NF-kappaB protects lung epithelium against hyperoxia-induced nonapoptotic cell death-oncosis

Prolonged exposure to hyperoxia induces pulmonary epithelial cell death and acute lung injury. Although both apoptotic and nonapoptotic morphologies are observed in hyperoxic animal lungs, nonapoptotic cell death had only been recorded in transformed lung epithelium cultured in hyperoxia. To test whether the nonapoptotic characteristics in hyperoxic animal lungs are direct effects of hyperoxia, the mode of cell death was determined both morphologically and biochemically in human primary lung epithelium exposed to 95% O(2). In contrast to characteristics observed in apoptotic cells, hyperoxia induced swelling of nuclei and an increase in cell size, with no evidence for any augmentation in the levels of either caspase-3 activity or annexin V incorporation. These data suggest that hyperoxia can directly induce nonapoptotic cell death in primary lung epithelium. Although hyperoxia-induced nonapoptotic cell death was associated with NF-kappaB activation, it is unknown whether NF-kappaB activation plays any causal role in nonapoptotic cell death. This study shows that inhibition of NF-kappaB activation can accelerate hyperoxia-induced epithelial cell death in both primary and transformed lung epithelium. Corresponding to the reduced cell survival in hyperoxia, the levels of MnSOD were also low in NF-kappaB-deficient cells. These results demonstrate that NF-kappaB protects lung epithelial cells from hyperoxia-induced nonapoptotic cell death.     

Hyperoxia-induced signal transduction pathways in pulmonary epithelial cells

Mechanical ventilation with hyperoxia is necessary to treat critically ill patients. However, prolonged exposure to hyperoxia leads to the generation of excessive reactive oxygen species (ROS), which can cause acute inflammatory lung injury. One of the major effects of hyperoxia is the injury and death of pulmonary epithelium, which is accompanied by increased levels of pulmonary proinflammatory cytokines and excessive leukocyte infiltration. A thorough understanding of the signaling pathways leading to pulmonary epithelial cell injury/death may provide some insights into the pathogenesis of hyperoxia-induced acute inflammatory lung injury. This review focuses on epithelial responses to hyperoxia and some of the major factors regulating pathways to epithelial cell injury/death, and proinflammatory responses on exposure to hyperoxia. We discuss in detail some of the most interesting players, such as NF-kappaB, that can modulate both proinflammatory responses and cell injury/death of lung epithelial cells. A better appreciation for the functions of these factors will no doubt help us to delineate the pathways to hyperoxic cell death and proinflammatory responses.     

Necrotic cell death in response to oxidant stress involves the activation of the apoptogenic caspase-8/bid pathway

Human epithelial (A549) cells exposed to hyperoxia die by cellular necrosis. In the current study, we demonstrated the involvement of apoptogenic factors in epithelial cell necrosis in response to hyperoxia, including the formation of the Fas-related death-inducing signaling complex and initiation of mitochondria-dependent apoptotic pathways. We showed increased activation of both Bid and Bax in A549 cells subjected to hyperoxia. Bax activation involved a Bid-assisted conformational change. We discovered that the response to hyperoxia in vivo predominantly involved the activation of the Bid/caspase-8 pathway without apparent increases in Bax expression. Disruption of the Bid pathway by gene deletion protected against cell death in vivo and in vitro. Likewise, inhibition of caspase-8 by Flip also protected against cell death. Taken together, we have demonstrated the involvement of apoptogenic factors in epithelial cell responses to hyperoxia, despite a final outcome of cellular necrosis. We have, for the first time, identified a predominant role for the caspase-8/Bid pathway in signaling associated with hyperoxic lung injury and cell death in vivo and in vitro.     

The Role of Angiopoietin 2 in Hyperoxia-Inuduced Acute Lung Injury

Hyperoxia-induced acute lung injury (HALI) is characterized by an influx of inflammatory cells, increased pulmonary permeability, endothelial and epithelial cell death. In a murine model and in vitro, we found Angiopoietin 2 to be a critical mediator of lung oxidant injury, inflammation, edema, and regulator of necrotic cell death pathways. The clinical significance of our findings was highlighted by the detection of increased Angiopoietin 2 levels in patients with ALI.     

Responses of baboons to prolonged hyperoxia: physiology and qualitative pathology.

