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Inhibition of Nitric Oxide Restores Surfactant Gene Expression following Nickel-Induced Acute Lung Injury

University of Cincinnati; and Children's Hospital Medical Center, Cincinnati, Ohio

Address correspondence to: George D. Leikauf, Ph.D., Department of Environmental Health, University of Cincinnati, P.O. Box 670056, Cincinnati, OH 45267-0056. E-mail: leikaugd{at}ucmail.uc.edu

	Abstract

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The role of nitric oxide (NO) in acute lung injury remains controversial. Although inhaled NO increases oxygenation in clinical trials, inhibiting NO-synthase (NOS) can be protective. To examine the latter, nickel-exposed mice were treated with saline or NOS inhibitor, N^G-nitro-L-arginine methyl ester (L-NAME). Initial microarray analysis of nickel-induced gene expression of saline-treated mice revealed increased inflammatory mediator, matrix injury-repair, and hypoxia-induced factor-mediated sequences and decreased lung-specific (e.g., surfactant-associated protein B and C) sequences. Compared with saline control, L-NAME–treated mice had enhanced survival with attenuated serum nitrate/nitrite, endothelial NOS activity, and lavage neutrophils and protein. Although initial cytokine (i.e., interferon-, interleukins-1ß and -6, macrophage inflammatory protein-2, monocyte chemotactic protein-1, and tumor necrosis factor-) gene expression was similar between groups, subsequent larger cytokine increases only occurred in saline-treated mice. Similarly, surfactant protein gene expression decreased initially in both groups yet was restored subsequently with L-NAME treatment. Interestingly, the role of inducible NOS (iNOS) in these responses seems minimal. iNOS gene expression was unaltered, iNOS activity and nitrotyrosine residues were undetectable, and an iNOS antagonist, aminoguanidine, failed to increase survival. Rather, systemic L-NAME treatment appears to attenuate pulmonary endothelial NOS activity, subsequent cytokine expression, inflammation, and protein permeability, and thereby restores surfactant gene expression and increases survival.

Abbreviations: Dulbecco's phosphate-buffered saline, D-PBS • endothelial NOS, eNOS • expressed sequence tag, EST • interferon, IFN • interleukin, IL • inducible NOS, iNOS • N^G-nitro-L-arginine methyl ester, L-NAME • monocyte chemotactic protein, MCP • macrophage migration inhibitory factor, Mif • macrophage inflammatory protein, MIP • nitric oxide, NO • NO synthase, NOS • polymorphonuclear leukocyte, PMN • surfactant-associated protein, SP • tumor necrosis factor, TNF

	Introduction

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Acute lung injury commonly develops in critically ill patients as a response to a number of insults including sepsis, trauma, pneumonia, and inhalation of toxicants (1). Although initially described over 30 yr ago, mortality remains high (30–40%), with an estimated annual incidence of acute lung injury in the United States exceeding 150,000. Pathogenesis is dependent upon the initiating insult as well as the host response and involves multiple factors. In many cases, release of inflammatory mediators leads to polymorphonuclear (PMN) leukocyte infiltrates, although acute lung injury can develop in neutropenic patients. In many cases, deterioration of the alveolar capillary–epithelial barrier results in proteinaceous edema that accompanies disruption of surfactant homeostasis. These events, in turn, can lead to atelectasis, hypoxia, and multiple organ failure, culminating in death. Although complete resolution can occur, the prognosis for survivors of acute lung injury is often complicated by the development of fibrosis and decreased lung compliance.

Inhaled nitric oxide (NO) has been proposed as a therapeutic approach for ventilation-perfusion mismatching in acute lung injury, with the administration of low levels of NO preferentially dilating vessels within well-ventilated regions of the lung to improve oxygenation. However, whether NO is protective or deleterious during acute lung injury continues to be debated. Although clinical trials with inhaled NO have found minimal improvements in oxygenation and pulmonary artery pressure, overall mortality is not reduced in adults. Alternatively, recent pediatric clinical trials suggest that inhaled NO may be more efficient, possibly due to the limited involvement of other organ systems in this subpopulation.

