Modulatory effects of ozone on THP-1 cells in response to SP-A stimulation

Ozone (O(3)), a major component of air pollution and a strong oxidizing agent, can lead to lung injury associated with edema, inflammation, and epithelial cell damage. The effects of O(3) on pulmonary immune cells have been studied in various in vivo and in vitro systems. We have shown previously that O(3) exposure of surfactant protein (SP)-A decreases its ability to modulate proinflammatory cytokine production by cells of monocyte/macrophage lineage (THP-1 cells). In this report, we exposed THP-1 cells and/or native SP-A obtained from bronchoalveolar lavage of patients with alveolar proteinosis to O(3) and studied cytokine production and NF-kappaB signaling. The results showed 1) exposure of THP-1 cells to O(3) significantly decreased their ability to express TNF-alpha in response to SP-A; TNF-alpha production, under these conditions, was still significantly higher than basal (unstimulated) levels in filtered air-exposed THP-1 cells; 2) exposure of both THP-1 cells and SP-A to O(3) did not result in any significant differences in TNF-alpha expression compared with basal levels; 3) O(3) exposure of SP-A resulted in a decreased ability of SP-A to activate the NF-kappaB pathway, as assessed by the lack of significant increase and decrease of the nuclear p65 subunit of NF-kappaB and cytoplasmic IkappaBalpha, respectively; and 4) O(3) exposure of THP-1 cells resulted in a decrease in SP-A-mediated THP-1 cell responsiveness, which did not seem to be mediated via the classic NF-kappaB pathway. These findings indicate that O(3) exposure may mediate its effect on macrophage function both directly and indirectly (via SP-A oxidation) and by involving different mechanisms.     

Inhibition of apoptosis by 60% oxygen: a novel pathway contributing to lung injury in neonatal rats

During early postnatal alveolar formation, the lung tissue of rat pups undergoes a physiological remodeling involving apoptosis of distal lung cells. Exposure of neonatal rats to severe hyperoxia (≥95% O(2)) both arrests lung growth and results in increased lung cell apoptosis. In contrast, exposure to moderate hyperoxia (60% O(2)) for 14 days does not completely arrest lung cell proliferation and is associated with parenchymal thickening. On the basis of similarities in lung architecture observed following either exposure to 60% O(2), or pharmacological inhibition of physiological apoptosis, we hypothesized that exposure to 60% O(2) would result in an inhibition of physiological lung cell apoptosis. Consistent with this hypothesis, we observed that the parenchymal thickening induced by exposure to 60% O(2) was associated with decreased numbers of apoptotic cells, increased expressions of the antiapoptotic regulator Bcl-xL, and the putative antiapoptotic protein survivin, and decreased expressions of the proapoptotic cleaved caspases-3 and -7. In summary, exposure of the neonatal rat lung to moderate hyperoxia results in an inhibition of physiological apoptosis, which contributes to the parenchymal thickening observed in the resultant lung injury.     

Apoptosis induced by ozone and oxysterols in human alveolar epithelial cells

The mechanism of ozone-induced lung cell injury is poorly understood. One hypothesis is that ozone induces lipid peroxidation and that these peroxidated lipids produce oxidative stress and DNA damage. Oxysterols are lipid peroxides formed by the direct effects of ozone on pulmonary surfactant and cell membranes. We studied the effects of ozone and the oxysterol 5β,6β-epoxycholesterol (β-epoxide) and its metabolite cholestan-6-oxo-3,5-diol (6-oxo-3,5-diol) on human alveolar epithelial type I-like cells (ATI-like cells) and type II cells (ATII cells). Ozone and oxysterols induced apoptosis and cytotoxicity in ATI-like cells. They also generated reactive oxygen species and DNA damage. Ozone and β-epoxide were strong inducers of nuclear factor erythroid 2-related factor 2, heat shock protein 70, and Fos-related antigen 1 protein expression. Furthermore, we found higher sensitivity of ATI-like cells compared to ATII cells exposed to ozone or treated with β-epoxide or 6-oxo-3,5-diol. In general the response to the cholesterol epoxides was similar to the effect of ozone. Understanding the response of human ATI-like cells and ATII cells to oxysterols may be useful for further studies, because these compounds may represent useful biomarkers in other diseases.     

