INTRODUCTION

TOP ABSTRACT INTRODUCTION METHODS RESULTS DISCUSSION REFERENCES

Nitric oxide (NO) is a pleiotropic mediator formed from L-arginine by nitric oxide synthase (NOS). Several major isoforms of NOS have been identified. The constitutively expressed (cNOS) enzyme releases NO under physiologic conditions in numerous cells, including endothelial cells and neurons. During inflammation and shock, proinflammatory cytokines and bacterial-wall components stimulate the production of NO by inducible NOS (iNOS) in endothelial and smooth-muscle cells, macrophages, and other cell types (1, 2). In rodents, the production of NO by cNOS is calcium-dependent, whereas the production of NO by iNOS does not require increases in intracellular Ca²⁺ concentration. iNOS can be induced in a variety of cells by proinflammatory agents such as endotoxin (bacterial lipopolysaccharide [LPS]), interleukin-1, tumor necrosis factor-, and interferon-. Enhanced formation of NO following the induction of iNOS has been implicated in the pathogenesis of shock and inflammation (2). Recent data suggest that some of the cytotoxic effects of NO are related to the production of peroxynitrite, a reactive oxidant formed by the rapid reaction of NO and superoxide (3).

The biologic activity and decomposition of peroxynitrite are highly dependent on the cellular or chemical environment (presence of proteins, thiols, glucose, the ratio of NO to superoxide, and other factors), and these factors influence its toxic potential (5, 6). In a number of pathophysiologic conditions associated with inflammation or oxidant stress, peroxynitrite has been proposed to mediate cell damage (3, 7, 8). Peroxynitrite is cytotoxic via a number of mechanisms, including: (1) the initiation of lipid peroxidation; (2) the inactivation of a variety of enzymes (most notably mitochondrial respiratory enzymes and membrane pumps) (3); and (3) depletion of glutathione (9). Moreover, peroxynitrite can also cause DNA damage (10, 11) resulting in activation of the nuclear enzyme poly (adenosine diphosphate [ADP]-ribose) synthetase (PARS), in depletion of nicotinamide adenine dinucleotide (NAD⁺) and adenosine triphosphate (ATP), and ultimately in cell death (12). The overproduction of reactive oxygen species (ROS) in inflammation leads to considerable oxidant stress, as indicated by lipid peroxidation, high blood levels of malondialdehyde (MDA) and conjugated dienes, and consumption of the endogenous antioxidant vitamins C and E (13).

Injection of carrageenan into the pleural space leads to pleurisy, infiltration by polymorphonuclear leukocytes (PMN), and lung injury. Models of carrageenan-induced pleurisy have been widely used to investigate the pathophysiology of acute inflammation and also to evaluate the efficacy of drugs in inflammation. In this study we investigated the role of iNOS in a model of carrageenan-induced pleurisy, using iNOS-knockout (iNOS-KO) mice and iNOS-wild type (iNOS-WT) mice. In order to characterize the role of iNOS in this model of acute inflammation, we determined the following endpoints of the inflammatory response in iNOS-KO mice and in the corresponding WT mice: (1) exudate formation; (2) PMN infiltration; (3) peroxynitrite formation (by immunohistochemistry); (4) activation of the nuclear enzyme poly (ADP-ribose) polymerase (PARP); (5) lipid peroxidation; and (6) lung injury.

	METHODS

TOP ABSTRACT INTRODUCTION METHODS RESULTS DISCUSSION REFERENCES

Animals

Male iNOS-KO and iNOS-WT mice (weight: 20 to 25 g) were used to assess the role of iNOS in the pathogenesis of carrageenan-induced pleurisy. All animals were allowed access to food and water ad libitum. Animal care was in compliance with Italian regulations on the protection of animals used for experimental and other scientific purposes (D.M. 116192) as well as with the regulations of the European Economic Community (O.J. of E.C. L358/1 12/18/1986)

Carrageenan-Induced pleurisy

Mice were anesthetized with isoflurane and submitted to a skin incision at the level of the left sixth intercostal space. The underlying muscle was dissected, and saline (0.2 ml) or saline containing 1% -carrageenan (0.2 ml) was injected into the pleural cavity. The skin incision was closed with a suture and the animals were allowed to recover. At 4 h after the injection of carrageenan, the animals were killed by inhalation of CO₂. The chest was carefully opened and the pleural cavity was rinsed with 2 ml of saline solution containing heparin (5 U/ml) and indomethacin (10 µg/ml). The exudate and washing solution were removed by aspiration, and the total volume of this fluid was measured. Any exudate that was contaminated with blood was discarded. The amount of exudate was calculated by subtracting the volume of washing fluid injected (2 ml) from the total volume recovered. The leukocytes in the exudate were suspended in phosphate-buffered saline (PBS) and counted with an optical microscope in a Burker's chamber after staining with vital trypan blue.