Cardiopulmonary responses to prolonged hyperoxia and their relationships to the development of lung pathology have not been fully characterized in primates. In this study, circulatory hemodynamics and pulmonary function, vascular permeability, and leukocyte sequestration were measured in male baboons after 100% O2 exposure and related to ultrastructural changes of lung injury by electron microscopy. Three groups of animals were exposed to 100% O2 in an exposure cage for 40, 66, and 80 h, respectively. A fourth group of animals was exposed in a cage for 80 h and then anesthetized and ventilated with 100% O2 for additional time. These animals were exposed for a total duration of 110 h or until death from the injury. Physiological responses to hyperoxia were characterized by decreases in total lung capacity and inspiratory capacity at 80 and 110 h. A significant increase in pulmonary leukocyte accumulation was noted by 80 h. Extravascular lung water and permeability surface-area product increased at 80 and 110 h. Cardiac output and stroke volume also decreased, and systemic vascular resistance increased after 80 and 110 h of hyperoxia. Histopathological changes were present in the lungs of all but the 40-h exposure group. Animals exposed for 66 h showed endothelial injury and neutrophil accumulation. By 80 h, animals showed endothelial cell destruction, interstitial edema, and type I cell injury. At 110 h, animals showed substantial destruction of endothelial and type I epithelial cells, exposure of alveolar basement membrane, congestion of capillaries, and substantial interstitial edema. The data indicate that histological changes by electron microscopy precede physiological responses to hyperoxic pulmonary injury in baboons by as much as 14 h and that the physiological responses to early hyperoxic injury are relatively insensitive to the pathological injury.     

GM-CSF and the impaired pulmonary innate immune response following hyperoxic stress

We have previously demonstrated that mice exposed to sublethal hyperoxia (an atmosphere of >95% oxygen for 4 days, followed by return to room air) have significantly impaired pulmonary innate immune response. Alveolar macrophages (AM) from hyperoxia-exposed mice exhibit significantly diminished antimicrobial activity and markedly reduced production of inflammatory cytokines in response to stimulation with LPS compared with AM from control mice in normoxia. As a consequence of these defects, mice exposed to sublethal hyperoxia are more susceptible to lethal pneumonia with Klebsiella pneumoniae than control mice. Granulocyte/macrophage colony-stimulating factor (GM-CSF) is a growth factor produced by normal pulmonary alveolar epithelial cells that is critically involved in maintenance of normal AM function. We now report that sublethal hyperoxia in vivo leads to greatly reduced alveolar epithelial cell GM-CSF expression. Systemic treatment of mice with recombinant murine GM-CSF during hyperoxia exposure preserved AM function, as indicated by cell surface Toll-like receptor 4 expression and by inflammatory cytokine secretion following stimulation with LPS ex vivo. Treatment of hyperoxic mice with GM-CSF significantly reduced lung bacterial burden following intratracheal inoculation with K. pneumoniae, returning lung bacterial colony-forming units to the level of normoxic controls. These data point to a critical role for continuous GM-CSF activity in the lung in maintenance of normal AM function and demonstrate that lung injury due to hyperoxic stress results in significant impairment in pulmonary innate immunity through suppression of alveolar epithelial cell GM-CSF expression.     

NALP-3 inflammasome silencing attenuates ceramide-induced transepithelial permeability

The hallmark of acute lung injury (ALI) is the influx of proinflammatory cytokines into lung tissue and alveolar permeability that ultimately leads to pulmonary edema. However, the mechanisms involved in inflammatory cytokine production and alveolar permeability are unclear. Recent studies suggest that excessive production of ceramide has clinical relevance as a mediator of pulmonary edema and ALI. Our earlier studies indicate that the activation of inflammasome promotes the processing and secretion of proinflammatory cytokines and causes alveolar permeability in ALI. However, the role of ceramide in inflammasome activation and the underlying mechanism in relation to alveolar permeability is not known. We hypothesized that ceramide activates the inflammasome and causes inflammatory cytokine production and alveolar epithelial permeability. To test this hypothesis, we analyzed the lung ceramide levels during hyperoxic ALI in mice. The effect of ceramide on activation of inflammasome and production of inflammatory cytokine was assessed in primary mouse alveolar macrophages and THP-1 cells. Alveolar transepithelial permeability was determined in alveolar epithelial type-II cells (AT-II) and THP-1 co-cultures. Our results reveal that ceramide causes inflammasome activation, induction of caspase-1, IL-1β cleavage, and release of proinflammatory cytokines. In addition, ceramide further induces alveolar epithelial permeability. Short-hairpin RNA silencing of inflammasome components abrogated ceramide-induced secretion of proinflammatory cytokines in vitro. Inflammasome silencing abolishes ceramide-induced alveolar epithelial permeability in AT-II. Collectively, our results demonstrate for the first time that ceramide-induced secretion of proinflammatory cytokines and alveolar epithelial permeability occurs though inflammasome activation.     