Rad9 is upregulated and plays protective roles in an acute lung injury model

The Rad9-Hus1-Rad1 protein complex is believed to respond to DNA damage and play important roles in the cell cycle. We studied the role of Rad9 protein in alveolar epithelial cells in the pathogenesis of acute lung injury. In a mouse model of lung injury induced by bleomycin or lipopolysaccharide, Rad9 expression is increased in type II alveolar epithelial cells from the early stage of lung injury. A549 cells and mouse primary alveolar epithelial cells also upregulated Rad9 expression after exposure to bleomycin. Gene silencing of Rad9 using siRNA decreased the G2/M arrest in A549 cells induced by bleomycin and also decreased the survival of A549 cells following exposure to bleomycin and hydrogen peroxide. In conclusion, Rad9 is a signal in the earlier stage of epithelial cell cycle regulation and plays protective roles in alveolar epithelial cells in the pathogenesis of acute lung injury.     

GM-CSF provides autocrine protection for murine alveolar epithelial cells from oxidant-induced mitochondrial injury

Exposure of mice to hyperoxia induces alveolar epithelial cell (AEC) injury, acute lung injury and death. Overexpression of granulocyte-macrophage colony-stimulating factor (GM-CSF) in the lung protects against these effects, although the mechanisms are not yet clear. Hyperoxia induces cellular injury via effects on mitochondrial integrity, associated with induction of proapoptotic members of the Bcl-2 family. We hypothesized that GM-CSF protects AEC through effects on mitochondrial integrity. MLE-12 cells (a murine type II cell line) and primary murine type II AEC were subjected to oxidative stress by exposure to 80% oxygen and by exposure to H(2)O(2). Exposure to H(2)O(2) induced cytochrome c release and decreased mitochondrial reductase activity in MLE-12 cells. Incubation with GM-CSF significantly attenuated these effects. Protection induced by GM-CSF was associated with Akt activation. GM-CSF treatment also resulted in increased expression of the antiapoptotic Bcl-2 family member, Mcl-1. Primary murine AEC were significantly more tolerant of oxidative stress than MLE-12 cells. In contrast to MLE-12 cells, primary AEC expressed significant GM-CSF at baseline and demonstrated constitutive activation of Akt and increased baseline expression of Mcl-1. Treatment with exogenous GM-CSF further increased Akt activation and Mcl-1 expression in primary AEC. Conversely, suppression of AEC GM-CSF expression by use of GM-CSF-specific small interfering RNA resulted in decreased tolerance of oxidative stress, Furthermore, silencing of Mcl-1 prevented GM-CSF-induced protection. We conclude that GM-CSF protects alveolar epithelial cells against oxidative stress-induced mitochondrial injury via the Akt pathway and its downstream components, including Mcl-1. Epithelial cell-derived GM-CSF may contribute to intrinsic defense mechanisms limiting lung injury.     

Distinct patterns of insulin-like growth factor binding protein (IGFBP)-2 and IGFBP-3 expression in oxidant exposed lung epithelial cells

Oxygen (O(2)) species are involved in a large variety of pulmonary diseases. Among the various cell types that compose the lung, the epithelial cells of the alveolar structure appear to be a major target for oxidant injury. Despite their importance in the repair processes, the mechanisms which regulate the replication of the stem cells of the alveolar epithelium, the type 2 cells, remain poorly understood. Based on the results of several studies which have documented the involvement of the insulin-like growth factor (IGF) system in lung epithelial cell replication, and which have also suggested a role for IGF binding proteins (IGFBPs) in the control of cell proliferation, the aim of the present work was to determine whether IGFBPs could be involved in the modulation of growth of human lung epithelial cells exposed to oxidants. Experiments were performed using a human lung adenocarcinoma cell line (A549) which was exposed for various durations to hyperoxia (95% O(2)). We observed a rapid and reversible growth arrest of the cells after only 24 h of O(2) exposure. When oxidant injury was prolonged, growth arrest was followed by induction of apoptosis with activation of the Fas pathway. These effects were associated with an increased expression of IGFBP-2 and IGFBP-3. In addition, study of localization of these proteins revealed distinct patterns of distribution. IGFBP-3 was mainly present in the extracellular compartment. In comparison, the fraction of IGFBP-2 secreted was less abundant whereas the IGFBP-2 fraction in the intracellular compartment appeared stronger. In addition, analysis of the subcellular localization provided data indicating the presence of IGFBP-2 in the nucleus. Taken together these data support a role for IGFBP-2 and IGFBP-3 in the processes of growth arrest and apoptosis in lung epithelial cells upon oxidant exposure. They also suggest that distinct mechanisms may link IGFBP-2 and IGFBP-3 to the key regulators of the cell cycle.     