Cell Culture

Resident pleural macrophages were collected from mice at 4 h after carrageenan injection as previously described (14). The cells (10⁶/ml), consisting mainly (approximately 70%) of macrophages, were cultured in 12-well plates for 3 h in Dulbecco's modified Eagle's medium (DMEM) supplemented with L-glutamine (3.5 mM), penicillin (50 U/ml), streptomycin (50 µg/ml), and heparin sodium (10 U/ml), and were allowed to adhere at 37° C in a humidified 5% CO₂ incubator. Nonadherent cells were removed by rinsing the plates three times with 5% dextrose-in-water. After nonadherent cells (approximately 10%) were removed, adherent macrophages were scraped from the plates for the measurement of DNA strand breaks and cellular NAD⁺. Mitochondrial respiration and peroxynitrite formation were measured in the adherent cells in the subsequent 1-h period.

Measurement of Nitrite/nitrate

Nitrite + nitrate (NOx) production, an indicator of NO synthesis, was measured in the supernatant samples as previously described (16). Briefly, the nitrate in the supernatant was first reduced to nitrite by incubation with nitrate reductase (670 mU/ml) and nicotinamide adenine dinucleotide phosphate (NADPH) (160 µM) at room temperature for 3 h. The nitrite concentration in the samples was then measured through the Griess reaction by adding 100 µl of Griess reagent (0.1% naphthylethylenediamide dihydrochloride in H₂O, and 1% sulfanilamide in 5% concentrated H₂PO₄ in a ratio of 1:1 [vol/vol]) to 100-µl aliquots of the samples. The optical density of the reaction product at 550 nm (OD₅₅₀) was measured with an enzyme-linked immunosorbent assay microplate reader (SLT-Labinstruments, Salzburg, Austria). Nitrate concentrations were calculated by comparison with the OD₅₅₀ of standard solutions of nitrate in saline.

NOS Assay

Lungs taken 4 h after carrageenan injection were placed into a homogenization buffer composed of: 50 mM Tris-HCl, 0.1 mM ethylene glycol-bis-(-amino-ethyl ether)-N,N,N',N'-tetraacetic acid (EGTA), and 1 mM phenylmethylsulfonyl fluoride (pH 7.4). Tissues were then homogenized in the buffer on ice, using a Tissue Tearor 985-370 homogenizer (Biopsies Products, Racine, WI). Conversion of [³H]L-arginine to [³H]L-citrulline was measured in the homogenates as described (16). Briefly, homogenate (30 µl) was incubated for 30 min at 22° C in the presence of [³H]L-arginine (10 µM, 5 kBq/tube), NADPH (1 mM), calmodulin (30 nM), tetrahydrobiopterin (5 µM), and calcium (2 mM). Reactions were stopped by dilution with 0.5 ml of ice-cold 4-(2-hydroxyethyl)-1-piperazine-N'-2-ethanesulfonic acid buffer (pH 5.5) containing EGTA (2 mM) and ethylenediamine tetraacetic acid (EDTA) (2 mM). Experiments performed in the absence of NADPH determined the extent of [³H]L-citrulline formation independent of a specific NOS activity. Experiments in the presence of NADH without calcium or EGTA (5 mM) determined the calcium-independent (i.e., induced) NOS activity. Reaction mixtures were applied to Dowex 50W (Na⁺ form) columns, and the eluted [³H]L-citrulline activity was measured with a Wallac scintillation counter (Wallac, Gaithersburg, MD).

