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American Journal of Respiratory Cell and Molecular Biology. Vol. 28, pp. 199-207, 2003
© 2003 American Thoracic Society
DOI: 10.1165/rcmb.4899

Vitamin E Reduces Transendothelial Migration of Neutrophils and Prevents Lung Injury in Endotoxin-Induced Airway Inflammation

David Rocksén, Barbro Ekstrand-Hammarström, Lenore Johansson and Anders Bucht

Department of Medical Countermeasures, Divison of NBC Defence, Swedish Defence Research Agency, Umeå; Department of Respiratory Medicine and Allergy, University Hospital Umeå, Sweden; and Department of Medicine, Karolinska Institute, Stockholm, Sweden

Address correspondence to: David Rocksén, M.Sc., Dept. of Medical Countermeasures, FOI NBC Defence, SE-901 82, Umeå, Sweden. E-mail: david.rocksen{at}foi.se


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
We investigated the pharmacologic effects of the antioxidant Vitamin E ({alpha}-tocopherol [{alpha}-toc]) in airway inflammation induced by inhaled endotoxin. A preparation of {alpha}-toc incorporated in liposomes was administered intraperitoneally in mice 1 h after exposure of aerosolized endotoxin. Injection of 50 mg {alpha}-toc/kg significantly decreased the number of neutrophils in airspaces and prevented lung injury, monitored both as decreased lactate dehydrogenase activity in airways and reduced lung edema when compared with animals treated with plain liposomes. Immunofluorescence staining of lung tissue revealed that treatment with {alpha}-toc decreased the number of neutrophils in lung interstitium, whereas the number in lung blood vessels and peripheral blood did not differ between mice treated with {alpha}-toc and control mice. Our results indicate that {alpha}-toc downmodulates the migration of neutrophils across the endothelial barrier, but in contrast to strong anti-inflammatory drugs such as corticosteroids, without inhibition of transcription factors involved in the early inflammatory response (nuclear factor-{kappa}B/activator protein-1). Neither was the endotoxin-induced expression of proinflammatory cytokines in lung tissue downregulated. Treatment with a combination of {alpha}-toc and a suboptimal dose of 0.5 mg/kg dexamethasone enhanced the effect, suggesting that {alpha}-toc, in combination with low doses of corticosteroids, might be effective for therapeutic treatment of acute lung injury.

Abbreviations: activator protein-1, AP-1 • {alpha}-tocopherol, {alpha}-toc • bronchoalveolar lavage fluid, BALF • electrophoretic mobility shift assay, EMSA • glyceraldehydes-3'-phosphate dehydrogenase, GAPDH • Hanks' balanced salt solution, HBSS • interleukin, IL • lactate dehydrogenase, LDH • lipopolysaccharide, LPS • macrophage inflammatory protein, MIP • nuclear factor-{kappa}B, NF-{kappa}B • reactive oxygen species, ROS • tumor necrosis factor-{alpha}, TNF-{alpha}


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
Inhalation of bacterial endotoxin (lipopolysaccharide, LPS) evokes a well-defined acute airway inflammation (1). In lungs, alveolar macrophages promote the inflammatory response through the production of a variety of inflammatory mediators, for example cytokines and chemoattractants, which results in an accumulation of neutrophils in lung tissue and airspaces (2). Moreover, macrophages and recruited neutrophils produce a plethora of reactive oxygen species (ROS), i.e., H2O2, O2-, OH, HOCl, NO, and singlet oxygen, for the purpose of killing bacteria and other microorganisms. Conversely, excessive production of ROS may lead to acute tissue injury and organ failure (3). The lung is also at potentially higher risk of injury mediated by ROS and lipid peroxidation as compared with other organs, due to high exposure to oxygen and the fact that lung tissue contains unsaturated fatty acids that are substrates of lipid peroxidation (4). Pharmacologic intervention of inflammation-induced lung injury can be performed at different levels, for example, through downregulation of proinflammatory cytokines, blocking of neutrophil infiltration, or inhibition of ROS formation. In a mouse model of endotoxin-induced airway inflammation, we have previously demonstrated powerful anti-inflammatory effects of the corticosteroid dexamethasone, but poor effects of the antioxidant N-acetylcysteine (5).

Vitamin E ({alpha}-tocopherol [{alpha}-toc]) is considered the most important, naturally occurring, antioxidative defense against lipid peroxidation in the cell membrane of mammals. Initiated by ROS, fatty acids of the membranic phospholipids undergo peroxidation, but {alpha}-toc interrupts this chain reaction by donating hydrogen either to the lipid or to the lipid peroxyl radical (6). Plasma levels of {alpha}-toc have been reported to be decreased after infection, trauma, burns, and inflammatory reactions, indicating that this antioxidant is exhausted during acute tissue injury (7). Aside from its antioxidative properties, further biological effects of {alpha}-toc are under investigation. {alpha}-toc is thought to modulate the inflammatory response, although the mechanism of this action remains to be clarified. Treatment with high doses of {alpha}-toc have been shown to be beneficial in animal models of acute respiratory distress syndrome (8–10), and has also been shown to have some protective effects against chemical insults such as sulfur and nitrogen mustards (11), as well as in bleomycin-induced lung injury (12, 13).

