INTRODUCTION

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Nitric oxide (NO) may either contribute to or protect against inflammatory lung injury. NO can react with O₂ to form the cytotoxic compound, nitrogen dioxide (NO₂), and with superoxide to form a more potent molecule, peroxynitrite, which causes tyrosine nitration in lung tissue (1). Thus, when superoxide is produced at high rates during inflammatory lung injury, inhaled NO might accelerate the formation of peroxynitrite and aggravate lung damage.

Myeloperoxidase-dependent pathways also contribute to the formation of tyrosine nitration, because a recent study showed that the myeloperoxidase of polymorphonuclear neutrophils converts nitrite (NO₂) into NO₂Cl and NO₂, resulting in tyrosine nitration, and that the tyrosine nitration is enhanced by addition of NO₂ or by flux of NO (2) during leukocyte accumulation.

Nitration of tyrosine located at the key positions in a protein may alter protein structure and function in vivo (2, 3). Nitrotyrosine is known to be reduced to aminotyrosine. Therefore, the amount of nitrotyrosine is determined by the balance between the rate of nitrotyrosine production (tyrosine nitration) and the rate of nitrotyrosine reduction to aminotyrosine. Therefore, aminotyrosine should be evaluated as well as nitrotyrosine to detect the degree of tyrosine nitration, and aminotyrosine itself may also change the protein structure and function.

On the other hand, the results of several studies performed under various conditions have suggested that NO may act as an antioxidant to counteract the cytotoxic effects of reactive oxygen species. In vitro, NO was found to inhibit lipid peroxidation caused by oxygen radicals and to protect against cellular damage (4). In in vivo experiments, hyperoxic lung injury (5) and the damage induced by ischemia and reperfusion (6) were found to be reduced by inhaled NO. The reduction of damage by NO is thought to be attributable to the antiadhesion effect of NO, which prevents leukocyte infiltration. Inhibition of leukocyte recruitment in the lungs will reduce the amount of myeloperoxidase, and it may reduce the degree of tyrosine nitration.

However, the potential of inhaled NO to affect tyrosine nitration and alveolar damage during pulmonary inflammation in the lungs has not been extensively investigated. Furthermore, although nitrotyrosine was detected in septic lung injury (7) and idiopathic pulmonary fibrosis (8), aminotyrosine, a reduced form of nitrotyrosine, has not been investigated.

The specific questions we attempted to answer in this study were: (1) Does NO inhalation affect tyrosine nitration in control and in lipopolysaccharide (LPS)-instilled lung tissue? (2) Does NO inhalation affect pulmonary leukocyte infiltration and edema after LPS instillation? and (3) Does leukocyte accumulation affect tyrosine nitration in LPS-instilled lungs tissue? To answer these questions, we (1) quantitatively measured nitrotyrosine in lung tissue and stained for nitrotyrosine immunohistochemically; (2) newly developed an antiaminotyrosine antibody and immunohistochemically stained aminotyrosine; (3) evaluated the degree of pulmonary edema and cell accumulation by determining lung water, by measuring cell count and albumin content in bronchoalveolar lavage fluid (BALF), and by measuring myeloperoxidase activity in lung tissue; and (4) instilled LPS in leukocyte-depleted rats and stained nitrotyrosine and aminotyrosine.

In this study we chose LPS instillation rather than intraperitoneal or intravenous LPS injection to produce pulmonary inflammation in an attempt to focus on pulmonary inflammation without causing systemic inflammation and multi-organ failure.

	METHODS

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General Protocol

Animal care was in accordance with the guidelines of the Animal Care Committee of Kitasato University, and the conduct of these studies conformed to the Guide for the Care and Use of Laboratory Animals published by the U.S. National Institutes of Health.

Male Sprague-Dawley rats, 7 wk old, weighing 200 to 250 g (Clea Japan, Tokyo), were randomly assigned to four groups: (A) room air after 0.4 ml saline instillation (control group); (B) 20 parts per million (ppm) NO inhalation after 0.4 ml saline instillation (NO group); (C) room air after instillation of 40 µg LPS (Escherichia coli, 0127:B8, phenol extract, L-3129; Sigma, St. Louis, MO) in 0.4 ml saline (LPS group); and (D) 20 ppm NO inhalation after instillation of 40 µg LPS in 0.4 ml saline (LPS + NO group). After instillation, the animals were exposed to either room air or 20 ppm NO in air for 16 h. Leukocyte count in BALF after LPS instillation was reported to increase gradually over 12 h (9), and decrease at 24 h after intraperitoneal LPS treatment (10). We therefore chose the time point of 16 h, and could obtain sufficient leukocyte accumulation in the lungs after LPS instillation. There was no mortality associated with LPS and/or NO exposure.