Ozone causes lipid peroxidation but little antioxidant depletion in exercising and nonexercising hamsters.

Ozone (O(3)), a major component of urban air pollution, is a strong oxidizing agent that can cause lung injury and inflammation. In the present study, we investigated the effect of inhalation of O(3) on levels of F(2)-isoprostanes in bronchoalveolar lavage fluid (BALF) and on levels of antioxidants in the BALF and plasma of hamsters. Because antioxidants, including urate, ascorbate, GSH, and vitamin E, defend the lungs by reacting with oxidizing agents, we expected to find a decrease in antioxidant levels after O(3) exposure. Similarly, we expected an increase in the levels of F(2)-isoprostanes, which are lipid peroxidation products. Exposure to 1.0 or 3.0 parts/million (ppm) O(3) for 6 h resulted in an increase in BALF neutrophil numbers, an indicator of acute inflammation, as well as elevation of BALF F(2)-isoprostanes. The higher dose of O(3) caused an increase in the BALF level of urate and a decrease in the plasma level of ascorbate, but 1.0 ppm O(3) had no effect on BALF or plasma antioxidant levels. Exposure to 0.12 ppm O(3) had no effect on BALF neutrophils or F(2)-isoprostanes nor on BALF and plasma antioxidants. We also investigated the effect of O(3) exposure of hamsters during exercise on F(2)-isoprostane and antioxidant levels. We found that exposure to 1.0 ppm O(3) during 1 h of exercise on a laddermill increased BALF levels of F(2)-isoprostanes but had no effect on BALF neutrophils or on BALF and plasma antioxidants. These results indicate that O(3) induces inflammation and biomolecule oxidation in the lungs, whereas extracellular antioxidant levels are relatively unchanged.     

Effects of hypoxia and hypercapnia on surfactant protein expression proliferation and apoptosis in A549 alveolar epithelial cells

During lung injury alveolar epithelial cells are directly exposed to changes in PO(2) and PCO(2). Integrity of alveolar epithelial type II cells (AECII) is critical in lung injury but the effect of hypoxia and hypercapnia on AECII function, viability and proliferation has not been clearly investigated. Aim of the present work was to determine the direct effect of hypoxia and hypercapnia on surfactant protein expression, proliferation and apoptosis of lung epithelial cells in vitro. A549 alveolar epithelia cells were subjected to hypoxia (1%O(2)-5% CO(2)) or hypercapnia (21% O(2-) 15% CO(2)) and expression of surfactant protein C was measured and compared to normal conditions (21% O(2)- 5% CO(2)). Cell cycle progression and apoptosis were measured by flow cytometric analysis. RESULTS: A549 alveolar epithelial cells produce surfactant proteins, including surfactant protein C, when cultured under normal conditions, which is reduced under hypoxic conditions. Specifically, pro-SpC expression is moderately decreased after 8 h of culture in hypoxia, and is completely attenuated after 48 h. Hypercapnia decreases pro-SpC expression only after 48 h of exposure. Stimulation with TNF-alpha partly reverses pSPC decrease observed under hypoxic and hypercapnic conditions. Hypoxic culture of A549 cells results in progressive arrest of cells in the G1 phase of the cell cycle and increased apoptosis first observed 4 h following exposure and peaking at 24 h. In contrast hypercapnia has no significant effect on alveolar epithelial cell proliferation or apoptosis. CONCLUSIONS: Taken together we can conclude that hypoxia rapidly and severely affects AECII function and viability while hypercapnia has an inhibitory effect on pro-SpC production only after prolonged exposure.     