Liposomes have been widely investigated in recent years as carriers for drugs, and lipophilic molecules such as {alpha}-toc can be incorporated in the lipid bilayer. In the present study, we used a mouse model of endotoxin-induced acute airway inflammation (14) to examine whether liposome-incorporated {alpha}-toc blocks infiltration of neutrophils into the lungs. Most previous studies of {alpha}-toc treatment have used a pretreatment approach, i.e., administration before induction of injury. In this study, however, we used a therapeutic approach and thus investigated the effects of the drug when given after onset of the inflammatory reaction. We also examined the antioxidative properties of {alpha}-toc by measuring the effects on oxidative burst in neutrophils, and the protective effects on lung cell injury and edema formation. We then investigated the mechanism(s) for the treatment effects of {alpha}-toc. Our results demonstrated that {alpha}-toc, in contrast to dexamethasone, does not inhibit the early activation of nuclear factor-{kappa}B (NF-{kappa}B) or activated protein-1 (AP-1). Immunohistochemical staining of lung tissue sections and flow cytometric analysis of peripheral blood demonstrated that {alpha}-toc reduces the transendothelial migration of neutrophils. These data indicate that {alpha}-toc exerts anti-inflammatory activity through pathways clearly different from those of corticosteroids. This motivated us to perform combined treatment with {alpha}-toc and dexamethasone to study synergistic or additive effects.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Induction of Acute Lung Inflammation
Female C57BL/6JBom mice (aged 10–12 wk) (M&B A/S, Ry, Denmark) were used in all experiments. Animals were fed with standard chow and water ad libitium and allowed to acclimatize for at least 7 d. The study was approved by the local ethical committee in Umeå, Sweden. Acute lung inflammation was induced by exposure of aerosolized Escherichia coli LPS (serotype: 0128:B12; Sigma, St. Louis, MO) for 15 min (7 liters/min) using a nose-only exposure chamber (Batelle, Geneva, Switzerland). The aerosol was generated by a compressed air nebulizer (Collision 6-jet; BGI Inc., Waltham, MA) using 0.1 mg/ml LPS dissolved in endotoxin-free distilled water. In one experiment, the LPS dose was increased 10-fold to provoke epithelial cell damage. Previous experiments in our laboratory have demonstrated a particle size distribution ranging between 0.1 and 0.3 µm, with a total deposition of 18.3 ± 1.23% for small laboratory animals. The deposition was determined with 125I-labeled particles and the size of aerosolized particles was analyzed using a scanning mobility particle sizer (Model 3934; TSI, Shoreview, MN) (15). Control animals were exposed to endotoxin-free distilled water alone.

Drug Treatment
{alpha}-toc and dexamethasone 21-phosphate were purchased from Sigma. In initial dose–response experiments {alpha}-toc incorporated in liposomes was administered at four different doses (2, 10, 50, and 125 mg/kg body weight). In subsequent experiments, the 50 mg/kg {alpha}-toc dose was chosen.

{alpha}-toc–encapsuled liposomes were prepared as described by Suntres and Shek (9) with some minor modifications. The mixture of dipalmitoylphosphatidylcholine and {alpha}-toc (7:3 molar ratio) was dissolved in a glass vessel with chloroform and methanol (2:1 vol/vol). The solvent was removed in an evaporator (Buchi, Flawil, Switzerland) under argone to form a thin film coating the inner wall of the glass vessel. The lipid film was placed under vacuum for 2 h to remove all contaminating solvent. The lipid mixture was hydrated with phosphate-buffered saline (PBS) and sonicated in a cell disruptor (Sonifier B-15; Branson, Danbury, CT) with pulses for 10 min to form liposomes. The multilamellar vesicles were extruded 10 times with an extruder (Avestin; Ottawa, ON, Canada) through polycarbonate filters of 400 nm pore size to remove lipid aggregates. One milliliter of {alpha}-toc liposomes hydrated in PBS was injected intraperitoneally into each animal 1 h after LPS exposure. For the mechanistic studies (analysis of NF-{kappa}B/AP-1 activation and cytokine/chemokine gene expression), {alpha}-toc was administered 1 h before LPS exposure. Combination treatment of {alpha}-toc (50 mg/kg) and a suboptimal dose of dexamethasone (0.5 mg/kg) were performed by intraperitoneal injection of 400 µl dexamethasone dissolved in PBS (0.025 mg/ml) together with 1 ml {alpha}-toc liposome. A control group of LPS-exposed animals injected intraperitoneally with empty liposomes were included in all experiments.

Flow Cytometric Analysis of Neutrophils in Bronchoalveolar Lavage Fluid
Analysis of neutrophil accumulation in airspaces and the production of reactive oxygen species in neutrophils were performed essentially as previously described (5).

Mice were killed by cervical dislocation 18 h after LPS exposure. The trachea was cannulated with polyethene tubing followed by introduction and withdrawal of bronchoalveolar lavage fluid (BALF) by repeated 1-ml aliquots of Hanks' balanced salt solution (HBSS) to a total volume of 4 ml. Total leukocytes in BALF were counted and 2 x 105 leukocytes from each animal were used. The cells were pre-warmed at 37°C for 5 min in a waterbath before adding 1 µl of the oxygen radical sensitive probe dichlorodihydrofluorescein diacetate (H2DCFDA; Molecular Probes, Eugene, OR) to a final concentration of 20 µM, followed by incubation at 37°C for 15 min. After incubation, the cell suspension was immediately washed in pre-cooled HBSS and resuspended in a buffer consisting of PBS (without Ca2+ and Mg2+), 1% fetal calf serum, and 0.1% sodium-azide. Nonspecific binding was blocked using 4 µl rat-serum and 4 µl (1:5) Fc-block (PharMingen, San Diego, CA). After 5 min incubation, 6 µl (1:10) of a phycoerythrin (PE)-conjugated granulocyte-specific antibody were added (GR-1; PharMingen) and allowed to bind for 30 min at 4°C in the dark. At least 10,000 cells were analyzed with a FACSort (Becton-Dickinson, San Jose, CA). The number of neutrophils in BALF was determined by analyzing the percentage of positive cells in FL2. Because, in addition to neutrophils, the GR-1 MAb might bind to eosinophils and bone marrow monocytes, we performed a standard cytospin staining (May-Grünewald Giemsa) confirming that the gated GR-1+ cells were neutrophils. A PE-conjugated isotype-matched control antibody (IgG2b; PharMingen) typically stained < 1% of collected cells. The production of oxygen radicals in gated neutrophils was determined by calculating the median fluorescence in FL1.