Measurement of Oxidation of Dihydrorhodamine 123 to Rhodamine 123

The formation of peroxynitrite was measured by the peroxynitrite- dependent oxidation of dihydrorhodamine 123 to rhodamine 123, as previously described (15). Cells were rinsed with PBS and the medium was then replaced with PBS containing 5 µM dihydrorhodamine 123. After a 60-min incubation at 37° C, the fluorescence of rhodamine 123 was measured with a fluorometer at an excitation wavelength of 500 nm and emission wavelength of 536 nm (slit widths: 2.5 and 3.0 nm, respectively). The rate of peroxynitrite formation was calculated by using a standard curves obtained with authentic peroxynitrite, injected into PBS containing 5 µM dihydrorhodamine 123. This method therefore provides an indirect measure of peroxynitrite production, since other oxidant species can also induce oxidation of dihydrorhodamine 123.

Measurement of Cell Energy Status

Cell energy status was assessed by measuring the mitochondrial- dependent reduction of 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) to formazan (15). Harvested macrophages were incubated at 37° C with MTT (0.2 mg/ml) in 96-well plates for 1 h. Culture medium was removed by aspiration and the cells were solubilized in dimethylsulfoxide (DMSO) (100 µl). The extent of reduction of MTT to formazan within cells was quantified by the measurement of OD₅₅₀. As previously discussed (15), the reduction of MTT appears to be accomplished mainly by mitochondrial complexes I and II, but may also involve reduced nicotinamide adenine diphosphate (NADH)- and NADPH-dependent energetic processes that occur outside the mitochondrial inner membrane. The method of reduction of MTT to formazan therefore cannot be used to separate the effect of free radicals, oxidants, or other factors on the individual enzymes in the mitochondrial respiratory chain, but is useful to monitor changes in the general energy status of cells.

Determination of DNA Single-Strand Breaks

The formation of DNA strand breaks in double-stranded DNA was determined by the alkaline unwinding method as previously described (15). Cells in 12-well plates were scraped into 0.2 ml of Solution A buffer (myoinositol 250 mM, NaH₂PO₃ 10 mM, MgCl₂ 1 mM; pH 7.2). The cell lysate was then transferred into plastic tubes designated T (maximum fluorescence), P (fluorescence in sample used to estimate extent of DNA unwinding), or B (background fluorescence). To each tube, 0.2 ml of Solution B (alkaline lysis solution: NaOH 10 mM, urea 9 M, EDTA 2.5 mM, sodium dodecyl sulfate 0.1%) was added, and the mixture was incubated at 4° C for 10 min to allow cell lysis and chromatin disruption. Following this, 0.1 ml each of Solutions C (0.45 volume Solution B in 0.2 N NaOH) and D (0.4 volume Solution B in 0.2 N NaOH) were then added to the P and B tubes. A volume of 0.1 ml of solution E (neutralizing solution: glucose 1 M, mercaptoethanol 14 mM) was then added to the T tubes before Solutions C and D were added. From this point onward, all incubations were done in the dark. A 30-min incubation period at 0° C was then allowed, during which the alkali diffused into the viscous lysate. Because the neutralizing solution, Solution E, was added to the T tubes before addition of the alkaline Solutions C and D, the DNA in the T tubes was never exposed to a denaturing pH. At the end of the 30-min incubation period, the contents of the B tubes were sonicated for 30 s to ensure rapid denaturation of DNA in the alkaline solution. All tubes were then incubated at 15° C for 10 min. Denaturation was stopped by chilling to 0° C and adding 0.4 ml of Solution E to the P and B tubes. A volume of 1.5 ml of Solution F (ethidium bromide 6.7 µg/ml in 13.3 mM NaOH) was added to all the tubes, and fluorescence (excitation: 520 nm, emission: 590 nm) was measured with a fluorometer. Under the conditions used, in which ethidium bromide binds preferentially to double-stranded DNA, the percentage of double-stranded DNA (D) may be determined by using the equation: % D = 100 × [F(P)  F(B)]/[F(T)  F(B)]; where F(P) is the fluorescence of the sample, F(B) is the background fluorescence (i.e., fluorescence due to all cell components other than double stranded DNA), and F(T) is the maximum fluorescence.