In each group of animals, we performed: (1) total cell count, cell differential count, and measurement of albumin content in BALF; (2) myeloperoxidase activity in lung homogenates; (3) nitrotyrosine analysis of lung homogenates; (4) immunohistochemical staining for nitrotyrosine and aminotyrosine; and (5) determinations of extravascular lung water weight (EVLW) and extravascular dry lung weight (EVDW).

To evaluate the role of leukocytes in the production of nitrotyrosine and aminotyrosine in LPS-instilled rats, we depleted leukocytes by treating rats with cyclophosphamide, and stained nitrotyrosine and aminotyrosine 16 h after LPS instillation.

LPS Instillation

After anesthesia with diethyl ether (Wako Pure Chemical Industries, Ltd., Osaka, Japan) 400 µl of endotoxin-free saline or LPS solution was injected into the trachea via an endotracheal polyethylene tube with an outer diameter of 1.67 mm (No. 5; Hibiki Co., Tokyo, Japan). In a few minutes rats recovered from the anesthesia, and were immediately placed in an exposure chamber.

NO Gas Delivery

NO was supplied from a nitrogen-balanced NO gas cylinder (20,000 ppm ultra-high pure grade; Sumitomo Seika Chemicals Co., Osaka, Japan) through thermal mass-flow controllers (SEC-4400; Estec, Kyoto, Japan). NO gas was diluted just before the exposure chamber (20 L) with filtered air (25° C, relative humidity 50 to 55%) to obtain 20 ppm NO. The flow rate was set at 10 L/min for each chamber. Three to four animals were housed in each chamber. NO and NO₂ levels were monitored by electrochemical sensors (TM-100 and TM-1002, respectively; Saan, Osaka, Japan), and the NO₂ level in the chamber was always less than 1.0 ppm. For animals without NO inhalation, only filtered air was introduced at 10 L/min to the chamber.

Bronchoalveolar Lavage (BAL)

Eight animals were studied in each group. The animals were anesthetized by intraperitoneal injection of sodium pentobarbital (50 mg/kg body weight) and killed by severing the abdominal aorta. The lungs were exposed, and lavaged with endotoxin-free saline (Otsuka Pharm, Naruto, Japan) 5 times. The volume of saline used in each wash was 0.035 (ml/g) multiplied by body weight (g). BAL fluid was placed in conical polypropylene tubes in ice. The recovered amount of BAL fluid was always over 90% of the injected fluid, and LPS instillation did not significantly decrease the recovery rate. The total nucleated cells in the tube were counted immediately with a hemocytometer, and the differential cell count was performed on a cytocentrifuged smear stained with Diff-Quik (Kokusai Shiyaku Co., Kobe, Japan).

Myeloperoxidase Activity in the Lung

Five animals were studied in each group. The myeloperoxidase activity in the lung was assayed according to Hirano (10). Briefly, a portion of lung was homogenized in nine volumes of 50 mM phosphate buffer (pH 6.0) containing 0.5% hexadecyltrimethyl ammonium bromide and 5 mM ethylenediaminetetraacetic acid (EDTA) on ice using a Polytron homogenizer (Kinematica, Luzern, Switzerland). The homogenate was centrifuged at 3,000 g for 15 min at 4° C. The supernatant was centrifuged again. Then 10 µl of the clear supernatant, 200 µl of the homogenization buffer, 2 ml of Hanks' balanced salt solution at pH 6.2, and 100 µl of O-dianisidine · 2 HCl solution (1.25 mg/ml) (Sigma, St. Louis, MO) were mixed and preincubated at 37° C. To start the reaction, 100 ml of 0.05% H₂O₂ was added. After 15 min of incubation, 100 µl of 1% NaN₃ was added to stop the reaction, and the optical density of the mixture at 460 nm was measured.