Stat3 is required for cytoprotection of the respiratory epithelium during adenoviral infection

The role of Stat3 in the maintenance of pulmonary homeostasis following adenoviral-mediated lung injury was assessed in vivo. Stat3 was selectively deleted from bronchiolar and alveolar epithelial cells in Stat3(DeltaDelta) mice. Although lung histology and function were unaltered by deletion of Stat3 in vivo, Stat3(DeltaDelta) mice were highly susceptible to lung injury caused by intratracheal administration of AV1-GFP, an early (E) region 1- and E3-deleted, nonproliferative adenovirus. Severe airspace enlargement, loss of alveolar septae, and sloughing of the bronchiolar epithelium were observed in Stat3(DeltaDelta) mice as early as 1 day after exposure to the virus. Although surfactant protein A, B, and C content and surfactant protein-B mRNA expression in Stat3(DeltaDelta) mice were similar, TUNEL staining and caspase-3 were increased in alveolar type II epithelial cells of Stat3(DeltaDelta) mice after exposure to virus. RNA microarray analysis of type II epithelial cells isolated from Stat3(DeltaDelta) mice demonstrated significant changes in expression of numerous genes, including those genes regulating apoptosis, supporting the concept that the susceptibility of Stat3-deficient mice to adenovirus was related to the role of Stat3 in the regulation of cell survival. AV1-Bcl-x(L), an E1- and E3-deleted, nonproliferative adenovirus expressing the antiapoptotic protein Bcl-x(L), protected Stat3(DeltaDelta) mice from adenoviral-induced lung injury. Adenoviral infection of the lungs of Stat3-deficient mice was associated with severe injury of the alveolar and bronchiolar epithelium. Thus, Stat3 plays a critical cytoprotective role that is required for epithelial cell survival and maintenance of alveolar structures during the early phases of pulmonary adenoviral infection.     

Inhalation of ozone induces DNA strand breaks and inflammation in mice

Ozone (O3) is a well-known oxidant pollutant present in photochemical smog. Although ozone is suspected to be a respiratory carcinogen it is not regulated as a carcinogen in most countries.The genotoxic and inflammatory effects of ozone were investigated in female mice exposed to ozone for 90 min. The tail moment in bronchoalveolar lavage (BAL) cells from BALB/c mice was determined by the comet assay as a measure of DNA strand breaks. Within the first 200 min after exposure, the BAL cells from the mice exposed to 1 or 2 ppm ozone had 1.6- and 2.6-fold greater tail moments than unexposed mice. After 200 min there was no effect. It could be ruled out that the effect during the first 200 min was due to major infiltration of lymphocytes or neutrophils. Unexpectedly, ozone had no effect on the content of 8-oxo-deoxyguanosine (8-oxo-dG) in nuclear DNA or on oxidised amino acids in the lung tissue. The mRNA level of the repair enzyme ERCC1 was not increased in the lung tissue. Inflammation was measured by the cytokine mRNA level in lung homogenates. An up to 150-fold induction of interleukin-6 (IL-6) mRNA was detected in the animals exposed to 2 ppm ozone compared to the air-exposed control mice. Also at 1 ppm ozone, the IL-6 mRNA was induced. The large induction of IL-6 mRNA in the lung took place after DNA strand breaks were induced in BAL. This does not support the notion that inflammatory reactions are the cause of DNA damage. To determine whether these exposures were mutagenic, Muta Mice were exposed to 2 ppm ozone, 90 min per day for 5 days. No treatment-related mutations could be detected in the cII transgene.These results indicate that a short episode of ozone exposure at five times the threshold limit value (TLV) in US induces lung inflammatory mediators and DNA damage in the cells in the lumen of the lung. This was not reflected by an induction of mutations in the lung of Muta Mice.     

Antioxidants preserve macrophage phagocytosis of Pseudomonas aeruginosa during hyperoxia