In contrast, NO formation within the lung may be detrimental because treatment with nitric oxide synthase (NOS) inhibitors can restore pulmonary function (2, 3). NO formation occurs during the oxidation of L-arginine to L-citrulline, catalyzed by three NOS isoforms: neuronal (NOS1), inducible (iNOS or NOS2) and endothelial (eNOS or NOS3) (4). Both iNOS and eNOS can be present in the lung and have been implicated in the pathogenesis of acute lung injury. The major source of iNOS includes activated neutrophils and macrophages that form NO as part of respiratory burst. The major source of eNOS includes endothelial cells, and increased vascular permeability, a component of acute lung injury, has been attributed primarily to eNOS rather than iNOS activity (5, 6).

Acute lung injury develops in inbred mice during continuous exposure to nickel (submicrometer NiSO₄ aerosol) as indicated by the development of pulmonary edema, focal hemorrhage, and neutrophilic infiltration (7). To initially examine the pathogenesis of acute lung injury, mice were exposed and differential gene expression was evaluated by cDNA microarray. From this analysis, specific genes and pathways were pursued in mice treated with N^G-nitro-L-arginine methyl ester (L-NAME), a competitive inhibitor of all three NOS isoforms (8), and the underlying mechanism of protection from NO inhibition examined.

	Materials and Methods

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Experimental Design
To investigate changes in gene expression in response to moderate nickel exposure, microarray analysis was performed with lung mRNA from A/J mice (6–8 wk; Jackson Laboratory, Bar Harbor, ME) before or 72 h after the initiation of 24 h exposure. These results coupled with our previous observation that mice deficient in functional Ron tyrosine kinase receptor (which may compromise the regulation of NO formation) had heightened susceptibility to nickel-induced acute lung injury (9), lead us to postulate that NOS activity may influence survival. To test this hypothesis, the effects of treatment with two NO inhibitors, L-NAME or aminoguanidine, were compared with those produced with saline (vehicle control). The A/J mouse strain was selected because these mice had been identified as a sensitive strain in response to continuous nickel exposure (approximate mean survival time of 60 h versus 120 h for resistant C57BL/6 strain mice) (7). The hypothesis for the current work was that inhibition of NO would increase survival; therefore, the most sensitive strain was used to increase the likelihood for detection of a difference in mortality. In addition to survival, serum nitrate/nitrite, protein nitrotyrosine formation, NOS activity, cytokine and lung-specific gene expression, pulmonary inflammation (differential cell counts) and protein levels in bronchoalveolar lavage were assessed with and without L-NAME treatment.

Nickel Aerosol Generation
Nickel aerosol was generated from 50 mM NiSO₄ hexahydrate, (Sigma, St. Louis, MO) using a modified Collison three-jet nebulizer (3.5 liters/min; BGI Incorporated, Waltham, MA) placed inside a glass tube (24 mm inner diameter) and introduced 0.3 m³ stainless steel inhalation chamber as described previously (7). The particle size was determined using a differential mobility analyzer consisting of an electrostatic classifier (Model 3071A; Thermo-Systems, Inc., St. Paul, MN), condensation nucleus counter (Model 3022A; Thermo-Systems, Inc.), and scanning mobility particle size fast-scanning software (Thermo-Systems, Inc.). The aerosol had a mass median aerodynamic diameter of 0.2 µm and a geometric standard deviation (g) of 1.9. The chamber nickel concentration was 108 ± 2 µg Ni/m³ determined by the dimethylglyoxime method (10).

Microarray Analysis
To examine differential gene expression of over 8,700 cDNAs, A/J mice were exposed to nickel for 24 h and total lung RNA isolated at 72 h from the initiation of exposure. Mice were anesthetized (sodium pentobarbital, 5 mg intraperitoneally; Abbott, Chicago, IL), ventricular blood was obtained for serum nitrate/nitrite analysis, and lobes of lung removed and immediately frozen in liquid nitrogen and stored at -70°C until analysis. The lung was placed in TRIZOL solution (Life Technologies, Gaithersburg, MD), homogenized using a Tissumizer (Tekmar Co., Cincinnati, OH), and total RNA isolated by centrifugation. The microarray was fabricated by the Genomic and Microarray Laboratory, Center for Environmental Genetics, University of Cincinnati (http://microarray.uc.edu/). Briefly, clones from the Incyte Genomics mouse GEM1 Library were amplified by PCR and printed onto glass slides (Omnigrid Microarrayer; GeneMachines, San Carlos, CA). This set of 8,734 cDNAs contains 3,205 clones for known genes, 2,045 for RIKEN cDNAs, 2,103 nonannotated expressed sequenced tags (ESTs), 1,066 for annotated ESTs, and 315 DNA segments or hypothetical proteins. Six microarrays were compared using 20 µg total RNA (n = 6 slides; 3–4 mice/slide; total n = 20 unexposed control or 20 nickel-exposed, saline-treated A/J mice). Each sample of pooled RNA was reverse transcribed and tagged with either the fluorescent Cy3 (A/J control) or Cy5 (A/J exposed and collected at 72 h). Cy3 and Cy5 samples were co-hybridized with the printed cDNAs. Following hybridization, slides were washed and scanned at 635 (Cy5) and 532 (Cy3) nm (GenePix 4000B; Axon Instruments, Inc., Union City, CA). A balance coefficient was calculated for each slide by determining the inverse of the median of all the Cy3 to Cy5 ratios. The average balance coefficient was 0.80 ± 0.03 SEM. Each Cy3 value (minus background) was multiplied by the corresponding balance coefficient and results expressed as a ratio. For inclusion in analysis, a cDNA covered its grid location on a slide by a minimum of 50% and a maximum of 400%. The average number of cDNAs passing these criteria per slide exceeded 8,700. A mean and SEM (n = 6) was calculated for each balanced differential expression value using GeneSpring (Silicon Genetics, Redwood City, CA).