Hydrolysis of Lung Tissue and Nitrotyrosine Analysis

Six animals were studied in each group. The animals were anesthetized by intraperitoneal injection of sodium pentobarbital (50 mg/kg body weight) and killed by severing the abdominal aorta. The amount of nitrotyrosine was measured by using high-performance liquid chromatography (HPLC) according to Fukuyama and colleagues (11), which is a slight modification of the procedure described in a previous report by Kaur and Halliwell (12). Briefly, approximately 20 mg of lung tissue was homogenized with 500 µl of Milli-Q water and hydrolyzed, and the homogenized tissue was incubated at 110° C for 24 h in a vessel rack (aminochrome 593R; Jasco Ltd., Tokyo, Japan) containing 6N HCl and 0.1% phenol, which was added to rule out artifactual nitrotyrosine formation during acid hydrolysis in the presence of nitrite or nitrate. Separation of nitrotyrosine was achieved by HPLC on a Nucleocil 5-µg C-18 reverse-phase column (15 cm × 4.6 cm) with a guard column (Crest C18T-5P; Jasco Ltd., Tokyo, Japan). The column was eluted with 50 mmol/L KH₂PO₄-H₃PO₄ (pH 3.01) containing 10% methanol (vol/vol) at a flow rate of 1 ml/min through an isocratic pump, and peaks were measured with an ultraviolet detector set at 274 nm (UV-970; Jasco Ltd., Tokyo, Japan). The nitrotyrosine peak was identified by comparison of its retention time with that of authentic nitrotyrosine, and the identification was confirmed by reduction with excess Na₂S₂O₄ to yield aminotyrosine. The nitrotyrosine detection limit in this system was 0.6 µmol/L. Tyrosine and nitrotyrosine recovery rates after the hydrolysis procedure, determined by using authentic tyrosine and nitrotyrosine, were over 80% in both cases. Nitrotyrosine data were expressed as ratios of nitrotyrosine to total tyrosine to normalize the values with respect to lung concentration of tyrosine.

Tissue Fixation and Immunohistochemistry of Nitrotyrosine and Aminotyrosine

Three animals were studied in each group. They were anesthetized by intraperitoneal injection of sodium pentobarbital (50 mg/kg body weight), and the heart and lungs were exposed via a midline incision through the sternum. The trachea and main pulmonary artery were cannulated with polyethylene catheters, and the pulmonary veins were ligated. The pulmonary artery and trachea were distended to 100 cm and 23 cm of water pressure, respectively, with 4% paraformaldehyde (PFA) in phosphate-buffered saline (PBS), and the heart and lungs were removed en bloc and fixed for an additional 3 h in 4% PFA in PBS. The technique we used to distend lungs has been well characterized (13). We then selected tissue from the right cardiac and diaphragmatic lobes and from the single-lobed left lung, to confirm even distribution of the histological findings, and stored them in 4% PFA in PBS at 4° C overnight. After washing in water for 2 h, the tissue was dehydrated in 70%, 80%, 90%, 99%, and 100% ethanol (2.5 h each), cleared in toluene (1.5 h 3 times), and embedded in paraffin wax. Sections (4 µm thick) were obtained, and a Histostain SP kit (Zymed Laboratories Inc., South San Francisco, CA) appropriate for a polyclonal antibody was used for the immunohistochemical studies. This provides normal goat serum, biotinylated goat anti-rabbit IgG, an enzyme complex, components to prepare chromogen (aminoethylcarbazole), and hematoxylin for counterstaining. The paraffin sections were quenched in 3% hydrogen peroxide in absolute methanol (10 min), washed in PBS, treated with normal serum (10 min), and incubated overnight at 4° C with antinitrotyrosine antibody (rabbit polyclonal IgG diluted to 1:100 in PBS) or antiaminotyrosine antibody (rabbit polyclonal IgG diluted to 1:800 in PBS). The antinitrotyrosine polyclonal antibody was commercially available (Upstate Biotechnology, Lake Placid, NY), but the antiaminotyrosine polyclonal antibody was raised in rabbits by injecting them with aminotyrosine conjugated with bovine serum albumin (BSA), using Freund's complete adjuvant. No cross-reaction with BSA was observed in the immunoprecipitation method after purification of the antibody through a BSA column.

Three negative controls were performed for staining. Preadsorption tests were performed: the antinitrotyrosine antibody was incubated with 20 mM authentic nitrotyrosine, and antiaminotyrosine antibody was incubated with 20 mM authentic aminotyrosine. Nonimmune IgG instead of the primary antibody was applied to the lung sections. Before incubation with antinitrotyrosine antibody, tissue sections were flooded with three washes of 1 M sodium hydrosulfite for 20 s each. The 1 M sodium hydrosulfite solution was prepared just before use, its pH was adjusted at 9 to 9.5 with 2 N NaOH, and it was kept in a sealed vacutainer tube.