Pseudomonas. aeruginosa (PA) is a leading cause of nosocomial pneumonia in patients receiving mechanical ventilation with hyperoxia. Exposure to supraphysiological concentrations of reactive oxygen species during hyperoxia may result in macrophage damage that reduces their ability to phagocytose PA. We tested this hypothesis in cultured macrophage-like RAW 264.7 cells and alveolar macrophages from mice exposed to hyperoxia. Exposure to hyperoxia induced a similarly impaired phagocytosis of both the mucoid and the nonmucoid forms of PA in alveolar macrophages and RAW cells. Compromised PA phagocytosis was associated with cytoskeleton disorganization and actin oxidation in hyperoxic macrophages. To test whether moderate concentrations of O(2) limit the loss of phagocytic function induced by > or =95% O(2), mice and RAW cells were exposed to 65% O(2). Interestingly, although the resulting lung injury/cell proliferation was not significant, exposure to 65% O(2) resulted in a marked reduction in PA phagocytosis that was comparable to that of > or =95% O(2). Treatment with antioxidants, even post hyperoxic exposure, preserved actin cytoskeleton organization and phagocytosis of PA. These data suggest that hyperoxia reduces macrophage phagocytosis through effects on actin functions which can be preserved by antioxidant treatment. In addition, administration of moderate rather than higher concentrations of O2 does not improve macrophage phagocytosis of PA.     

Effect of endotoxin pretreatment on the pulmonary vascular response to hypoxia in O2-exposed lambs

We recently reported that endotoxin infusion before O2 exposure significantly reduced or delayed the onset of pulmonary edema formation and respiratory failure by reducing the oxidant stress of O2 exposure. Despite these beneficial effects of endotoxin treatment, lung microvascular permeability eventually increased, but postmortem lung water content was less than expected. Prolonged O2 breathing blunts or abolishes the pulmonary constrictor response to alveolar hypoxia in some species, and it is possible that the loss of this response could contribute further to edema formation. To determine whether the reduction in lung edema observed in endotoxin-treated, O2-exposed lambs was linked to the preservation of hypoxic pulmonary vasoconstriction (HPV), we measured pulmonary vascular resistance before and after 8 min of isocarbic hypoxia (inspired O2 fraction 0.12) during each day of O2 exposure. In six control lambs, the pressor response to hypoxia was abolished after 72 h in O2, and the lambs developed respiratory failure shortly thereafter. In six endotoxin-treated lambs, HPV was preserved for as long as 144 h of O2 exposure. In two control O2-exposed lambs in whom HPV was abolished, the infusion of either angiotensin or prostaglandin H2 analogue increased pulmonary vascular resistance by greater than 75%. We conclude that in lambs 1) hyperoxia abolishes the pulmonary vascular response to hypoxia, 2) endotoxin pretreatment reduces acute O2-induced lung injury and preserves the pulmonary constrictor response to hypoxia, and 3) the loss of HPV during O2 exposure may be the result of oxidant-mediated injury to the hypoxia response itself and not the result of diffuse damage to the vasoconstrictor effector mechanism.     

Effects of transgene expression of superoxide dismutase and glutathione peroxidase on pulmonary epithelial cell growth in hyperoxia

Prolonged exposure to supraphysiological oxygen concentrations results in the generation of reactive oxygen species, which can cause significant lung injury in critically ill patients. Supplementation with human recombinant antioxidant enzymes (AOE) may mitigate hyperoxic lung injury, but it is unclear which combination and concentration will optimally protect pulmonary epithelial cells. First, stable cell lines were generated in alveolar epithelial cells (MLE12) overexpressing one or more of the following AOE: Mn superoxide dismutase (MnSOD), CuZnSOD, or glutathione peroxidase 1. Next, A549 cells were transduced with 50-300 particles/cell of recombinant adenovirus containing either LacZ or each of the three AOE (alone or in combination). Cells were then exposed to 95% O(2) for up to 3 days, with cell number and viability determined daily. Overexpression of either MnSOD (primarily mitochondrial) or CuZnSOD (primarily cytosolic) reversed the growth inhibitory effects of hyperoxia within the first 48 h of exposure, resulting in a significant increase in viable cells (P < 0.05), with 1.5- to 3-fold increases in activity providing optimal protection. Protection from mitochondrial oxidation was confirmed by assessing aconitase activity, which was significantly improved in cells overexpressing MnSOD (P < 0.05). Data indicate that optimal protection from hyperoxic injury occurs in cells coexpressing MnSOD and glutathione peroxidase 1, with prevention of mitochondrial oxidation being a critical factor. This has important implications for clinical trials in preterm infants receiving SOD supplementation to prevent acute and chronic lung injury.     