L-NAME Treatment
A/J mice were pretreated with L-NAME (40 mg/kg in saline, intraperitoneal), an inhibitor of all three NOS isoforms, and compared with aminoguanidine (100 mg/kg in saline, intraperitoneal), an inhibitor primarily of iNOS, or with an equal volume of saline 3 h before nickel exposure. L-NAME, aminoguanidine, or saline treatment was repeated every 24 h for 9 d, terminating at Day 9 (L-NAME treatment group) as mice that survived were regaining original body weight. Survival was monitored up to 2 wk (L-NAME, n = 15; aminoguanidine, n = 9; saline, n = 20).

Nitrate/Nitrite Analysis
Nitrate/nitrite was measured in serum and bronchoalveolar lavage fluid. Blood was allowed to coagulate (22°C, 30 min) and following centrifugation (5,000 x g, 10 min) the supernatant (serum) was isolated and frozen. To reduce nitrate to nitrite, 25 µl nitrate reductase (0.6 U/ml of 40 mM Tris pH 7.6) and 25 µl NADPH (0.1 mg/ml of 40 mM Tris pH 7.6) were added to 50 µl thawed serum or lavage and incubated (22°C, 3 h). Nitrate/nitrite was measured using the Griess Reagent System (Promega, San Luis Obispo, CA) that uses sulfanilamide and N-1-napthylethylenediamine dihydrochloride under acidic (phosphoric acid) conditions to produce an azo-compound (absorbance 520–550 nm).

Measurement of NOS Activity and Nitrotyrosine Residue Immunohistochemistry
To assess NOS activity, Ca²⁺-dependent and Ca²⁺-independent conversion of L-arginine to L-citrulline, indicators of eNOS and iNOS activity, respectively (11), were determined in lung homogenates obtained from mice before and at selected times after the initiation of a 24 h nickel exposure. Lungs were homogenized on ice in a buffered solution containing 50 mM Tris-HCl, 0.1 mM EDTA and 1 mM phenylmethylsulfonyl fluoride (pH 7.4) and the conversion of [³H]-L-citrulline was measured as described previously (11). Briefly, homogenates (30 µl) were incubated (22°C, 20 min) in the presence of 10 µM [³H]-L-arginine (5 kBq/sample), 1 mM NADPH, 30 nM calmodulin, 5 µM tetrahydrobiopterin, and 2 mM EGTA. Reactions were stopped by dilution with 0.5 ml of ice-cold HEPES buffer (pH 5.5) containing 2 mM EGTA and 2 mM EDTA. Reaction mixtures were applied to Dowex 50W (Na⁺ form) columns and the eluted [³H]-L-citrulline activity was measured by a scintillation counter (Wallac, Gaithersburg, MD). Activity of the enzyme is expressed as fmol/mg tissue protein/min.