As a positive control for antinitrotyrosine antibody staining, after quenching with 3% hydrogen peroxide and washing three times with PBS, the sections were covered with a 1 mM peroxynitrite solution (Upstate Biotechnology, NY) and incubated for 2 h at 37° C. As a positive control for antiaminotyrosine antibody staining, the peroxynitrite-treated tissue sections were flooded with three washes of 1 M sodium hydrosulfite for 20 s each to reduce nitrotyrosine into aminotyrosine. To exclude the cross-reactivity of newly developed antiaminotyrosine antibody with nitrotyrosine, the antiaminotyrosine antibody was incubated with 20 mM authentic nitrotyrosine and used in the tissue treated with peroxynitrite and hydrosulfite.

To further confirm the specificity of antibodies and existence of aminotyrosine and nitrotyrosine in lung tissue, we converted aminotyrosine back to nitrotyrosine by incubating the tissue sections with 10 µM copper sulfate and 100 µM hydrogen peroxide.

Leukocyte Depletion

In four rats, 100 mg/kg cyclophosphamide (Cytoxan; Bristol-Myers, Syracuse, NY) was injected intraperitoneally 6 d before LPS instillation. On the day of LPS instillation, a second injection of 50 mg/kg cyclophosphamide was given. After LPS instillation, rats were housed in the filtered-air chamber for 16 h, and thereafter lung samples were obtained. Nitrotyrosine and aminotyrosine were stained in the tissue sections as previously described. Leukocyte count in blood was confirmed to be less than 400/mm³ on the day of lung fixation.

EVLW and EVDW

Eight animals were studied in each group. EVLW and EVDW were measured according to Pearce and coworkers (14). Briefly, a double amount of water was added to excised lung fragments (about 1 g), and they were homogenized. Blood (approximately 5 ml) was sampled from the abdominal aorta. Aliquots of the homogenate and 1 ml of whole blood were weighed and evaporated in an oven at 60° C for 72 h. Thereafter, the dry weight of the lung aliquots and blood was measured. Other aliquots of the homogenate were centrifuged at 15,000 rpm for 30 min, and the supernatant was obtained and cleared with sodium lauryl sulfate (Sigma) at the final concentration of 1%. Hemoglobin concentrations of the supernatant and whole blood were measured with a spectrophotometer (UV-2200; Shimadzu, Tokyo, Japan) using a commercial kit (HbWako; Wako, Osaka, Japan) based on the cyanomethemoglobin method. The EVLW (difference between whole lung water weight and blood water weight), EVDW (difference between whole lung dry weight and blood dry weight), and the blood-free wet-to-dry weight ratio (the ratio of EVLW plus EVDW to EVDW itself) were calculated according to Pearce and coworkers (14).

Statistical Analysis

Values are expressed as means ± SD unless otherwise stated. Two-way analysis of variance (ANOVA) with category "LPS instillation" and category "NO inhalation" was used to detect the effects of LPS instillation and NO inhalation, and one-way ANOVA with Scheffe's test was employed to detect the difference among the four groups. The chi-square test was applied to the results of quantitative nitrotyrosine measurement using HPLC to examine differences in the amount of nitrotyrosine between the LPS group and the LPS + NO group, because nitrotyrosine was detectable only in the LPS group by HPLC. p Values less than 0.05 were considered significant.

	RESULTS

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Findings in BALF

Inhalation of NO increased the numbers of total cells in BALF (p < 0.01) compared with the control group (Table 1). Two-way ANOVA revealed a significant contribution of "NO" to the increase in pulmonary alveolar macrophages (PAMs) (p < 0.01) and a decrease in polymorphonuclear leukocytes (PMNs) (p < 0.0001). Two-way ANOVA also revealed that there was a significant contribution of "LPS" to increases in PAMs (p < 0.01) and PMNs (p < 0.0001). LPS increased the total cell count (p < 0.0001), mainly due to an increased total number of PMN (Figure 1), but PAMs decreased as a fraction of all cells (Table 1), while the total number of PAMs in the LPS group was increased (Figure 1). Inhalation of NO after LPS instillation suppressed the increase in PMNs (p < 0.0001), but PAMs were increased (p < 0.01) (Figure 1). LPS increased the albumin content in BALF (p < 0.0001). However, inhalation of NO did not affect the albumin content (Table 1).