Analysis of Blood Neutrophils and Cell Surface Expression of CD11b+
The proportion of neutrophils in peripheral blood and the expression of CD11b/18 were studied by flow cytometric analysis. Peripheral blood was withdrawn 6 h after LPS exposure (0.1 mg/ml) directly into heparin tubes to prevent blood coagulation. Three hundred microliters of blood from each animal was washed in 4 ml of PBS containing 0.1% bovine serum albumin. After centrifugation for 10 min at 250 x g, the pellet was dissolved in 300 µl PBS (0.1% bovine serum albumin) containing 18 µl (1:5) Fc-block (PharMingen) and 18 µl rat serum. Each sample was divided into three separate 5-ml falcon tubes, followed by 5-min incubation. After incubation, 6 µl (1:10) of a PE-conjugated GR-1 antibody (PharMingen) was added to each tube together with 10 µl FITC-labeled CD 11b mAb (Immunotech, Marseille, France). The antibodies were allowed to bind in the dark for 30 min (4°C). After one wash with PBS, 1 ml (1:25) of whole blood lysing reagent (Coulter, Miami, CA) was added to each sample, followed by 250 µl fixative after 1 min incubation. After lysing erythrocytes the remaining white blood cells were washed and the double-stained neutrophils were analyzed for GR-1+/CD11b+ and GR-1+/CD11b- populations in the FL-1 channel (FITC).

Analysis of Lung Cell Injury
To monitor lung cell injury we used a lactate dehydrogenase (LDH) assay (Sigma). This method determines LDH activity based upon the oxidation of lactate to pyruvate and reduced nicotinamide adenine dinucleotide as end-products. Formation of nicotinamide adenine dinucleotide results in an increase in absorbance at 340 nm, which is directly proportional of the LDH activity in the sample. Mice were killed by cervical dislocation 22 h after LPS exposure (1 mg/ml), a time-point corresponding to the maximal LDH levels in this model. BAL fluid was withdrawn by 0.5-ml aliquots to a final volume of 1 ml and directly centrifuged to form a cell pellet. Supernatant of each sample was used for LDH analysis. Each vial of LDH reagent was mixed with 10 ml of deionized water. Supernatant and reagent were pre-warmed separately at 37°C for 1 h before analysis. Thereafter, 1 ml of LDH reagent was added to a cuvette with 50 µl sample. The absorbance was measured at 340 nm after 30 s and after 90 s. The difference (A90 sec - A30 sec) was multiplied by 3,376 according to the manufacturer's instructions.

Analysis of Edema Formation
Edema formation was measured 18 h after inhalation of LPS (0.1 mg/ml), a time-point that corresponds to the maximal edema formation in our model. After killing by cervical dislocation, the whole lung was dissected and the trachea was removed. Lungs were rinsed in PBS to remove contaminating blood. After removal of excessive PBS by careful drying on tissue paper, the lung tissue was weighed (wet weight). The lungs were then dried overnight in an oven at 50°C, followed by a second weighing (dry weight). The ratio of wet/dry lung weight was calculated as a marker for the degree of edema formation.

Electrophoretic Mobility Shift Assay
Nuclear extracts were prepared from 100–150 mg of frozen lung tissue dissected 2 h after LPS exposure, essentially according to the method of Deryckere and Gannon (16). Briefly, tissue was ground to powder in liquid nitrogen using a mortar and pestle. The ground tissue was then homogenized in 4 ml of solution A (0.6% Nonidet P-40, 150 mM NaCl, 10 mM HEPES, pH 7.9, 1 mM EDTA, and 0.5 mM phenylmethylsulfonylfluoride).

Centrifuging at 380 x g for 30 s pelleted unbroken tissue. The supernatant was incubated on ice for 5 min, and centrifuged for 10 min at 2,380 x g at 4°C. The nuclear pellets were resuspended in 100 µl of ice-cold solution B (25% glycerol, 20 mM HEPES, pH 7.9, 420 mM NaCl, 1.2 mM MgCl2, 0.2 mM EDTA, 0.5 mM DTT, 0.5 mM phenylmethylsulfonylfluoride, Complete protease inhibitors (Roche, Basel, Switzerland), and left on ice for 30 min. After centrifugation at 16,000 x g for 1 min at 4°C, aliquots of supernatant containing the nuclear proteins were collected and stored at –70°C. The nuclear protein content of the supernatant was determined by Coomassie Blue reaction using a Bio-Rad kit (Richmond, CA).

For detection of NF-{kappa}B and AP-1 DNA binding activities in nuclear extracts, electrophoretic mobility shift assays (EMSAs) were performed. The binding reaction was performed in a total volume of 20 µl, containing 5 µg nuclear extract, 4 µl 5x binding buffer (50 mM Tris-HCl, pH 7.5, 250 mM NaCl, 2.5 mM DTT 25 mM EDTA, 20% glycerol), 2 µg poly (dI-dC) (Amersham Biosciences, Uppsala, Sweden) as nonspecific competitor DNA and 32P-labeled oligonucleotides. After 30 min incubation at room temperature, samples were loaded into native 6% precast polyacrylamide gels (BMA; Bio Whittaker Molecular Applications, Rockland, ME), electrophoresed in 0.25x TBE (2.2 mM Tris borate, 2.2 mM boric acid, 0.5 mM EDTA) at 120 V for 1 h to separate DNA–protein complexes from unbound DNA probe. Gels were vacuum dried and exposed to Amersham Biosciences MP film at –70°C overnight. Intensity of bands corresponding to NF-{kappa}B and AP-1 were determined using an image analyzing system. Competition was performed by adding a molar excess of unlabeled probe. The sequences of the oligonucleotides used to detect the DNA-binding activity of NF-{kappa}B and AP-1 in this study were as follows (only the upper strands are indicated): NF-{kappa}B: 5'-AGTTGAGGGGACTTTCCCAGGC-3'; AP-1: 5'-CGCTTGATGAGTCAGCCGGAA-3'. The oligonucleotides were end labeled using {gamma}-[32P] ATP (3,000 Ci/mmol; NENLife Science Products, Boston, MA) and T4 polynucleotide kinase (Promega, Madison, WI). The labeled probes were purified from free nucleotides on a Sephadex G-50 M column (Amersham Biosciences).