To assess nitrotyrosine residues by immunohistochemistry, mice were anesthetized and exsanguinated. The trachea was exposed and cannulated using 0.58-mm (inner diameter) polyethylene tubing (Clay Adams, Parsippany, NJ) inserted through a slit below the larynx. The diaphragm was punctured and the lung infused (1.0 ml 4% paraformaldehyde in phosphate-buffered saline; 30 cm H₂O). After infusion, the chest wall was opened, trachea ligated, heart, lung, and trachea removed and immersed in fixative (16 h; 4°C). The lower half of each lobe was paraffin embedded following dehydration in ethanol, and sectioned (5 µm). Slides were stained with hematoxylin and eosin. Immunohistochemical analysis for antinitrotyrosine was performed as previously described (13). Paraffin was removed using xylene, endogenous peroxidase activity depleted with H₂O₂, and nonspecific binding inhibited with normal goat serum (Vector Laboratories, Burlingame, CA). Nitrotyrosine residues were recognized using an antinitrotyrosine, rabbit polyclonal antibody (Upstate Biotechnology, Lake Placid, NY) at a dilution of 1:500 and detected using a biotinylated anti-rabbit antibody and avidin-coupled horseradish peroxidase (Vectastain ABC kit; Vector Laboratories) reacting with H₂O₂ and 3,3'-diaminobenzidine (Sigma) to form a colored precipitate.

Pulmonary iNOS and Cytokine mRNA Analysis
To examine whether iNOS and cytokine mRNA expression differed with L-NAME treatment, RNase protection assays were performed (Ambion, Austin, TX) using the following murine riboprobes (PharMingen, San Diego, CA): inducible nitric oxide synthase (iNOS or Nitric oxide synthase 2, inducible, macrophage: Nos2), interleukin (IL)-1ß, IL-6, IL-10, IL-12, macrophage inflammatory protein-2 (MIP-2, or Small inducible cytokine subfamily, member 2: Scyb2), monocyte chemoattractant protein-1 (MCP-1 or Small inducible cytokine A2: Scya2), tumor necrosis factor- (TNF-), macrophage migration inhibitory factor (Mif), Regulated upon Activation, Normally T-Expressed, and presumably Secreted (Small inducible cytokine A5: Scya5), transforming growth factor- (TGF-), granulocyte macrophage-colony-stimulating factor [Colony stimulating factor 2 (granulocyte-macrophage): Csf2], interferon (IFN)-, and ribosomal protein L32 (L32 or mitochrondrial ribosomal protein L32: Mrpl32). Antisense, radiolabeled RNA probes were generated from these cDNAs using 40 U RNasin, 0.14 mM each GTP/CTP/ATP, 0.003 mM UTP, 10 mM DTT, 1x Transcription Buffer, 20 U T7 RNA polymerase (PharMingen), and 0.1 mCi -[³²P]-uridine 5'-triphosphate (37°C, 1 h; NEN, Boston, MA). The reaction was terminated (2 U RNase-free DNase; 37°C; 30 min), 2 µg tRNA and 4 µl of 5x stop buffer (5x stop: 1 M EDTA, 10% Ficoll, 0.1% bromophenol blue) were added and the radiolabeled probes column purified (G-50; Roche, Indianapolis, IN). Total RNA (10 µg) was denatured (95°C, 5 min) and hybridized by incubation (56°C, 16 h) in solution containing 2 µl [³²P]-labeled probes (800,000 cpm), 16 µl deionized formamide, 0.4 M NaCl, 2.0 mM EDTA, and 0.04 M 1,4-piperazinediethanesulfonic acid (pH 6.6). Nonhybridized RNA was digested by incubation (37°C, 1 h) in a solution containing 3.8 U RNase A/152 U RNase T1/µl RNase Digestion III Buffer (Ambion). Double-stranded RNA was precipitated with 225 µl RNase Inactivation/Precipitation III Solution (Ambion) and 100 µl 100% ethanol and then dried. The resulting pellet was resuspended in 95% formamide solution containing 0.025% xylene cyanol and bromophenol blue, 18 mM EDTA, 0.025% SDS. Fragments were separated by denaturing electrophoresis (6% polyacrylamide gel containing 8 M urea) and quantified by PhosphorImager analysis (ImageQuant; Molecular Dynamics PhosphorImager, Sunnyvale, CA). Each cytokine was normalized to the expression of ribosomal protein L32 mRNA for each mouse.

Surfactant Gene Expression
To determine whether surfactant gene expression differed with L-NAME treatment, total lung RNA was hybridized to S1 probes for murine surfactant protein A (SP-A), SP-B, SP-C, and L32 as previously described (12). Briefly, fragments containing cDNA sequence for each surfactant were end-labeled with ³²P-ATP, denatured and hybridized by incubation (55°C, 16 h) of 3 µg total lung RNA. S1 nuclease (110 U; Life Technologies) digestion was performed in the presence of excess unlabeled salmon sperm DNA (22°C, 1 h). Fragments were separated by electrophoresis (6% polyacrylamide; 8 M urea gels) and quantitated by phosphorimaging (ImageQuant; Molecular Dynamics PhosphorImager). Surfactant expression was normalized to L32 for each mouse.