View larger version (27K): [in this window] [in a new window]   Figure 1.   Cells in BALF. Control: Saline instillation and room air; NO: Saline instillation and inhalation of 20 ppm NO; LPS: lipopolysaccharide instillation and room air; LPS + NO: LPS instillation and inhalation of 20 ppm NO. Inhalation of NO after LPS instillation suppressed the increase in PMNs (p < 0.0001), but PAMs were increased (p < 0.05).

Myeloperoxidase Activity in Lung Homogenate

Inhalation of NO without LPS instillation did not increase myeloperoxidase activity in lung homogenate, but inhalation of NO after LPS instillation suppressed the increase in myeloperoxidase activity (p < 0.01) (Table 1).

Quantitation of Nitrotyrosine in Lung Homogenate

Nitrotyrosine was only detectable in lung homogenate in the LPS-treated group, with the nitrotyrosine concentrations in the other three groups being below the detection limit of 0.6 µmol/L in our HPLC system. Total tyrosine concentrations were similar in all the four groups. The nitrotyrosine level in the LPS-treated group was 0.16 ± 0.15% (mean ± SD, n = 6) with a total tyrosine concentration of 150.4 ± 56.4 µmol/g wet weight, whereas the nitrotyrosine level in the LPS + NO group was below the detection limit. Chi-square test revealed a significant reduction of the detection of nitrotyrosine in the LPS + NO group compared with the LPS group (₀² = 12, p < 0.005).

Nitrotyrosine Staining in the Tissue Sections

In the control group, nitrotyrosine was weakly stained in bronchial epithelial cell surface (Figure 2) and alveolar cuboid cells (because it was difficult to distinguish alveolar Type 2 cells from interstitial macrophages or macrophages attached to the alveolar capillary membrane in our stained sections, we simply described them as "cuboid cells"), but not in alveolar capillaries (Figure 3). Some bronchial vascular cells were stained (because it was difficult to differentiate vascular endothelial cells and perivascular cells, we simply described them as vascular cells, Figure 2). In the NO group, nitrotyrosine in bronchial epithelial cell surface was stained more homogeneously and strongly than in the control group (Figure 2), but not in bronchial vascular cells (Figure 2) or alveolar capillary cells (Figure 3). Bronchial muscle cells were stained slightly in the control group (Figure 2). In the LPS group, nitrotyrosine was stained clearly in the bronchial epithelial cells, some peribronchial and perivascular inflammatory cells, some bronchial vascular cells (Figure 2), and in alveolar macrophages, inflammatory cells, and capillary cells (Figure 3). In the LPS + NO group, fewer inflammatory cells were observed, and the nitrotyrosine staining was similar to the NO group (Figures 2 and 3). Treatment of copper sulfate and hydrogen peroxide in LPS-instilled lung section increased the staining of nitrotyrosine. In LPS-instilled rats with leukocyte depletion, nitrotyrosine was stained clearly in the bronchial epithelial cells (Figure 2), and in alveolar macrophages and alveolar cuboid cells, but not in capillary cells (Figure 3). There was not a prominent leukocyte accumulation in lung tissue (Figures 2 and 3) in the leukocyte-depleted rats.

View larger version (123K): [in this window] [in a new window]   Figure 2.   Nitrotyrosine staining of a large airway and a large vessel. (A) Control group (saline instillation and room air); (B) NO group (saline instillation and inhalation of 20 ppm NO); (C ) LPS group (lipopolysaccharide instillation and room air); (D) LPS + NO group (LPS instillation and inhalation of 20 ppm NO); (E ) Cu + H2O2: Treatment with copper sulfate and hydrogen peroxide of a lung section of the LPS group; (F ) Leukocyte-depleted rats treated with LPS. In the control group, nitrotyrosine was weakly stained in bronchial epithelial cell (be) surface, but not in cell bodies. Some bronchial vascular cells (vc) were also stained. In the NO group, nitrotyrosine in bronchial epithelial cell (be) surface was stained more homogeneously and strongly than in the control group. Some bronchial vascular cells (vc) were also stained. Bronchial muscle cells (mc) stained slightly in the control group. In the LPS group nitrotyrosine stained clearly in the bronchial epithelial cells (be), some peribronchial and perivascular inflammatory cells (*), and some bronchial vascular cells (vc). In the LPS + NO group, only a few inflammatory cells were observed, and the nitrotyrosine staining was similar to the NO group. Treatment of copper sulfate and hydrogen peroxide in a LPS-instilled lung section increased the staining of nitrotyrosine. In the leukocyte-depleted rats, nitrotyrosine stained clearly in the bronchial epithelial cells (be), but there was not a prominent leukocyte accumulation in peribronchial and perivascular tissue in the leukocyte-depleted rats. The calibration bars represent 50 µm.