Measurement of Cellular NAD⁺ Levels

Cell respiration was assessed by measuring the mitochondrial-dependent reduction of MTT to formazan (15). Harvested macrophages were incubated at 37° C with MTT (0.2 mg/ml) in 96-well plates for 1 h. Culture medium was removed by aspiration and the cells were solubilized in DMSO (100 µl). The extent of reduction of MTT to formazan within cells was quantified by the measurement of OD₅₅₀. As discussed previously (15), measurement of the reduction of MTT appears to be accomplished mainly by mitochondrial complexes I and II, but may also involve NADH- and NADPH-dependent energetic processes that occur outside the mitochondrial inner membrane. The method of reduction of MTT to formazan therefore cannot be used to separate the effect of free radicals, oxidants, or other factors on the individual enzymes in the mitochondrial respiratory chain. It is, however a useful technique that allows monitoring of changes in the general energy status of cells (15).

Immunohistochemical Localization of Nitrotyrosine

Tyrosine nitration, an index of the nitrosylation of proteins by peroxynitrite and/or oxygen-derived free radicals, was determined immunohistochemically as previously described (16). At the end of the experiment, the relevant organs were fixed in 10% buffered formaldehyde, and 8-µm sections were prepared from paraffin-embedded tissues. After deparaffinization of the tissue sections, endogenous peroxidase was quenched with 0.3% H₂O₂ in 60% methanol for 30 min. The sections were then permeabilized with 0.1% Triton X-100 in PBS for 20 min. Nonspecific adsorption was minimized by incubating the sections in 2% normal goat serum in PBS for 20 min. Endogenous biotin- or avidin-binding sites were blocked by sequential incubation for 15 min with avidin and biotin. The sections were then incubated overnight with a 1:1,000 dilution of primary antinitrotyrosine antibody or with control solutions. Some sections were also incubated with the primary antibody (antinitrotyrosine) in the presence of excess nitrotyrosine (10 mM) to verify the binding specificity of the antibody. Controls included buffer alone or nonspecific purified rabbit IgG. Specific labeling was detected with a biotin-conjugated goat antirabbit IgG and avidin-biotin-peroxidase complex (DBA, Milan, Italy). Photomicrographs (n = 5) of immunocytochemistry results were assessed densitometrically. Analysis of the photomicrographic data was done by using Optilab Graftek software on a Macintosh personal computer (CPU G3-266; Apple, Inc., Cupertino, CA).

Immunohistochemical Localization of PARP

Formation of PARP was determined immunohistochemically as previously described (17). At 4 h after carrageenan injection, lung tissues were fixed in 10% buffered formalin, and 8-µm sections were prepared from paraffin-embedded tissues. After deparaffinization of the tissue sections, endogenous peroxidase was quenched with 0.3% H₂O₂ in 60% methanol for 30 min. The sections were permeabilized with 0.1% Triton X-100 in PBS for 20 min. Nonspecific adsorption was minimized by incubating the sections in 2% normal goat serum in PBS for 20 min. Endogenous biotin- or avidin-binding sites were blocked by sequential incubation for 15 min with avidin and biotin, using a biotin blocking kit (DBA). The sections were then incubated overnight with a 1:500 dilution of primary anti-PARP antibody (DBA) or with control solutions. Controls included buffer alone or nonspecific, purified rabbit IgG. Specific labeling was detected with a biotin-conjugated goat antirabbit IgG (DBA) and avidin-biotin-peroxidase (DBA). Photomicrographs of (n = 5) immunocytochemistry results were assessed densitometrically. Analysis of the photomicrographic data was done by using Optilab Graftek software on a Macintosh personal computer (CPU G3-266).

Light Microscopy

Lung biopsies were taken at 4 h after injection of carrageenan. The biopsies were fixed for 1 wk in buffered formaldehyde solution (10% in PBS) at room temperature, dehydrated in graded solutions of ethanol, and embedded in Paraplast (Sherwood Medical, Mahwah, NJ). Tissue sections (7 µm thick) were deparaffinized with xylene, stained with trichromic Van Gieson's stain, and studied under light microscopy (Dialux 22; Leitz, Milan, Italy).