Bronchoalveolar Lavage
To examine whether PMN cell counts or protein differed with L-NAME treatment, bronchoalveolar lavage was performed. Control mice (no exposure, no treatment), saline-treated, and L-NAME–treated mice (n = 2–9/treatment/exposure) were anesthetized and exsanguinated. The trachea was exposed and a bent, blunt-end 20-gauge needle inserted at the larynx, and secured with suture. A syringe containing 1 ml Ca²⁺, Mg²⁺-free Dulbecco's phosphate-buffered saline (D-PBS; Life Technologies) was connected to the needle, D-PBS was infused into the lung and recovered, and placed immediately on ice. A total of three 1-ml aliquots were pooled, mixed, and 300 µl removed. Cells from this sample were placed on a microscopic slide (Cytospin 3; Shandon, Sewickley, PA), stained (Hema 3; Fisher, Pittsburgh, PA), and differential cell count performed with at least 300 cells identified. The remainder was centrifuged (150 g; 10 min). The supernatant retained for protein analysis using a Bradford assay (Bio-Rad, Hercules, CA) and cell pellet resuspended in 300 µl D-PBS for total cell count using a hemocytometer.

Statistical Analysis
Two-way ANOVA followed by Student-Newman-Keuls Method for Multiple Comparisons was performed on normally distributed data from differential cell counts, protein, and RNase and S1 nuclease protection assays. For microarray analysis, P values were derived from t test analysis using the default statistical comparison provided by GeneSpring (Silicon Genetics, Redwood City, CA). One-way ANOVA was performed followed by Dunnett's Method for Multiple Comparisons for comparison of L-NAME or saline treatment to baseline (no exposure, no treatment). Log-rank was used for survival analysis. NOS activity data were analyzed by t test. Values are expressed as mean ± SEM and P < 0.05 was used as threshold of significance.

	Results

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Differential Gene Expression in Saline-Treated A/J Mice
To examine the gene expression profile from a mouse strain sensitive to irritant-induced acute lung injury (7) in response to moderate nickel exposure, microarray analysis was performed with lung mRNA from A/J mice exposed to nickel for 24 h and then examined at 72 h and compared on the same slide to mRNA from unexposed A/J mice (control). The total number of genes differing between exposed and unexposed mice equaled 2,463 (28% of the total number of sequences read). Nearly an equivalent number of clones exhibited increased (1,183) as those that exhibited decreased (1,280) expression.

The results for known genes and annotated ESTs that increased the most are present in Table 1. The largest increase was S100 calcium binding protein A8 (Calgranulin A) (S100a8), a secreted chemotactic protein. Several cDNAs (including metallothionein 1; lysyl oxidase; lectin, galactose binding, soluble 3; nuclear factor light chain gene enhancer B-cells inhibitor, ; interleukin 4 receptor, ; and elastin) were increased in the sensitive A/J strain following limited nickel exposure (24 h) much as they were in the irritant resistant C57BL/6 strain with continuous nickel exposure (14). The cDNAs for known genes and annotated ESTs that were decreased the most are present in Table 2. The largest decrease was surfactant-associated protein C (Sftpc). Again, several cDNAs (including cytochrome P450, 2f2; fatty acid synthase; hemolytic complement; surfactant-associated protein B) were decreased in the sensitive A/J strain with a limited duration of nickel exposure much as they were in the resistant C57BL/6 mice with continuous nickel exposure (14).

View larger version (10K): [in this window] [in a new window]   Figure 1. NOS inhibitor, L-NAME treatment increases survival in response to nickel-induced acute lung injury. Mice pretreated (3 h) with L-NAME (filled circles; n = 15), an iNOS inhibitor (aminoguanidine [filled squares; n = 9] or saline [open triangles; n = 20]), were exposed to nickel sulfate aerosol (108 ± 2 µg Ni/m3; 0.2 µm MMAD, g = 1.9) for 24 h, and survival was monitored. L-NAME, aminoguanidine, and saline treatment were repeated every 24 h until Day 9. L-NAME–treated mice survived longer than saline-treated mice, P < 0.05 by log-rank analysis. Survival was not different between aminoguanidine- and saline-treated mice, P > 0.05.