View larger version (107K): [in this window] [in a new window]   Figure 3.   Nitrotyrosine staining of alveoli. For definition of Groups A-F, see Figure 2 and METHODS. In the control group nitrotyrosine did not stain clearly in alveolar cells except some cuboid cells (cub). In the NO group, nitrotyrosine did not stain clearly in alveolar cells. In the LPS groups, nitrotyrosine stained in alveolar macrophages (am), inflammatory cells in interstitial tissue, and capillary cells (cap). In the LPS + NO group, nitrotyrosine stained in some cuboid cells (cub). Treatment of copper sulfate and hydrogen peroxide in a LPS-instilled lung section showed the staining of nitrotyrosine. In the leukocyte-depleted rats, nitrotyrosine stained clearly in alveolar macrophages (am) and cuboid cells (cub), but not in capillary cells. There was not a prominent leukocyte accumulation in alveoli, and the staining of capillary cells was not obvious in the leukocyte-depleted rats compared with the LPS-instilled rats. The calibration bars represent 50 µm.

Aminotyrosine Staining in the Tissue Sections and Comparison with Nitrotyrosine Staining

In the control group, aminotyrosine was weakly stained in bronchial epithelial cell surface and some cell bodies (Figure 4), and alveolar capillary endothelial cells (Figure 5). Aminotyrosine staining in bronchial epithelial cell surface was also observed in the NO group (Figures 4 and 5). In the LPS group, aminotyrosine staining increased in bronchial epithelial cell surface, some bronchial epithelial cell bodies, some bronchial vascular cells, and inflammatory cells in peribronchial and alveolar interstitial tissues (Figures 4 and 5). Alveolar capillary cells, alveolar macrophages, some alveolar cuboid cells, and interstitial inflammatory cells were stained in the LPS group (Figure 5), whereas in the LPS + NO group fewer inflammatory cells were observed, and aminotyrosine staining was similar to the NO group (Figures 4 and 5). Treatment of copper sulfate and hydrogen peroxide in LPS-instilled lung section decreased the staining of aminotyrosine. In LPS-instilled rats with leukocyte depletion, aminotyrosine was clearly stained in bronchial epithelial cells, (Figure 4) and in alveolar macrophages, cuboid cells, and capillary cells (Figure 5). There was not a prominent leukocyte accumulation in lung tissue (Figures 4 and 5) in the leukocyte-depleted rats.

View larger version (111K): [in this window] [in a new window]   Figure 4.   Aminotyrosine staining of a large airway and a large vessel. For definition of Groups A-F, see Figure 2 and METHODS. In the control group aminotyrosine was weakly stained in bronchial epithelial cell surface (be). Aminotyrosine staining in bronchial epithelial cell (be) surface was also seen in the NO group. In the LPS group, aminotyrosine staining increased in bronchial epithelial cell (be) surface and cell bodies, vascular cells (vc), and inflammatory cells (*) in peribronchial interstitial tissues. In the LPS + NO group aminotyrosine staining was weak in intensity, but diffuse in distribution. Treatment of copper sulfate and hydrogen peroxide in LPS-instilled lung section decreased the staining of aminotyrosine. In LPS-instilled rats with leukocyte depletion, aminotyrosine was clearly stained in bronchial epithelial cells (be), and bronchial and vascular smooth muscle cells (mc). There was not a prominent leukocyte accumulation in peribronchial and perivascular tissue in the leukocyte-depleted rats. The calibration bars represent 50 µm.

View larger version (102K): [in this window] [in a new window]   Figure 5.   Aminotyrosine staining of alveoli. For definition of Groups A-F, see Figure 2 and METHODS. In contrast to nitrotyrosine staining aminotyrosine stained in some alveolar capillary cells (cap) in all four groups (see also Figure 3). In the LPS group alveolar capillary cells (cap), alveolar macrophages (am), some cuboid cells (cub), and interstitial inflammatory cells in interstitial tissue were stained, whereas in the LPS + NO group fewer inflammatory cells were observed, and aminotyrosine staining was similar to the NO group. Treatment of copper sulfate and hydrogen peroxide in LPS-instilled lung section decreased the staining of aminotyrosine. In the LPS-instilled rats with leukocyte depletion, aminotyrosine was stained in alveolar macrophages (am), cuboid cells (cub), and in capillary cells (cap). There was not a prominent leukocyte accumulation in lung tissue in the leukocyte-depleted rats. The aminotyrosine staining differed from nitrotyrosine staining in that aminotyrosine was detected in alveolar capillary cells in all four groups, whereas nitrotyrosine was detected in capillary cells only in the LPS group. The calibration bars represent 50 µm.