Determination of Myeloperoxidase Activity

Myeloperoxidase (MPO) activity, an indicator of PMN accumulation, was determined as previously described (18). At 4 h after intrapleural injection of carrageenan, lung tissues were obtained and weighed. Each piece of tissue was homogenized in a solution containing 0.5% hexadecyl-trimethylammonium bromide dissolved in 10 mM potassium phosphate buffer (pH 7), and was centrifuged for 30 min at 20,000 × g at 4° C. An aliquot of the supernatant was then allowed to react with a solution of tetramethylbenzidine (1.6 mM) and 0.1 mM H₂O₂. The rate of change in absorbance was measured spectrophotometrically at 650 nm. MPO activity was defined as the quantity of enzyme degrading 1 µmol of peroxide per minute at 37° C, and was expressed in milliunits per gram weight of wet tissue.

Determination of Malondialdehyde Levels

The levels of malondialdehyde (MDA) in lung tissue were determined as an indicator of lipid peroxidation (19). Lung tissue collected at the specified time was homogenized in 1.15% KCl solution. An aliquot (100 µl) of the homogenate was added to a reaction mixture containing 200 µl of 8.1% sodium dodecyl sulfate, 1,500 µl of 20% acetic acid (pH 3.5), 1,500 µl of 0.8% thiobarbituric acid, and 700 µl distilled water. Samples were then boiled for 1 h at 95° C and centrifuged at 3,000 × g for 10 min. The absorbance of the supernatant was measured spectrophotometrically at 650 nm.

Materials

All reagents and compounds not obtained from DBA were obtained from Sigma Chemical Company (Milan, Italy).

Data Analysis

All values in the figures and text are expressed as mean ± SEM of n observations. For the in vitro studies, data represent the number of wells studied (six to nine wells from two or three independent experiments). For the in vivo studies, n represents the number of animals studied. In the experiments involving histology or immunohistochemistry, the figures shown are representative of at least three experiments performed on different experimental days. The results were analyzed by one-way analysis of variance followed by Bonferroni's post hoc test for multiple comparisons. A value of p < 0.05 was considered significant

	RESULTS

TOP ABSTRACT INTRODUCTION METHODS RESULTS DISCUSSION REFERENCES

The Development of Carrageenan-Induced Pleurisy is Attenuated in iNOS-KO Mice

All WT mice that received carrageenan developed an acute pleurisy, producing 1.1 ± 0.08 (mean ± SEM) ml of turbid exudate (Figure 1A). Injection of carrageenan induced a significant increase in the number of PMN (66 ± 1.3 × 10⁶/mouse; Figure 1B) as compared with the number of cells collected from the pleural space of sham-operated mice (1.2 ± 0.9 × 10⁶/mouse; Figure 1B). iNOS-KO mice showed a significant attenuation of the volume of their pleural exudate as well as of the number of PMN in the exudate (Figures 1A and 1B). No significant exudate and no significant increase in the number of PMN was observed in the pleural cavity of sham-operated mice.

View larger version (31K): [in this window] [in a new window]   Figure 1.   (A) Exudate volume and (B) leukocyte accumulation. iNOS-KO mice showed a significant decrease in peritoneal exudate, leukocyte migration, and nitrate/nitrite levels. Responses in iNOS-WT controls and iNOS-KO animals were compared. Data are mean ± SEM of 10 mice for each group. *p < 0.01 versus vehicle; °p < 0.01 versus iNOS-WT mice.

MPO Activity, MDA, and Histologic Evaluation of Lung Injury

All iNOS-WT mice that were treated with carrageenan exhibited a substantial increase in the activities of MPO and MDA in the lung (Figures 2A and 2B). iNOS-KO mice showed a significant attenuation of the increase in MPO and MDA caused by carrageenan in the lung (Figures 2A and 2B). There was no increase in either MPO activity or MDA level in sham-operated animals. Histologic examination of lung sections of iNOS-WT mice treated with carrageenan showed edema, tissue injury, and infiltration of tissue with PMN (Figure 3B1, arrows), lymphocytes, and plasma cells (Figure 3B). iNOS-KO mice showed a significant reduction of lung injury as well as of infiltration of tissue by white blood cells (Figure 3C). No histologic alteration was found in sham-operated mice (Figure 3A).

View larger version (38K): [in this window] [in a new window]   Figure 2.   Myeloperoxidase (MPO) activity (A) and MDA (B) in the lungs of carrageenan-treated mice killed at 4 h. MPO activity and MDA levels were significantly increased in the lungs of carrageenan-treated iNOS-WT mice in comparison with sham-operated mice. iNOS-KO mice showed a significant reduction of the carrageenan-induced increase in MPO activity and MDA levels. Data are means ± SEM of 10 mice for each group. *p < 0.01 versus vehicle; °p < 0.01 versus iNOS-WT mice.