L-NAME Reduces eNOS Activity
Because L-NAME could be inhibiting either constitutive or inducible nitric oxide synthases, eNOS and iNOS activity assays were performed with homogenates isolated from control (unexposed), and saline-treated or L-NAME–treated mouse lung after initiation of a 24 h nickel exposure. With nickel exposure, eNOS (Ca²⁺-dependent) activity increased ( 2.5-fold) in saline-treated mice as compared with control (Figure 2) . With L-NAME treatment, eNOS activity was not different from control but was less than the activity of saline-treated mice. This attenuation was significant at 8, 24, and 48 h after the initiation of a 24-h nickel exposure. Ca²⁺-independent activity (iNOS) activity was undetectable in control, or in saline- or L-NAME–treated mouse lung homogenates at any time. Nitrotyrosine residues were not detected in lung sections by immunohistochemistry.

View larger version (18K): [in this window] [in a new window]   Figure 2. L-NAME attenuates eNOS activity. Enzymatic conversion of L-arginine to L-citrulline was assayed with and without Ca2+ in homogenates isolated from control (unexposed), and saline-treated or L-NAME–treated mouse lung after initiation of nickel exposure (NiSO4: 108 ± 2 µg Ni/m3; 0.2 µm MMAD, g = 1.9). With nickel exposure, eNOS (Ca2+-dependent) activity increased ( 2.5-fold) in saline-treated mice as compared with control. With L-NAME treatment, eNOS activity was not different from control, but was less than the activity of saline-treated mice. Calcium-independent (representative of inducible nitric oxide synthase, iNOS) activity was undetectable in control or in saline- or L-NAME–treated mouse lung homogenates at any time. Mice were pretreated (3 h) with L-NAME or saline and exposed to nickel for 24 h. L-NAME and saline treatment were repeated every 24 h (L-NAME treatment terminated at Day 9). Samples were pooled in pairs and each pooled sample was done in triplicate. Values are mean ± SEM and were analyzed by t test. *Significantly (P < 0.05) increased in saline-treated animals versus unexposed controls (Oh = 0.52 ± 0.27 fmol/mg/min). **Significantly (P < 0.05) different when L-NAME–treated mice were compared with saline-treated mice at the same time.

View larger version (20K): [in this window] [in a new window]   Figure 3. L-NAME treatment attenuates cytokine mRNA expression. RNase protection analysis was performed on total lung RNA from mice pretreated (3 h) with L-NAME (open bars; n = 4/time) or saline (filled bars; n = 4/time) and exposed to nickel for 24 h (NiSO4: 108 ± 2 µg Ni/m3; 0.2 µm MMAD, g = 1.9). L-NAME and saline treatment were repeated every 24 h. Expression of interleukin (IL)-6, macrophage inflammatory protein-2 (MIP-2, or Small inducible cytokine subfamily, member 2: Scyb2), monocyte chemoattractant protein-1 (MCP-1 or Small inducible cytokine A2: Scya2), IL-1ß, tumor necrosis factor (TNF)-, or IFN- was normalized to ribosomal protein L32 (L32 or mitochondrial ribosomal protein L32: Mrpl32) Values (mean ± SEM) are presented as fold of baseline (unexposed and untreated). *Significantly (P < 0.05) less expression in L-NAME–treated when compared with saline-treated mice as assessed by two-way ANOVA.

View larger version (18K): [in this window] [in a new window]   Figure 4. Mif mRNA expression increases with nickel-induced acute lung injury. Total lung RNA was assessed for Mif expression by RNase protection analysis. Mice were pretreated with L-NAME (open circles; n = 4/time) or saline (filled circles; n = 4/time) for 3 h, exposed to nickel for 24 h (NiSO4: 108 ± 2 µg Ni/m3; 0.2 µm MMAD, g = 1.9), and treated with L-NAME or saline repeatedly every 24 h for 9 d. Mif expression was greater than baseline (unexposed and untreated) throughout 48 h in both the L-NAME– and saline- treated mice (P < 0.05), but the groups were not significantly different (P > 0.05), as assessed by two-way ANOVA.