Analysis of Cytokine and Chemokine mRNA Expression in Lung Tissue
Lung tissue was removed 2 h after LPS exposure and immediately frozen in liquid nitrogen. The frozen lung tissue (100–200 mg) was homogenized in 2 ml TRIzol (GIBCO BRL, Gaithersburg, MD). Total cytoplasmic RNA and first-strand cDNA synthesis of mRNA was performed as described earlier (5). Two microliters of cDNA was added to a 23-µl PCR reaction mixture consisting of 12.5 µl SYBRgreen mix including AmpliTaq gold and cybergreen (Applied Biosystem, Warrington, UK), 0.4 µM sense and antisense primer, and 9.5 µl sterile distilled water. Target cDNA was amplified using primers specific for glyceraldehyde-3-phosphate dehydrogenase (GAPDH), tumor necrosis factor (TNF)-{alpha}, interleukin (IL)-1ß, and macrophage inflammatory protein (MIP)-1{alpha} (5). Amplification of cDNA was performed by a two-step PCR protocol (95°C, 10 min, followed by 40 cycles of 95°C for 15 s and 60°C for 60 s) using amplification equipment with a real-time monitor system (iCycler; Biorad). The reaction was followed in real time by recording the increase in fluorescence when cybergreen was incorporated in the double-stranded PCR product. The number of cycles needed to increase the fluorescence intensity above the background threshold was calculated using the icycler software (cycle threshold [CT]). This enables evaluation of the results in the early phase of the PCR reaction, i.e., when the amplification yields an exponential increase in PCR product. Because the CT value is inversely proportional to the number of target cDNA copies in the sample, we calculated a residual CT by subtracting the measured CT value from the total number of amplification cycles, giving an adjusted CT value which is directly proportional to the amount of target cDNA. The relative amount of cytokine cDNA in each sample was calculated by dividing the residual CT value by the corresponding value of the housekeeping gene GAPDH. Cytokine transcripts with no detectable fluorescence intensity after 40 cycles were assigned the value 0. Negative controls were included in each experiment to ensure that the reagents were free of contamination.

Immunofluorescence Staining
Mice were killed by cervical dislocation 6 h after LPS challenge (0.1 mg/ml) and the left lung lobe was dissected and immediately frozen in liquid petroleum gas. Frozen tissue was thereafter serially sectioned and mounted on superfrost slides (Menzel-Gläser, Braunschweig, Germany) and fixed in acetone for 10 min. After washing in PBS, the sections were incubated with the primary antibodies, rat anti-mouse CD11b (PharMingen) diluted 1:500 in PBS and rat anti-mouse Ly-6G (GR-1; PharMingen) diluted 1:300 in PBS for 1 h.

After rinsing in PBS, the sections were incubated with AlexaFluor 568 goat anti-rat and AlexaFluor 488 goat anti-rat (Molecular Probes) diluted 1:1,000 in PBS for 30 min.

Control sections incubated without primary antibody showed low background and no nonspecific staining. Scoring was based on the infiltration of GR-1+ and CD11b+ cells in lung tissue. Classification of slides was performed in a blinded manner, using a scale ranging between 1 and 5. The scale criteria were: (1) occasional positive cells in lung tissue (< 50 cells/high-power field); (2) moderate staining of positive cells (50–100 cells/high-power field); (3) intermediate infiltration of positive cells (100–150 cells/high-power field); (4) marked staining of positive cells (150–200 cells/high-power field); (5) intense fluorescence of positive cells (> 200 cells/high-power field). The scoring procedure was conducted with three serial slides from each animal and the average scoring number of the three slides was calculated. The interior surface of lung blood vessels was counted for GR-1+ and CD11b/18+ cells. Blood vessel area on each slide was scored in a scale between 1–5 as follows: (1) small blood vessel area; (2) moderate blood vessel area; (3) intermediate blood vessel area; (4) large blood vessel area; and (5) very large blood vessel area. Number of positive cells in the lung blood vessels was divided with the scoring number of the lung blood vessel area. Results were presented as the number of positive cells/area unit (a.u) (GR-1+/a.u and CD11b/18+/a.u).