View larger version (152K): [in this window] [in a new window]   Figure 3.   (A) Representative lung sections from sham-operated animals, demonstrating a normal alveolar architecture. Representative lung sections from a carrageenan-treated iNOS-WT mouse demonstrate inflammatory infiltration by PMN (arrows) (B1) and lymphocytes and plasma cells (B). Lung sections from a carrageenan-treated iNOS-KO mouse (C ) demonstrate reduced inflammatory infiltration. A, B, C, original magnification: ×125; B1, original magnification: ×370. Figure is representative of at least three experiments performed on different experimental days.

NO Production

Levels of nitrite/nitrate were significantly (p < 0.01) increased in the exudate from carrageenan-treated WT mice (133 ± 9 nmol/mouse, versus 14 ± 2 nmol/sham-operated mouse) (Figure 4). In contrast no increase of nitrite/nitrate was found the exudate of carrageenan-treated iNOS-KO mice (Figure 4).

View larger version (30K): [in this window] [in a new window]   Figure 4.   Nitrite and nitrate exudate levels (A), lung iNOS activity (B), and lung cNOS activity (C ) at 4 h after carrageenan administration. Nitrite and nitrate exudate levels and lung iNOS activity in carrageenan-treated iNOS-WT mice were significantly increased versus by comparison with those in the sham-operated group. No significant increase in nitrite and nitrate exudate levels or lung iNOS activity was found in iNOS-KO mice subjected to carrageenan-induced pleurisy. cNOS activity was unaffected by carrageenan challenge, and was not altered in iNOS-KO mice. Data are mean ± SEM of 10 mice for each group. *p < 0.01 versus vehicle; °p < 0.01 versus iNOS-WT mice.

In the lungs obtained from iNOS-WT mice subjected to carrageenan-induced pleurisy, a significant increase in iNOS activity was detected at 4 h (180 ± 11 fmol/mg/min) (Figure 4B). iNOS activity was absent in iNOS-KO mice (Figure 4B). Constitutive (calcium-dependent) NOS activity, however, was unaffected by carrageenan challenge, and was not altered in iNOS-KO mice (Figure 4C). This latter finding further indicated, as found in earlier studies (16, 20), that inhibition of iNOS in the present study did not interfere with constitutive production of NO.

Nitrotyrosine and PARP Formation

Immunohistochemical analysis of lung sections obtained from iNOS-WT mice treated with carrageenan revealed positive staining for nitrotyrosine (12.2 ± 1.4% of total tissue area), which was primarily localized in alveolar macrophages (Figure 5B, arrows) and in airway epithelial cells (Figure 5B). In contrast, less positive nitrotyrosine staining (1.43 ± 0.4% of total tissue area) was found in the lungs of carrageenan-treated iNOS-KO mice (Figure 5C). Immunohistochemical analysis of lung sections obtained from iNOS-WT mice treated with carrageenan revealed positive staining (6.8 ± 0.5% of total tissue area) for PARP, which was primarily localized in alveolar macrophages (Figure 6B, arrows) and in airway epithelial cells (Figure 6B). In contrast, less positive PARP staining (1.8 ± 0.9% of total tissue area) was found in the lungs of carrageenan-treated iNOS-KO mice (Figure 6C). There was no staining for either nitrotyrosine or PARP in lungs obtained from sham-operated mice (Figures 5A and 6A).

View larger version (155K): [in this window] [in a new window]   Figure 5.   Immunohistochemical localization of nitrotyrosine. Staining was absent in control tissue (A). Four hours after carrageenan injection, nitrotyrosine immunoreactivity was present in the lungs from iNOS-WT mice (B). Positive staining was primarily localized in alveolar macrophages (arrows) (B1). In the lungs of the carrageenan-treated iNOS-KO mice (C ), significantly less positive staining was found. A, B, C, original magnification: ×125; B1, original magnification: ×370. Figure is representative of at least three experiments performed on different experimental days.