View larger version (13K): [in this window] [in a new window]   Figure 5. L-NAME treatment restores SP gene expression. Total lung RNA was analyzed by S1 nuclease protection assays for the expression of SP-A, -B, and -C in mice pretreated (3 h) with L-NAME (open circles; n = 4/time) or saline (filled circles; n = 4/time), exposed to nickel for 24 h (NiSO4: 108 ± 2 µg Ni/m3; 0.2 µm MMAD, g = 1.9). Treatments with L-NAME or saline were repeated daily for 9 d. Expression of SP-A, -B, or -C was normalized to ribosomal protein L32 gene expression and values (mean ± SEM) are presented as percent of control (unexposed and untreated). *Significantly (P < 0.05) greater expression with L-NAME treatment than with saline treatment as assessed by two-way ANOVA.

	Discussion

Top Abstract Introduction Materials and Methods Results Discussion References

Interestingly, L-NAME treatment apparently had little protective effect on the initial increase in cytokine expression. Early (8–48 h) increases in IL-6, MIP-2, MCP-1, IL-1ß, TNF-, and IFN- were similar in saline- and L-NAME–treated mice. However, by 72 h, a large increase in the expression of each of these cytokines in the saline-treated mice was attenuated in the L-NAME–treated mice. The cellular source of subsequent increase in cytokines is unknown; however, additional PMNs infiltrating into the lung at 72 h in saline-treated mice also may have contributed to the increased cytokine expression. The inhibition of the induction of eNOS activity by L-NAME and subsequent attenuation of IL-6, MIP-2, and MCP-1 are consistent with our previous findings in which a loss of the tyrosine kinase domain of Ron was associated with a loss in NO regulation and large increased expression in each of these cytokines (9). L-NAME treatment prevented enhanced cytokine expression and possibly cytokine-induced pathogenesis.

A unique pattern of gene expression was displayed by Mif in comparison to the expression of the 12 other cytokines examined. Similar to IL-6, MIP-2, MCP-1, IL-1ß, TNF-, and IFN-, Mif expression increased early with or without L-NAME treatment. However, augmented Mif expression reversed rapidly after cessation of exposure, and returned to near baseline levels by 72 h in both L-NAME– and saline-treated mice. This pattern suggests that continuous nickel exposure is required to maintain Mif expression. Previously associated with acute lung injury, Mif exhibits a number of unusual properties that distinguish it from other cytokines (15–17). It is glucocorticoid-induced, has enzymatic thiol-protein oxidoreductase and a tautomerase/isomerase activity, and can stimulate release of TNF-, MIP-2, IL-8, and augmenting lipopolysachrride-induced neutrophil chemoattractant in the lung. It seems to have a lesser role in this model of acute lung injury because it did not display a secondary increase in the saline-treated mice.

The initial response to nickel-induced acute lung injury included a large decrease in expression of SP-B and SP-C as noted by microarray and S1-nuclease protection assays. L-NAME treatment had no apparent protective effect in this initial decrease of surfactant protein gene expression. However, surfactant gene expression was returning to baseline levels at 72 h with L-NAME treatment. That surfactant gene expression is essential for survival has been demonstrated both clinically and by SP-B gene-targeted mice. Mice lacking both alleles of SP-B (null) mice succumb to atelectatic respiratory failure within 20 min of birth, yet mice with one SP-B (hemizygotes) allele are viable, but susceptible to lung injury (18). In response to nickel, SP-B gene expression decreased to 50%, and SP-C expression declined to nearly 10% of pre-exposure control levels in the saline-treated mice. Loss of SP-C and SP-B, in conjunction with proteinaceous edema in the alveolar compartment, may have been sufficient for the progression of acute lung injury to death.

It is important to note the limitations in cDNA microarray. Although the functional trends noted in the microarray analysis, including increased inflammatory and decreased constitutive lung gene expression, were essentially confirmed by additional assays, not all changes in expression were validated by additional tests in this study. In the past, we found that nickel-induced acute lung injury is accompanied by increases in metallothionein and heme oxygenase 1, as measured with microarray, and that these effects are similar to those measured by Northern analysis of the same mRNA (14). Nonetheless, caution should be given to any interpretation of the microarray results (other than the cytokine and surfactant genes) until these results are confirmed by additional methods.

In addition to surfactant protein mRNA, decreases in other mRNA levels were noted in genes associated with constitutive Clara (uteroglobin, Cyp2b13) and alveolar type II (fatty acid synthase) cell functions, suggesting that airway and alveolar epithelial cells are perturbed directly by nickel inhalation. These decreases in expression are concurrent with the large increase of lavage total protein, suggesting that alveolar epithelial dysfunction could be contributing to the increased epithelial permeability. However, with L-NAME treatment, large decreases in SP-C are still initially observed, suggesting that eNOS inhibition in other cells also may mediate edema formation.