Interfacial phospholipids inhibit ozone-reactive absorption-mediated cytotoxicity in vitro

The intrapulmonary distribution of inhaled ozone (O(3)) and induction of site-specific cell injury are related to complex interactions among airflow patterns, local gas-phase concentrations, and the rates of O(3) flux into, and reaction and diffusion within, the epithelial lining fluid (ELF). Recent studies demonstrated that interfacial phospholipid films appreciably inhibited NO(2) absorption. Because surface-active phospholipids are present on alveolar and airway interfaces, we investigated the effects of interfacial films on O(3)-reactive absorption and acute cell injury. Compressed films of dipalmitoyl-glycero-3-phosphocholine (DPPC) and rat lung lavage lipids significantly reduced O(3)-reactive absorption by ascorbic acid, reduced glutathione, and uric acid. Conversely, unsaturated phosphatidylcholine films did not inhibit O(3) absorption. We evaluated O(3)-mediated cell injury using a human lung fibroblast cell culture system, an intermittent tilting exposure regimen to produce a thin covering layer, and nuclear fluorochrome permeability. Exposure produced negligible injury in cells covered with MEM. However, addition of AH(2) produced appreciable (<50%) cell injury. Film spreading of DPPC monolayers necessitated the use of untilted regimens. Induction of acute cell injury in untilted cultures required both AH(2) plus very high O(3) concentrations. Addition of DPPC films significantly reduced cell injury. We conclude that acute cell injury likely results from O(3) reaction with ELF substrates. Furthermore, interfacial films of surface-active, saturated phospholipids reduce the local dose of O(3)-derived reaction products. Finally, because O(3) local dose and tissue damage likely correlate, we propose that interfacial phospholipids may modulate intrapulmonary distribution of inhaled O(3) and the extent of site-specific cell injury.     

H(2)O(2) inhibits alveolar epithelial wound repair in vitro by induction of apoptosis

Reactive oxygen species (ROS) are released into the alveolar space and contribute to alveolar epithelial damage in patients with acute lung injury. However, the role of ROS in alveolar repair is not known. We studied the effect of ROS in our in vitro wound healing model using either human A549 alveolar epithelial cells or primary distal lung epithelial cells. We found that H(2)O(2) inhibited alveolar epithelial repair in a concentration-dependent manner. At similar concentrations, H(2)O(2) also induced apoptosis, an effect seen particularly at the edge of the wound, leading us to hypothesize that apoptosis contributes to H(2)O(2)-induced inhibition of wound repair. To learn the role of apoptosis, we blocked caspases with the pan-caspase inhibitor N-benzyloxycarbonyl-Val-Ala-Asp (zVAD). In the presence of H(2)O(2), zVAD inhibited apoptosis, particularly at the wound edge and, most importantly, maintained alveolar epithelial wound repair. In H(2)O(2)-exposed cells, zVAD also maintained cell viability as judged by improved cell spreading and/or migration at the wound edge and by a more normal mitochondrial potential difference compared with cells not treated with zVAD. In conclusion, H(2)O(2) inhibits alveolar epithelial wound repair in large part by induction of apoptosis. Inhibition of apoptosis can maintain wound repair and cell viability in the face of ROS. Inhibiting apoptosis may be a promising new approach to improve repair of the alveolar epithelium in patients with acute lung injury.     

Antioxidants and antioxidant enzymes protect against pulmonary oxygen toxicity in the rabbit

Two major lines of defense exist against oxidant lung injury: tissue antioxidants and antioxidant enzymes. We studied pretreatment with the antioxidants, vitamin E and butylated hydroxyanisole (BHA), and the antioxidant enzymes, superoxide dismutase (SOD) and catalase, in rabbits exposed to 100% O2 for 48 h. BHA (200 mg/kg ip) or vitamin E (50-100 mg/kg po) were given for 2 or 3 days, respectively, before O2 exposure. Combined therapy with polyethylene glycol- (PEG) conjugated SOD (12 mg/kg) and catalase (200,000 U/kg) was given intraperitoneally 1 h before and 24 h after beginning 100% O2. Hyperoxia significantly increased the pulmonary content of malondialdehyde, indicating enhanced lipid peroxidation. One hundred percent O2 also increased lung weight gain and alveolar-capillary permeability to aerosolized 99mTc-labeled diethylenetriaminepentaacetate (99mTc-DTPA, 500 mol wt) and fluorescein isothiocyanate-labeled dextran (7,000 mol wt). Pretreatment with vitamin E, BHA, or the combination of PEG-SOD and PEG-catalase prevented the increase in malondialdehyde, lung weight gain, and alveolar-capillary permeability caused by hyperoxia. These results indicate that augmenting either tissue antioxidants or antioxidant enzymes can prevent the pulmonary injury caused by 48 h of 100% O2 in rabbits.     