View larger version (148K): [in this window] [in a new window]   Figure 6.   Immunohistochemical localization of PARS. Staining was absent in control tissue (A). Four hours after carrageenan injection, poly (ADP- ribose) polymerase immunoreactivity was found in the lungs from iNOS-WT mice (B). Positive staining was primarily localized in alveolar macrophages (arrows) (B1). In the lungs of the carrageenan-treated iNOS-KO mice (C ), no positive staining was found. A, B, C, original magnification: ×125; B1, original magnification: ×370. Figure is representative of at least three experiments performed on different experimental days.

Effect of iNOS Inhibition on the Increase in Peroxynitrite Formation, iNOS induction, DNA Damage and Injury of Macrophages Obtained from the Pleural Cavity of Carrageenan-Treated Mice

When compared with the supernatant of macrophages collected from the pleural cavity of sham-operated animals, the supernatant of macrophages obtained from carrageenan-treated iNOS-WT mice showed a significantly greater concentration of nitrite + nitrate (Figure 7A). No increase in nitrite/ nitrite was found in the supernatant of macrophages obtained from iNOS-KO mice (Figure 7A). When compared with the supernatant of macrophages collected from the pleural cavity of sham-operated animals, the supernatant of macrophages obtained from carrageenan-treated iNOS-WT mice showed a significant increase in the concentration of peroxynitrite (Figure 7B). This was associated with a significant increase in the occurrence of single-strand breaks in the DNA (Figure 8C), a reduction in cell energy status (Figure 7D), and a significant decrease in the intracellular levels of NAD (Figure 7E) in these cells. Macrophages obtained from iNOS-KO mice showed attenuation of the formation of peroxynitrite (Figure 7B) as well as of the associated DNA damage (Figure 7C), and a reduced impairment in cell energy status (Figure 7D) and smaller decrease in NAD levels (Figure 7E).

View larger version (46K): [in this window] [in a new window]   Figure 7.   Nitrate/nitrite production (A), peroxynitrite production (B), DNA single-strand breakage (C ), reduction of cell energy status (D), and cellular levels of NAD+ (E ) in pleural macrophages harvested 4 h after carrageenan administration. The cells (106/ml) were cultured for 3 h and allowed to adhere at 37° C in a humidified 5% CO2 incubator. *p < 0.01 versus macrophages from control mice; °p < 0.01 versus macrophages from iNOS-WT mice.

	DISCUSSION

TOP ABSTRACT INTRODUCTION METHODS RESULTS DISCUSSION REFERENCES

The cellular and molecular mechanisms of carrageenan-induced inflammation are well characterized, since the corresponding models of inflammation are standard models in screening for antiinflammatory compounds (21). It appears that the early phase (1 to 2 h) of carrageenan-induced inflammation is related to the production of histamine, leukotrienes, and platelet-activating factor, whereas the delayed phase (4 to 24 h) of the carrageenan-induced inflammatory response has been linked to neutrophil infiltration and the production of neutrophil-derived free radicals, such as hydrogen peroxide, superoxide, and hydroxyl radical, as well as to the release of other neutrophil-derived mediators (14, 22). The role of ROS in the pathophysiology of inflammation is well established. In addition to ROS, an overproduction of NO due to the expression of iNOS plays important role in various models of inflammation (1, 2, 16). Pharmacologic inhibitors of NOS, and also ablation of the gene for iNOS, have been shown to reduce development of the inflammatory response (26). In the present study we demonstrated that lack of the iNOS gene reduces: (1) the development of carrageenan-induced pleurisy; (2) the infiltration of the lung by PMN (as shown by histology and MPO activity); (3) the degree of lipid peroxidation in the lung; and (4) the degree of lung injury (as shown by histology) in mice treated with carrageenan. All of these findings support the view that NO plays an important role in the degree of inflammation and lung injury caused by carrageenan in the mouse. More recent studies, done with the model of carrageenan-induced paw edema, have shown the formation of peroxynitrite in this model of inflammation (14, 26). The early and delayed production of peroxynitrite in this model are respectively related to the constitutive and inducible isoforms of NOS (14, 26).