Statistical Analysis
Statistical significant differences were determined by ANOVA. This was followed by Dunnett's multiple comparison test if more than one treated group was compared with a control group, or Bonferroni's test if several crosswise comparisons were performed. The nonparametric Mann-Whitney U-test was used when comparing cytokine mRNA expression and immunhistochemistry scoring between control and treated groups. P < 0.05 (two-tailed) was regarded as significant.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Effect of {alpha}-toc on Neutrophil Accumulation in BALF and Oxidative Burst
Aerosol exposure of mice with a nebulizer concentration of 0.1 mg/ml LPS evoked a transient inflammatory response with a peak of recovered neutrophils in BALF after 12–24 h (14). The effect of {alpha}-toc on neutrophilic inflammation was studied by withdrawal and analysis of cells in BALF 18 h after LPS challenge (Figure 1) . The number of neutrophils accumulated in BALF and the oxidative burst was analyzed by flow cytometric double staining using a neutrophil-specific mAb (GR-1) and DCF-fluorescense intensity as a marker for intracellular production of ROS. Dose–response studies of liposome-incorporated {alpha}-toc given intraperitoneally 1 h after LPS exposure demonstrated a significant reduction of granulocytes in BALF when mice were treated with 50 mg/kg and 10 mg/kg compared with the control group (given plain liposomes only), but not when treated with 2 mg/kg (Figure 1A). In a separate experiment, the dose was increased to 125 mg/kg {alpha}-toc without any enhanced effect compared with 50 mg/kg (data not included). At 50 mg/kg, the production of ROS in lung neutrophils tended to decrease when compared with animals receiving empty liposomes (P = 0.07) (Figure 1B). Based on these results, the dose 50 mg/kg was used in subsequent experiments. One experiment was conducted to ensure that the liposomes did not induce an inflammation at the site of injection. Peritoneal lavage was analyzed 18 h after injection with empty liposomes or liposomes incorporated with {alpha}-toc. The number of neutrophils was compared with the number in control animals injected with saline, revealing no peritoneal inflammation (data not included).



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  		Figure 1. Effect of {alpha}-toc on granulocyte accumulation in BALF and oxidative burst. Liposomal {alpha}-toc or empty liposomes was administered by i.p injection 1 h after LPS exposure (0.1 mg/ml). One group of mice received an aerosol of water representing healthy animals. Withdrawal and analysis of granulocytes in BALF was performed 18 h after challenge. Total number of leukocytes was determined by cell counting, and the proportion of granulocytes was analyzed by flow cytometry staining using a PE-labeled GR-1 Mab. The total number of granulocytes accumulated in BALF is depicted in A. Production of oxygen radicals (DCF-fluorescence intensity) in neutrophils was analyzed by flow cytometry double staining using the GR-1 Mab combined with the ROS-sensitive probe DCF. The fluorescence of DCF was measured as the median FL1 intensity of gated GR-1+ cells (B). Data were collected from at least 10,000 cells and are expressed as mean ± SD (five animals in each group). P values are indicated when mean values for {alpha}-toc groups were significantly different from the control group (LPS + liposome). *P < 0.05 and **P < 0.01 (ANOVA + Dunnet's multiple comparison test).

 
Effect of {alpha}-toc on Lung Cell Injury and Edema Formation
Leakage of LDH to the airspaces indicates cell injury, which may occur as a consequence of the acute airway inflammation (17). A detectable increase of LDH activity in BALF was observed 22 h after provocation, but only when mice were exposed to a relatively high dose of LPS (nebulizer concentration 1 mg/ml). This encouraged us to study the protective effects of {alpha}-toc on lung cell injury caused by the exaggerated airway inflammation. Liposome-encapsuled {alpha}-toc was administered 1 h after LPS exposure, and the activity of LDH in BALF was analyzed 22 h after LPS exposure. The LDH activity was significantly lower in {alpha}-toc–treated animals compared with animals receiving plain liposomes (Figure 2A) .



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  		Figure 2. Effect of {alpha}-toc on lung cell injury and edema formation. Lung cell injury was monitored using a commercial assay for LDH. Mice were exposed for a relatively high dose of LPS (nebulizer concentration 1 mg/ml) and BALF was collected after 18 h. LDH activity in BALF was measured as the rate of increase in absorbance at 340 nm (A). Lung edema was measured as the ratio of wet to dry lung weight 18 h after the mice were exposed for aerosolized LPS (nebulizer concentration 0.1 mg/ml) (B). In both experiments, administration of {alpha}-toc (50 mg/kg) was performed intraperitoneally 1 h after LPS challenge. One group of mice receiving an aerosol of water represented healthy animals. One control group were exposed for LPS and injected with empty liposomes. Data are expressed as mean ± SD (five animals in each group). P values are indicated when mean {alpha}-toc values were significantly different from the control group (LPS + liposome). **P < 0.01 (ANOVA).

 
Mice exposed to aerosolized LPS develop lung edema, which can be detected as an increase of lung wet weight. In our experimental model, challenge with a nebulizer concentration of 0.1 mg/ml LPS induced an increase in the wet lung weight with a peak after 18 h and a decline to normal levels after 36 h (data not included). Mice were injected with {alpha}-toc liposomes 1 h after LPS inhalation (0.1 mg/ml), and the ratio of wet to dry lung weight was determined after 18 h. Treatment with {alpha}-toc almost completely inhibited lung edema when compared with animals treated with plain liposomes (Figure 2B), yielding a ratio of wet to dry lung weight comparable to that of healthy animals.

Effect of {alpha}-toc on the Translocation of NF-{kappa}B/AP-1 and on Cytokine/Chemokine mRNA Expression in Lung Tissue
We have previously demonstrated that airway inflammation induced by LPS (0.1 mg/ml) is preceeded by an early (2 h) and transient onset of proinflammatory cytokines and chemokines in lung tissue (14). In a subsequent study, we showed that the LPS-induced expression of TNF-{alpha}, IL-1{alpha}, IL-1ß, IL-6, IL12 (p40), and MIP-1{alpha} mRNA are strongly downregulated by pretreatment with dexamethasone, which was expected, because among other biological effects, this corticosteroid acts as an NF-{kappa}B inhibitor (5). In this study, we used the pretreatment protocol to investigate whether {alpha}-toc inhibits NF-{kappa}B/AP-1–dependent cytokine/chemokine expression and compared the effect with that of dexamethasone. NF-{kappa}B/AP-1 DNA-binding activities were analyzed in nuclear extracts from lung tissue 2 h after LPS exposure using an EMSA (Figure 3A) . As anticipated, pretreatment with dexamethasone 1 h before challenge almost completely inhibited the translocation of NF-{kappa}B to the nucleus. Moreover, the binding activity of AP-1 was also inhibited by dexamethasone. However, {alpha}-toc had no effect on either NF-{kappa}B or AP-1 binding activity compared with the group receiving LPS and empty liposomes (Figure 3B). Expression of proinflammatory cytokines (TNF-{alpha}, IL-1ß) and the chemokine MIP-1{alpha} was analyzed in lung tissue 2 h after LPS exposure. Results demonstrated an increased expression of TNF-{alpha}, IL-1ß, and the chemokine MIP-1{alpha} in the LPS + liposome group when compared with animals receiving a water aerosol. Although we detected a background cytokine/GAPDH value for TNF-{alpha} in the water + liposome group, the increased expression observed in the LPS + liposome group still reached the level of statistical significance. The LPS-induced expression of TNF-{alpha}, IL-1ß, and MIP-1{alpha} were not significantly downregulated by pretreatment with {alpha}-toc (Figure 4) .