The aminotyrosine staining patterns in the four groups were similar to nitrotyrosine staining, but aminotyrosine staining differed from nitrotyrosine staining in that aminotyrosine was detected in some alveolar capillary cells in all four groups, whereas nitrotyrosine was detected in capillary cells only in the LPS group. Leukocyte depletion in LPS-instilled rats reduced nitrotyrosine staining in alveolar capillary cells.

EVLW and EVDW

Total lung weight increased after LPS instillation (p < 0.01), but inhalation of NO had no effect on the total lung weight. The EVLW and EVDW levels after NO inhalation were similar to the control levels. The EVLW and EVDW levels increased after LPS instillation compared with the control levels (p < 0.01), but inhalation of NO (LPS + NO) did not change the increased EVLW and EVDW levels. The EVLW-to-EVDW ratios were similar among the four groups, and the blood-free wet-to-dry lung ratio did not statistically differ among the four groups (Table 2).

	DISCUSSION

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Main Findings of This Study

The main findings of this study were as follows: (1) LPS instillation increased nitrotyrosine and aminotyrosine. (2) LPS instillation increased the leukocyte count and albumin content in BALF, myeloperoxidase activity in lung homogenate, and increased EVLW and EVDW. (3) Inhalation of 20 ppm NO by rats treated with LPS instillation resulted in a significant decrease in nitrotyrosine and aminotyrosine. (4) Inhalation of 20 ppm NO decreased the leukocyte count in BALF and myeloperoxidase activity in lung homogenate. (5) Leukocyte depletion in LPS-instilled rats reduced interstitial inflammatory cells, which were stained with nitrotyrosine and aminotyrosine, and attenuated the nitrotyrosine staining of alveolar capillaries.

Tyrosine Nitration

Although we could not measure nitrotyrosine quantitatively by HPLC except in the LPS group, there was nitrotyrosine staining in bronchial muscle cells and bronchial epithelial cell surface even in control animals, and in the NO group nitrotyrosine was homogenously stained in bronchial epithelial cell surface. LPS instillation increased nitrotyrosine staining in peribronchial and perivascular inflammatory cells, and in alveolar macrophages, alveolar cuboid cells, leukocytes, and some capillary cells, and the nitrotyrosine in the lung tissue was detectable by HPLC, suggesting increased production of peroxynitrite by NO and superoxide in these cells or an increased concentration of NO and superoxide released by accumulated inflammatory cells. Increased NO production in rats subjected to LPS instillation was also observed by Li and coworkers (15). In addition to peroxynitrite-dependent nitrotyrosine, there may be myeloperoxidase-dependent formation of nitrotyrosine after LPS instillation, because leukocytes contain a large amount of myeloperoxidase (16). However, NO inhalation after LPS instillation suppressed nitrotyrosine staining in the tissue, and nitrotyrosine became undetectable by HPLC.

Nitrotyrosine is known to be reduced to aminotyrosine. We found clear aminotyrosine staining in alveolar capillary endothelial cells even in the control animals where nitrotyrosine was not stained. A certain amount of nitrotyrosine may be constantly produced in untreated tissue, possibly by NO via constitutive NO synthase, but the amount of nitrotyrosine produced may be small, and reducing substances in the tissues may be able to almost completely convert nitrotyrosine into aminotyrosine, except in bronchial epithelial cell surface and bronchial smooth muscle cells. NO inhalation alone enhanced nitrotyrosine or aminotyrosine staining only in bronchial epithelial cell surface without the destruction of the cells, suggesting that neither production of nitrotyrosine nor conversion into aminotyrosine was considerably increased by NO inhalation except bronchial epithelial cell surface. LPS instillation increased aminotyrosine staining in alveolar macrophages and alveolar cuboid cells rather than in bronchial epithelial cells, suggesting that aminotyrosine conversion increased particularly in peripheral lung regions, where blood circulation is sufficient and rich in reducing substances. NO inhalation after LPS instillation suppressed both nitrotyrosine and aminotyrosine staining, suggesting a reduction in production of nitrotyrosine rather than an increase in the conversion into aminotyrosine.