Taken together our data show that absence of the iNOS gene attenuates but does not fully abolish oxidation, nitration, and cytotoxicity in response to immunostimulation. Notably, both oxidation and tyrosine nitration that could be inhibited with a NOS inhibitor were observed in response to carrageenan challenge (14). Furthermore iNOS-KO mice maintained low levels of nitrite/nitrate in their pleural exudate: in these animals, NO is obviously produced from the cNOS. Constitutive (calcium-dependent) NOS activity, however, was unaffected by carrageenan challenge, and was not altered in iNOS-KO mice (not shown). This latter finding further indicates (16, 20) that inhibition of iNOS does not interfere with constitutive production of NO.

Peroxynitrite rapidly oxidized the fluorescent probe substance dihydrorhodamine 123 to rodamine 123 both in vitro and in vivo (30, 31).

The production of peroxynitrite can be evidenced by increased oxidation of dihydrorhodamine 123 to rodamine 123 in the supernatant from macrophages collected from the pleural cavity of carrageenan-treated animals (32). Our present results support this concept. Therefore, it is important to point out that caution should be exercised in using this method, since oxidation of dihydrorhodamine 123 can be triggered by oxidants other than peroxynitrite (e.g., hydrogen peroxyde and/or hypochlorous acid) (30, 31). However, a component of increased oxidation of dihydrorhodamine that is inhibitable with a NOS inhibitor can be taken as relatively specific evidence of an effect of peroxynitrite (30, 32). Since oxidation of dihydrorhodamine that was inhibitable with a NOS inhibitor occurred under our experimental conditions, the logical conclusion is that peroxynitrite is generated in iNOS-KO mice in response to carrageenan challenge, presumably by the reaction of superoxide (produced, for example, by activated neutrophils) with NO produced by cNOS. In fact, NO produced by cNOS has previously been shown to be produced in various pathophysiologic conditions, most notably in the early phase of reperfusion of various organs previously subjected to ischemia (30, 32, 33). In addition, we found that absence of the iNOS gene attenuated the nitrosylation of proteins in the lungs of mice treated with carrageenan. Nitrotyrosine formation, along with its detection by immunostaining, was initially proposed as a relatively specific marker for detecting the endogenous formation of peroxynitrite (34). Recent evidence, however, indicates that certain other reactions can also induce tyrosine nitration (e.g., the reaction of nitrite with hypoclorous acid and the reaction of MPO with hydrogen peroxide can lead to the formation of nitrotyrosine) (35). Increased staining for nitrotyrosine is therefore considered an indication of "increased nitrative stress," rather than a specific marker of the generation of peroxynitrite. On the basis of the present data, we cannot determine the mechanism of tyrosine nitration: inhibition of NOS by an NOS inhibitor would both inhibit NO formation (and thereby reduce the generation of peroxynitrite) and suppress nitrite formation (and thereby attenuate the peroxidase-dependent mechanisms of tyrosine nitration). Nevertheless, we can certainly conclude from the current data that the absence of iNOS does not abolish tyrosine nitration in vivo.

ROS and peroxynitrite produce cellular injury and necrosis via several mechanisms, including peroxidation of membrane lipids, protein denaturation, and DNA damage. ROS produce strand breaks in DNA, which trigger energy-consuming DNA repair mechanisms and activate the nuclear enzyme PARS, resulting in the depletion of its substrate, NAD⁺, in vitro, and a reduction in the rate of glycolysis. Because NAD⁺ functions as a cofactor in glycolysis and in the tricarboxylic acid cycle, depletion of NAD⁺ leads to a rapid decrease in intracellular ATP. This process has been termed "the PARS suicide hypothesis" (12). There is recent evidence that the activation of PARS may also play an important role in inflammation (32, 36). In the present study, we found that absence of the iNOS gene attenuates the increase in PARS activity caused by carrageenan in the lung. Similarly, in pleural macrophages collected from iNOS-KO mice, we observed a significant attenuation of the decrease in NAD⁺. We therefore propose that iNOS induction is also and at least in part important for the activation of PARS.

In conclusion, this study demonstrates that the degree of inflammation caused by injection of carrageenan into the pleural cavity of the mouse is significantly attenuated in iNOS-KO mice. These findings support the view that the induction of iNOS contributes to the extension of inflammation in the model of carrageenan-induced pleurisy used in our study. Our findings also suggest that interventions that may reduce the generation or the effects of iNOS may be useful in conditions associated with local or systemic inflammation.