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  		Figure 3. Effect of {alpha}-toc on the activation of NF-{kappa}B and AP-1. Mice were exposed for an LPS aerosol (0.1 mg/ml) and injected i.p with {alpha}-toc (50 mg/kg) 1 h prior to LPS challenge. Dexamethasone (Dex), a known inhibitor of NF-{kappa}B, was used as a positive control (10 mg/kg). Animals were killed after 2 h and lungs were removed and immediately frozen at –70°C, followed by preparation of cell nuclei. After extraction, DNA binding activities in nuclear extracts were analyzed using electrophoretic mobility shift assay (EMSA) (A). A control experiment using excess cold probe is depicted to the right, verifying that the binding of [32P]-labeled oligonucleotides was specific. Intensity of bands, corresponding to NF-{kappa}B (solid bars) and AP-1 (shaded bars), was determined using an image analysis system (B). Data are expressed as the relative change compared with animals receiving an aerosol of water (mean ± SD, two to three animals in each group).

 


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  		Figure 4. Effect of {alpha}-toc on cytokine/chemokine mRNA expression in lung tissue. Mice were exposed to an LPS aerosol (0.1 mg/ml) and injected intraperitoneally with {alpha}-toc (50 mg/kg) 1 h before LPS challenge (circles). Control groups were injected with liposome only (water + liposome [diamonds] and LPS + liposome [triangles], respectively). Animals were killed after 2 h, and lungs were removed and immediately frozen in liquid nitrogen. Expression of mRNA was analyzed using a semiquantitavie real-time PCR assay. The residual cycle threshold value (CT) was determined for each amplified transcript (see MATERIALS AND METHODS) and the relative levels of TNF-{alpha}, IL-1ß, and MIP-1{alpha} mRNA were calculated in relation to the expression of the housekeeping GAPDH gene. The residual CT ratio against GAPDH of all three cytokines was significantly increased in LPS-exposed mice compared with the unexposed control group (P < 0.05, Mann-Whitney U test). The cytokine/GAPDH ratios did not differ significantly between LPS-challenged mice receiving plain liposomes and the group treated with {alpha}-toc. Each dot in the scatter plot represents one animal.

 
Effect of {alpha}-toc on Neutrophils in Lung Tissue and Blood Vessels
Mice were exposed to an LPS aerosol (0.1 mg/ml) and treated with {alpha}-toc 1 h after LPS exposure. Peripheral blood was withdrawn and lung tissue dissected in separate experiments 6 h after LPS exposure. In a previous study, we have observed the first signs of neutrophil migration into airways at this time point (14). Lung tissue sections were stained for neutrophils (GR-1+) and expression of CD11b/18. Scoring in a blinded manner demonstrated intense neutrophil and CD11b staining of lung tissue infiltrates in mice receiving LPS (Table 1). The infiltration of neutrophils and CD11b+ cells into the lung interstitium was significantly blocked by {alpha}-toc compared with animals receiving LPS and empty lipsomes. Cell counting of GR-1+ and CD11b+ cells in lung blood vessels revealed no difference between animals receiving {alpha}-toc and animals receiving liposomes alone (LPS+liposome) (Table 1). This experiment was repeated in the late phase of the inflammatory course (18 h after LPS exposure), with results that were similar, but not as striking (data not included).


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  		TABLE 1 Effect of {alpha}-toc on neutrophil number and CD11b expression in lung tissue and blood vessels.

 
Flow cytometric analysis of peripheral blood from LPS-exposed animals demonstrated an increased percentage of neutrophils (GR-1+), compared with healthy animals receiving a water aerosol (Table 1). Expression of CD11b was measured on gated GR-1+ cells, revealing one CD11b+ and one CD11b- population among the neutrophils. The results indicated an increase of CD11b+ cells in LPS-exposed animals compared with healthy animals. Treatment with {alpha}-toc did not decrease the percentage of neutrophils in peripheral blood. Neither was the expression of CD11b/18 on the cell surface of neutrophils affected, as indicated by the equal proportion of CD11b+ neutrophils in both groups receiving {alpha}-toc and empty liposomes, respectively.

Combination Treatment with Dexamethasone and {alpha}-toc
Mice were treated with {alpha}-toc (50 mg/kg) together with a suboptimal dose of dexamethasone (0.5 mg/kg) 1 h after LPS exposure. This treatment protocol resulted in significantly enhanced inhibition of neutrophil recruitment into airspaces compared with animals receiving only {alpha}-toc, and furthermore, a tendency of improved effect when compared with the group receiving dexamethasone alone (Figure 5A) . A similar effect on neutrophil accumulation was observed when {alpha}-toc was combined with a higher dose of dexamethasone (1 mg/kg) (data not included). We have previously demonstrated that high-dose treatment with dexamethasone (10 mg/kg) inhibits spontaneous production of ROS in lung neutrophils (5). In the present study, we observed that also a relatively low dose of dexamethasone (0.5–1 mg/kg) downmodulates the oxidative burst (Figure 5B, P < 0.001 versus LPS-treated control group). Combination treatment with {alpha}-toc and a low dose dexamethasone did not improve the inhibition of ROS production compared with animals receiving dexamethasone alone.