Evaluating aminotyrosine staining as well as nitrotyrosine staining therefore confirmed that NO inhalation suppressed tyrosine nitration enhanced by LPS instillation. Modification of tyrosine may influence the activity of enzymes that contain tyrosine residues critical for catalytic activity.

Effects of NO Inhalation

Either reduction of superoxide production or reduction of endogenous NO production, or both, may cause the reduction in tyrosine nitration. NO inhibits neutrophil superoxide production via a direct action on the NADPH oxidase (17). NO is reported to exert negative feedback on constitutive (18) as well as inducible nitric oxide synthase (iNOS) activity in various tissue and cell preparations, including alveolar macrophages (19). There is evidence that NO may function as a negative feedback modulator of iNOS by interacting with enzyme-bound heme, which plays a mechanistic role in the catalytic conversion of L-arginine to NO plus L-citrulline (20). In clinical settings, interruption of NO inhalation causes a marked and rapid rebound increase in pulmonary vascular resistance and pressure (21), suggesting a feedback inhibition of endothelial NOS activity during NO inhalation.

Reduction of tyrosine nitration by inhaled NO may be caused by suppression of leukocyte accumulation in the lung by inhibiting their adherence (22, 23), inhibiting their migration through endothelial barrier (24), or inhibiting production of proinflammatory cytokines in vascular endothelial cells (22). Hickey and colleagues (25) reported that after LPS treatment iNOS-deficient mice have enhanced leukocyte-endothelium interactions and increased myeloperoxidase in the lungs. It is thus suggested that NO functions as an inhibitory regulator of leukocyte recruitment. The inhibition of leukocyte accumulation in the lungs reduces the amount of myeloperoxidase, thereby reducing tyrosine nitration.

Leukocyte depletion reduced numbers of inflammatory cells in pulmonary interstitial tissue of the LPS-instilled rats, thereby reducing nitrotyrosine and aminotyrosine, because nitrotyrosine and aminotyrosine were stained in accumulated leukocytes in the pulmonary interstitial tissue of leukocyte-rich LPS-instilled rats. Leukocyte depletion in LPS-instilled rats also decreased the nitrotyrosine staining of alveolar capillaries, suggesting that myeloperoxidase of leukocytes in pulmonary circulation produced a considerable amount of nitrotyrosine. However, leukocyte depletion did not affect the staining of bronchial epithelial cells, bronchial muscle cells, alveolar macrophages, and alveolar cuboid cells, suggesting that tyrosine was nitrated in these cells by peroxynitrite which was formed by superoxide and NOS-derived NO.

Inhaled NO may decrease the LPS-induced inflammatory cell accumulation in the lung by other mechanisms unrelated to tyrosine nitration. NO chelates iron, causing a decrease in iron-mediated generation of ·OH from hydrogen peroxide. Therefore, NO can block lipid peroxidation by preventing hydrogen peroxide reaction with heme proteins (26). NO can also inhibit lipoxygenase and cyclo-oxygenase enzymes that contain iron at their active sites (27).

EVLW and EVDW were increased after LPS instillation, but the blood-free wet-to-dry weight ratio was not increased after LPS instillation. The inflammation in the lung tissue in our study may have been modest, making cell extravasation dominant over interstitial edema, because severe alveolar flooding was not obvious in the tissue 16 h after LPS instillation. It has been reported that a large number of neutrophils migrate into the air spaces of sheep lungs in response to alveolar endotoxin, without altering the permeability of the epithelium (28). NO inhalation did not affect the increased EVLW and EVDW after LPS instillation or the wet-to-dry weight ratio.

These results indicate that inhaling 20 ppm NO during inflammatory cell accumulation in the lung induced by LPS instillation reduced rather than augmented the pulmonary inflammation. Comparable effects of inhaled NO have previously been reported in oxidant lung injury (29) and in lung injury caused by sublethal hyperoxia (30). Inhalation of 50 ppm NO prevented neutrophil-mediated lung injury by N-formyl-methionyl-leucyl-phenylanine in rats (29). Inhalation of 20 ppm NO attenuated the O₂-mediated lung endothelial injury and alveolar damage (30). Dose and time dependency of the effect of inhaled NO should be explored in detail to select the most effective timing (and dose) of NO inhalation to suppress pulmonary inflammatory cell accumulation, particularly in clinical settings.

In summary, the results of this study indicated that inhalation of 20 ppm NO decreased the leukocyte accumulation and tyrosine nitration caused by LPS instillation.