Microvascular permeability can be stimulated by NO (19). The proposed mechanism is that NO activates soluble guanylate cyclase forming of cyclic guanosine monophosphate from GTP, which in turn activates protein kinase G and alters Ca²⁺ levels (20). This signaling cascade may lead to changes in endothelial cell shape, providing passage for serum components (21). Thus, microvascular permeability increases as a response to a signaling cascade, rather than disruptions in the endothelial barrier. This concept is supported by histologic analysis of nickel-induced acute lung injury that revealed only slight endothelial disruption, yet marked increases in lung wet: dry weight, extensive extravascular fluid accumulation surrounding major blood vessels (perivascular edema), and interstitial thickening in the alveolar wall (7).

Previously, eNOS, but not iNOS, was found to mediate increases in cellular infiltration, vascular permeability, and edema formation induced by caveolin-1 peptide in Swiss mice (5). Furthermore, vascular endothelial growth factor–induced increases in vascular permeability appear to be primarily mediated by eNOS rather than iNOS from a study using gene-targeted mice (6). When iNOS is stimulated, NO levels can be elevated to concentrations readily detected in serum and lavage and that lead to the formation of nitrotyrosine residues. However, eNOS-catalyzed reactions can result in only slight, yet physiologically relevant, localized increases in NO (22). That iNOS appears to play a less significant role than eNOS during transient nickel exposure (24 h) was supported by the following: iNOS activity was not detectable, iNOS gene expression remained unchanged, aminoguanidine (an iNOS-specific inhibitor [23]) failed to protect mice from nickel-induced acute lung injury, and nitrotyrosine residues were not observed in lung sections. Rather, eNOS activity increased in saline-treated mice and this increase was attenuated with L-NAME treatment. Taken together, this evidence supports the concept that L-NAME appears to provide a critical turning point in the progression of nickel-induced acute lung injury by inhibiting NO-induced vascular permeability that is primarily mediated by eNOS. Although eNOS is important in the regulation of vascular homeostasis, the data suggest that a partial inhibition (or prevention of increase) has no deleterious effect. On the contrary, it may have a therapeutic effect during an acute injury response.

Previous studies of acute lung injury induced by N-nitroso-N-methylurethane (2) or neonatal hyperoxia (3) found that inhibiting NO formation restored pulmonary function. The current work builds upon these findings through the use of a transient insult (limited nickel exposure). In this study, acute lung injury progressed after the cessation of exposure through two phases distinguished by different responses to L-NAME. The first phase predominately involved the pulmonary epithelial function, with a large decrease in surfactant gene expression accompanied by a moderate increase in expression of several cytokines. During this initial stage, L-NAME treatment appears to have little effect upon surfactant and cytokine gene expression. The second phase was marked by further increases in cytokine expression, neutrophil infiltration, and protein in bronchoalveolar lavage. The changes noted during this phase were attenuated by L-NAME treatment. Together, these findings indicate that inhibition of NO formation during the second phase of acute lung injury may be protective, possibly by limiting eNOS-mediated vascular permeability, blunting cytokine formation, and enabling subsequent restoration of surfactant homeostasis.

	Acknowledgments

 
The authors thank Dr. Mario Medvedovic and Dr. Peter Gartside for statistical assistance; Lisa Warshawsky, Lisa Case, and Wayne Tsuang for exposure assistance; and Michael O'Connor for assistance in measuring NOS activity. This study was supported by HL65612, HL65213, AI4556, ES06096, and ES10562 from the National Institutes of Health, and by the Health Effects Institute. The Health Effects Institute is an organization jointly funded by the US Environmental Protection Agency, Assistance Agreement X-812059, and automotive manufacturers. The contents of this article do not necessarily reflect the views of the Health Effects Institute or the policies of the U.S. Environmental Protection Agency or automotive manufacturers. S. M. is a recipient of the U.S. Environmental Protection Agency Science to Achieve Results Graduate Fellowship and the Albert J. Ryan Fellowship, and this work was conducted in partial fulfillment of the requirements for the Ph.D. degree at the University of Cincinnati.

Received in original form May 28, 2002

Received in final form September 25, 2002

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