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  		Figure 5. Combination treatment with dexamethasone (Dex) and {alpha}-toc. The accumulation of neutrophils in BALF (A) and the oxidative burst (B) were studied when mice were treated with {alpha}-toc (50 mg/kg), dexamethasone (0.5 mg/kg), or both drugs in combination. One control group was exposed to LPS and injected with empty liposomes. When combining {alpha}-toc and dexamethasone, an enhanced treatment effect was observed compared with animals receiving {alpha}-toc alone. Data are derived from two pooled experiments. P values are indicated when the {alpha}-toc/Dex group was significantly different from groups receiving only one of the two drugs (LPS + {alpha}-toc and LPS + Dex). Data are expressed as mean ± SD (seven to nine animals in each group). **P < 0.01 and ***P < 0.001 (ANOVA + Bonferroni's multiple comparison test).

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
Previous studies have pinpointed that in addition to its antioxidative properties, {alpha}-toc also exerts anti-inflammatory effects (8–13, 18–20). However, the anti-inflammatory action of {alpha}-toc has not been rigorously defined in vivo. In the present investigation we used a mouse model of airway inflammation induced by inhaled endotoxin (LPS) to study the treatment effects of {alpha}-toc on the acute inflammatory response and lung injury. Our results clearly demonstrate that {alpha}-toc administered 1 h after exposure of aerosolized LPS significantly downmodulates the neutrophilia in airways in a dose-dependent manner, and also protects against lung cell injury and edema formation. To our knowledge, this is the first evidence that therapeutic treatment with {alpha}-toc is useful in counteracting neutrophilia and lung injury following inhalation of compounds activating an acute inflammatory response. These results are in agreement with previous studies of ischemic-reperfused injury (21) and in animal models of acute respiratory distress syndrome (8–10). We measured the release of LDH and edema formation in the lungs to evaluate the protective efficiency of {alpha}-toc on cell damage, which may occur in numerous inflammatory disorders, including inhalation of bacterial endotoxin. LDH is a cytoplasmatic enzyme present in essentially all major organ systems and is only detected extracellulary in BALF in the presence of dead or damaged lung cells (17). Measurement of the extracellular appearance of LDH in BALF and lung edema are useful parameters to determine pulmonary endothelial and epithelial cell injury, which may arise as a consequence of lipid peroxidation and release of other reactive oxygen species (22). Our results demonstrated that treatment with {alpha}-toc prevented lung injury, monitored both as decreased LDH activity in BALF and as reduced lung edema. It is thus possible that the decreased number of recovered neutrophils in airways after treatment with {alpha}-toc is due to protection against leakage of cells through the endothelial and epithelial layers.

Aside from the well-established fact that {alpha}-toc acts as an antioxidant by preventing lipid peroxidation, other studies suggest that this drug also may function as a direct antioxidant against singlet oxygen and superoxide anion (6). To investigate if treatment with {alpha}-toc could attenuate the intracellular oxidative burst, i.e., production of singlet oxygen, O2- and H2O2, we added a fluorescent probe (H2DCFDA) to neutrophils recovered from BALF and monitored the spontaneous oxidative activity ex vivo. This hydrophobic probe reaches the cytosol by diffusion and within the cytosol the probe is catalyzed by intracellular esterases to a hydrophilic metabolite (H2DCF), which is then trapped within the cell. The probe is thereafter oxidized to DCF by a variety of intracellular oxygen radicals, but predominantly by hydrogen peroxide (23). However, the inhibition of intracellular ROS formation was weak and did not reach the level of statistical significance, indicating that this antioxidative effect is not a major mechanism for the protective properties of {alpha}-toc on lung injury. It is thus probable that the main antioxidative effect of {alpha}-toc is defense against lipid peroxidation of the cell membrane.

An additional explanation for the downmodulation of neutrophil recruitment can be that {alpha}-toc inhibits the induction of the inflammatory cascade by blocking activation of NF-{kappa}B and/or AP-1, leading to decreased production of proinflammatory cytokines and chemokines. When alveolar macrophages are stimulated with inflammatory agents such as LPS, NF-{kappa}B dissociates from I{kappa}B and translocates to the nucleus, where it activates transcription of proinflammatory genes. This is commonly refered to as NF-{kappa}B activation, which is a key early event in a variety of lung disorders, including acute lung inflammation. The glucocorticoid dexamethasone has been shown to inhibit NF-{kappa}B activation through induction of I{kappa}B gene transcription (24–25). ROS have been suggested to be implicated in the stimulation of the signal transduction pathway involving NF-{kappa}B (26), and given the fact that {alpha}-toc is a powerful antioxidant, these properties might be a hypothetical explanation for the anti-inflammatory effects of {alpha}-toc. In vitro studies have indicated that {alpha}-toc inhibits NF-{kappa}B activation in rat Kupffer cells (20) and a human monocytic cell line (18, and our own unpublished observations). Furthermore, we have observed a downmodulative effect of {alpha}-toc on NF-{kappa}B activation when human alveolar type II cells (A549) and broncheal epithelial cells (BEAS-2B) are stimulated with TNF-{alpha} (data not included). Conversely, Faruqi and coworkers (27) have shown that {alpha}-toc has no effect on the activation of NF-{kappa}B in spite of a decreased adhesion of monocytes to endothelial cells. In this study we investigated the effect of {alpha}-toc on the activation of NF-{kappa}B and AP-1 in vivo using an EMSA. We also studied the subsequent mRNA expression of the cytokines TNF-{alpha}, IL-1ß, and the chemokine MIP-1{alpha} in lung tissue using a semiquantitative real-time PCR assay. We used a pretreatment protocol in both experiments to be able to compare our results with a previous study demonstrating powerful inhibition of TNF-{alpha}, IL-1{alpha}, IL-1ß, IL-6, IL-12, and MIP-1{alpha} mRNA by dexamethasone (5). In contrast to dexamethasone, treatment with {alpha}-toc did not inhibit the activation of NF-{kappa}B or AP-1. Neither did treatment with {alpha}-toc affect the mRNA expression of TNF-{alpha}, IL-1ß, or MIP-1{alpha}. It is thus unlikely that {alpha}-toc has any major effect on the signal transduction pathways leading to expression of proinflammatory cytokines or chemokines in lung tissue following LPS challenge.

