with the higher likelihood of arginine and/or tetrahydrobiopterin depletion later in the septic response, conditions that favor oxidant generation by NOS.

ICAM-1 expression was increased by E. coli challenge as expected, but L-NMMA treatment had very little effect on the response. However, addition of an NO donor substantially reduced the ICAM-1 response at 1 and 6 h after E. coli challenge. The effect of the NO donor is consistent with previous reports of the ability of NO to down-regulate ICAM-1 expression in endothelial cells (66, 67). Overall, the results can be explained by the dual nature of iNOS activity. Thus, generation of oxidants by iNOS during E. coli challenge stimulates ICAM-1 expression, whereas NO generation by the enzyme counteracts this effect. Therefore, the net result of iNOS activation on ICAM-1 expression is limited under these conditions.

The most striking results were obtained with MIP-2 expression. MIP-2 production is known to be stimulated by NF-{kappa}B activation, consistent with its rapid up-regulation after E. coli challenge in the present studies. However, changes in the levels of MIP-2 in response to L-NMMA and DETA NONOate administration were dramatic and quite different from those seen with NF-{kappa}B and either iNOS or ICAM-1. Thus, L-NMMA almost completely inhibited MIP-2 production, whereas administration of the NO donor in the presence of L-NMMA substantially restored the response. These data indicate that NO has a profound stimulatory effect on MIP-2 expression that predominates over or greatly potentiates the NF-{kappa}B-mediated response in this in vivo model system. In support of this conclusion, a very recent study of rat mesangial cells showed that L-NMMA inhibited IL-1{beta} induction of MIP-2 mRNA and protein expression (68). Conversely, the NO donor DETA NONOate stimulated MIP-2 expression and also greatly potentiated the IL-1{beta}-mediated response (68). This was due to increased MIP-2 gene transcription, as similar results were obtained using an artificial MIP-2 promoter luciferase construct. Furthermore, in an in vivo model of mesangioproliferative glomerulonephritis, administration of iNOS inhibitors resulted in a marked reduction in glomerular MIP-2 expression and a corresponding decrease in the number of infiltrating granulocytes (68).

The generally beneficial effects of L-NMMA on the parameters we measured seem to contradict the numerous reports of the generally deleterious effects of nonspecific NOS inhibitors in animal models of endotoxemia and sepsis (49, 69). However, the nature of the model system used has a significant impact on the effect of NOS inhibitors. In a recent extensive review of the literature in this area, Feihl et al. (69) concluded that in most studies of rodent models of endotoxemia (i.e., the injection of LPS) nonspecific NOS inhibitors generally worsen the outcome, whereas more specific iNOS inhibitors tend to have beneficial effects (69). However, in bacteremic models of sepsis (i.e., infection with live bacteria more similar to the model we used) the results were more heterogeneous, with a significant number of studies showing beneficial effects of L-NMMA and other nonspecific NOS inhibitors (69). The parameters measured can also influence the results. Whereas in many studies survival is used as an endpoint, a recent report investigating lung injury (edema and histologic changes) in rats in response to endotoxin administration found that both a nonspecific NOS inhibitor and a specific iNOS inhibitor had equally protective effects (26). Whether the beneficial effects of L-NMMA we found at 6 h would translate into improved survival is not known.

The rapid induction of MIP-2 (within 30 min) and the inhibition by L-NMMA even at early time points (before significant increases in iNOS expression) indicates that NO release by cNOS activity may play an important role in early MIP-2 expression. Thus, NO production during E. coli infection may proceed in two phases: an acute stimulation of cNOS in the early stages of infection followed by a higher and more sustained production by iNOS at later stages. The stimulation of cNOS activity in sepsis is quite likely, as LPS results in an immediate increase in NO generation in endothelial cells (70) and the pore-forming hemolysin of E. coli is also a potent acute stimulator of eNOS activity in isolated endothelial cells (71) and in perfused rabbit lungs (72). Furthermore, bradykinin, a peptide hormone generated during sepsis, is also a potent stimulator of eNOS in endothelial cells (73).

The ability of L-NMMA to block the increase in MIP-2 expression in response to E. coli was striking; however, the NOS inhibitor was less effective than MIP-2 Ab in reducing neutrophil migration and lung edema. This indicates that the proinflammatory ability of NO to increase MIP-2 expression is accompanied by anti-inflammatory actions as well, which may modulate its overall effect on lung neutrophil migration and injury. For example, NO itself can inhibit neutrophil-endothelial cell adhesion and migration in the absence of any effects on adhesion molecule expression (74, 75). In addition, it has anti-inflammatory effects via its ability to down-regulate NF-{kappa}B-mediated responses such as ICAM-1 expression (66, 67). NO is also antiapoptotic and can act as an antioxidant to inhibit the oxidation chemistry mediated by hydrogen peroxide and superoxide (50). Microarray analysis has also shown that NO can alter the expression of many genes, e.g., during endotoxin-induced hepatotoxicity (76). Thus, it is likely that NO regulates NF-{kappa}B and other inflammatory responses either positively or negatively at multiple steps in the activating pathway, depending on the cell type, cell stimulus, NO concentration, and the NO-related species generated. The ability of NO to enhance MIP-2 expression in vivo, as shown here, adds another important inflammatory mediator to the group of factors controlled by NO generation in sepsis.


   Acknowledgments
 
We gratefully acknowledge Dr. Khandakar Anwar for helpful technical assistance and Dr. Stephen Vogel for helpful advice.


   Footnotes
 
1 This study was supported by National Institutes of Health Grants HL60678 (to R.A.S.), 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. Randal A. Skidgel, Department of Pharmacology, University of Illinois College of Medicine, 835 South Wolcott Avenue (M/C 868), Chicago, IL 60612. E-mail address: rskidgel{at}uic.edu Back

3 Abbreviations used in this paper: PMN, polymorphonuclear leukocyte; NOS, NO synthase; cNOS, constitutive NOS; eNOS, endothelial NOS; iNOS, inducible NOS; L-NMMA, NG-methyl-L-arginine; DETA NONOate, (Z)-1-[N-(2-aminoethyl)-N-(2-ammonioethyl)amino]diazen-1-ium-1,2-diolate; D-NMMA; NG-methyl-D-arginine; COX-2, cyclooxygenase-2; MPO, myeloperoxidase; BAL, bronchoalveolar lavage; MIP-2, macrophage-inflammatory protein-2; mMIP, murine MIP. Back

Received for publication April 8, 2002. Accepted for publication June 12, 2002.


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 Discussion
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