AMP-activated protein kinase deficiency reduces ozone-induced lung injury and oxidative stress in mice

Background

Acute ozone exposure causes lung oxidative stress and inflammation leading to lung injury. At least one mechanism underlying the lung toxicity of ozone involves excessive production of reactive oxygen and nitrogen intermediates such as peroxynitrite. In addition and beyond its major prooxidant properties, peroxynitrite may nitrate tyrosine residues altering phosphorylation of many protein kinases involved in cell signalling. It was recently proposed that peroxynitrite activates 5''-AMP-activated kinase (AMPK), which regulates metabolic pathways and the response to cell stress. AMPK activation as a consequence of ozone exposure has not been previously evaluated. First, we tested whether acute ozone exposure in mice would impair alveolar fluid clearance, increase lung tissue peroxynitrite production and activate AMPK. Second, we tested whether loss of AMP-activated protein kinase alpha1 subunit in mouse would prevent enhanced oxidative stress and lung injury induced by ozone exposure.

Methods

Control and AMPKα1 deficient mice were exposed to ozone at a concentration of 2.0 ppm for 3 h in glass cages. Evaluation was performed 24 h after ozone exposure. Alveolar fluid clearance (AFC) was evaluated using fluorescein isothiocyanate tagged albumin. Differential cell counts, total protein levels, cytokine concentrations, myeloperoxidase activity and markers of oxidative stress, i.e. malondialdehyde and peroxynitrite, were determined in bronchoalveolar lavage (BAL) and lung homogenates (LH). Levels of AMPK-Thr¹⁷² phosphorylation and basolateral membrane Na(+)-K(+)-ATPase abundance were determined by Western blot.

Results

In control mice, ozone exposure induced lung inflammation as evidence by increased leukocyte count, protein concentration in BAL and myeloperoxidase activity, pro-inflammatory cytokine levels in LH. Increases in peroxynitrite levels (3 vs 4.4 nM, p = 0.02) and malondialdehyde concentrations (110 vs 230 μmole/g wet tissue) were detected in LH obtained from ozone-exposed control mice. Ozone exposure consistently increased phosphorylated AMPK-Thr¹⁷² to total AMPK ratio by 80% in control mice. Ozone exposure causes increases in AFC and basolateral membrane Na(+)-K(+)-ATPase abundance in control mice which did not occur in AMPKα1 deficient mice.

Conclusions

Our results collectively suggest that AMPK activation participates in ozone-induced increases in AFC, inflammation and oxidative stress. Further studies are needed to understand how the AMPK pathway may provide a novel approach for the prevention of ozone-induced lung injury.     

Type II pneumocyte changes during hyperoxic lung injury and recovery

Adult rabbits exposed to 100% O2 for 64 h and then returned to room air for up to 200 h, develop a lung injury characterized by decreased levels of alveolar surfactant followed by a rebound recovery. In the present study we isolated alveolar type II cells from rabbits at various times during hyperoxic exposure and recovery and measured rates of phosphatidylcholine (PC) synthesis, cellular lipid content, and the specific activity of glycerol 3-phosphate (G-3-P) acyltransferase, an enzyme that catalyzes one of the early reactions in phosphoglyceride biosynthesis. These biochemical parameters were compared with measurements of cell size and cell cycle phase by laser flow cytometry. Results showed that alterations in alveolar phospholipid levels in vivo correlated consistently with cellular lipid metabolic changes measured in isolated type II pneumocytes. In particular, alveolar pneumocytes isolated from lungs of rabbits exposed to 100% O2 for 64 h exhibited a 60% decrease in PC synthesis, cell lipid content, and G-3-P acyltransferase activity. All variables then followed a pattern of recovery to normal and ultimately supranormal levels beginning at approximately 3 days postexposure, at which point there was also a measured increase in the number of type II cells in S phase. These findings suggest that O2-induced changes in type II cell surfactant biosynthesis may account, at least in part, for observed changes in lung phospholipid levels in vivo.