One reason for the poor in vivo effect on NF-{kappa}B activation in our model might be a limited pulmonary uptake of {alpha}-toc early during the inflammatory course. Liposomes circulating in the blood vessels interact with endothelial cells and blood components such as high-density lipoproteins. Most of the {alpha}-toc transported in the bloodstream is bound to lipoproteins (28), suggesting that interactions of liposomes with lipoproteins might result in a transfer of {alpha}-toc to lipoproteins (29). Several studies indicate an augmented receptor-mediated uptake of {alpha}-toc to damaged tissues and various cell types (29–30). Although the time course of which intraperitoneally administered liposomal {alpha}-toc reach injured lung tissue has not been well investigated, it is reasonable to assume that such uptake occurs later in the inflammatory reaction after the initial activation of NF-{kappa}B and cytokine gene transcription. It is possible that our protocol for liposomal adminstration enables transfer of {alpha}-toc from the circulating liposomes to lipoproteins, resulting in an uptake of the drug by circulating inflammatory cells and endothelial cells in the alveolar capillary bed. It is well known that {alpha}-toc stabilizes the cell membrane and alters fluidity, and it is proposed that this changes the mobility of membrane components and augments the phagocytic properties of macrophages (31). Such an effect on cell membrane fluidity represents an additional hypothesis for the anti-inflammatory effect of {alpha}-toc. The change of membrane fluidity may alter the expression of adhesion molecules such as CD11b/18 on the surface of neutrophils and/or ICAM-1 on the endothelial cells, or affect the binding affinity between the cells. To investigate this hypothesis, we studied the appearance of neutrophils in lung tissue sections using immunohistochemistry and the proportion of neutrophils in peripheral blood using flow cytometry. Our results demonstrated a significant diminished neutrophilia in lung interstitium in mice treated with {alpha}-toc, whereas the proportion of neutrophils in lung blood vessels and peripheral blood was unaffected by the drug. This result clearly indicates that {alpha}-toc downmodulates the migration of neutrophils through the lung endothelium. Further support for this scenario are several in vitro studies demonstrating that {alpha}-toc blocks the adhesion of neutrophils to endothelial cells. Expression of the adhesion molecules ICAM-1 and VCAM-1 on endothelial cells as well as their counter-receptors CD11b/18 and VLA-4 on neutrophils and monocytes, respectively, have been reported to be inhibited by {alpha}-toc in vitro (18–19). We were not able to support such a specific downmodulation of adhesion molecules, however, because the expression of CD11b/18 on cells in the circulation and in lung blood vessels was unchanged after treatment with {alpha}-toc.

Our results indicate that {alpha}-toc downmodulates airway inflammation through mechanisms different from those of corticosteroids. Such differences in mode of action motivated us to perform combined treatment with the two drugs to investigate synergistic or additive effects. It has previously been reported that hydrocortisone in combination with {alpha}-toc exerts synergistic or additive protection against photodamage events in an ultraviolet-irradiation model (32). Our results indicate that a combination of {alpha}-toc (50 mg/kg) with a suboptimal dose of dexamethasone (0.5 mg/kg) improved the effect, monitored as neutrophil number in BALF, when compared with {alpha}-toc given alone. In a previous study, we concluded that high-dose treatment with dexamethasone (10 mg/kg) suppresses the production of ROS in neutrophils, probably through transcriptional inhibition of MIP-1{alpha} (5), a C-C chemokine involved in both neutrophil recruitment to inflamed tissue and in induction of the oxidative burst (33). In the present study, we demonstrated that a relatively low dose of dexamethasone (0.5 mg/kg) also exerts a downmodulatory effect on the oxidative burst produced by neutrophils. This antioxidative effect was not significantly enhanced when {alpha}-toc was added to the treatment protocol, however.

In conclusion, we have demonstrated that treatment with {alpha}-toc incorporated in liposomes dose-dependently downmodulates airway inflammation induced by inhaled endotoxin. In addition to the inhibition of airway neutrophilia, we demonstrate a significant protection against lung injury. This protective effect of {alpha}-toc is not due to inhibition of proinflammatory transcription factors or the subsequent production of cytokines/chemokines. Instead, our results indicate that {alpha}-toc reduces the transendothelial migration of neutrophils. We suggest that {alpha}-toc either decreases the expression of adhesion molecules on the endothelial cells or by some other mechanism affects the binding affinity between the neutrophil and the endothelial cell. Combination of {alpha}-toc with low-dose dexamethasone enhances the therapeutic effect, suggesting that {alpha}-toc, in combination with corticosteroids, might be effective for treatment of acute lung injury.


   Acknowledgments
 
The work was financially supported by the Swedish Heart Lung foundation and the Swedish Ministry of Defense. The authors thank Linda Svensson for technical assistance and Assoc. Prof. R.A Harris for linguistic advice.

Received in original form May 15, 2002

Received in final form September 13, 2002


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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