Recombinant human VEGF treatment transiently increases lung edema but enhances lung structure after neonatal hyperoxia

Recent studies suggest that VEGF may worsen pulmonary edema during acute lung injury (ALI), but, paradoxically, impaired VEGF signaling contributes to decreased lung growth during recovery from ALI due to neonatal hyperoxia. To examine the diverse roles of VEGF in the pathogenesis of and recovery from hyperoxia-induced ALI, we hypothesized that exogenous recombinant human VEGF (rhVEGF) treatment during early neonatal hyperoxic lung injury may increase pulmonary edema but would improve late lung structure during recovery. Sprague-Dawley rat pups were placed in a hyperoxia chamber (inspired O(2) fraction 0.9) for postnatal days 2-14. Pups were randomized to daily intramuscular injections of rhVEGF(165) (20 microg/kg) or saline (controls). On postnatal day 14, rats were placed in room air for a 7-day recovery period. At postnatal days 3, 14, and 21, rats were killed for studies, which included body weight and wet-to-dry lung weight ratio, morphometric analysis [including radial alveolar counts (RAC), mean linear intercepts (MLI), and vessel density], and lung endothelial NO synthase (eNOS) protein content by Western blot analysis. Compared with room air controls, hyperoxia increased pulmonary edema by histology and wet-to-dry lung weight ratios at postnatal day 3, which resolved by day 14. Although treatment with rhVEGF did not increase edema in control rats, rhVEGF increased wet-to-dry weight ratios in hyperoxia-exposed rats at postnatal days 3 and 14 (P < 0.01). Compared with room air controls, hyperoxia decreased RAC and increased MLI at postnatal days 14 and 21. Treatment with VEGF resulted in increased RAC by 181% and decreased MLI by 55% on postnatal day 14 in the hyperoxia group (P < 0.01). On postnatal day 21, RAC was increased by 176% and MLI was decreased by 58% in the hyperoxia group treated with VEGF. rhVEGF treatment during hyperoxia increased eNOS protein on postnatal day 3 by threefold (P < 0.05). We conclude that rhVEGF treatment during hyperoxia-induced ALI transiently increases pulmonary edema but improves lung structure during late recovery. We speculate that VEGF has diverse roles in hyperoxia-induced neonatal lung injury, contributing to lung edema during the acute stage of ALI but promoting repair of the lung during recovery.     

Deficiency in the c-Jun NH2-terminal kinase signaling pathway confers susceptibility to hyperoxic lung injury in mice

Hyperoxia generates an oxidative stress in the mouse lung, which activates the major stress-inducible kinase pathways, including c-Jun NH2-terminal kinase (JNK). We examined the effect of Jnk1 gene deletion on in vivo responses to hyperoxia in mice. The survival of Jnk1-/- mice was reduced relative to wild-type mice after exposure to continuous hyperoxia. Jnk1-/- mice displayed higher protein concentration in bronchoalveolar lavage (BAL) fluid and increased expression of heme oxygenase-1, a stress-inducible gene, after 65 h of hyperoxia. Contrary to other markers of injury, the leukocyte count in BAL fluid of Jnk1-/- mice was markedly diminished relative to that of wild-type mice. The decrease in BAL leukocyte count was not associated with any decrease in lung myeloperoxidase activity at baseline or after hyperoxia treatment. Pretreatment with inhaled lipopolysaccharide increased BAL neutrophil content and extended hyperoxia survival time to a similar extent in Jnk1-/- and wild-type mice. Associated with increased mortality, Jnk1-/- mice had increased pulmonary epithelial cell apoptosis after exposure to hyperoxia compared with wild-type mice. These results indicate that JNK pathways participate in adaptive responses to hyperoxia in mice.     

Aerosolized bovine lactoferrin reduces lung injury and fibrosis in mice exposed to hyperoxia

This study investigated the ability of aerosolized bovine lactoferrin (bLF) to protect the lungs from injury induced by chronic hyperoxia. Female CD-1 mice were exposed to hyperoxia (FiO₂ = 80 %) for 7 days to induce lung injury and fibrosis. The therapeutic effects of bLF, administered via an aerosol delivery system, on the chronic lung injury induced by this period of hyperoxia were measured by bronchoalveolar lavage, lung histology, cell apoptosis, and inflammatory cytokines in the lung tissues. After exposure to hyperoxia for 7 days, the survival of the mice was significantly decreased to 20 %. The protective effects of bLF against hyperoxia were further confirmed by significant reductions in lung edema, total cell numbers in bronchoalveolar lavage fluid, inflammatory cytokines (IL-1β and IL-6), pulmonary fibrosis, and apoptotic DNA fragmentation. The aerosolized bLF protected the mice from oxygen toxicity and increased the survival fraction to 66.7 % in the hyperoxic model. The results support the use of an aerosol therapy with bLF in intensive care units to reduce oxidative injury in patients with severe hypoxemic respiratory failure or chronic obstructive pulmonary disease.     

Mitochondrial aldehyde dehydrogenase attenuates hyperoxia-induced cell death through activation of ERK/MAPK and PI3K-Akt pathways in lung epithelial cells

Oxygen toxicity is one of the major risk factors in the development of the chronic lung disease or bronchopulmonary dysplasia in premature infants. Using proteomic analysis, we discovered that mitochondrial aldehyde dehydrogenase (mtALDH or ALDH2) was downregulated in neonatal rat lung after hyperoxic exposure. To study the role of mtALDH in hyperoxic lung injury, we overexpressed mtALDH in human lung epithelial cells (A549) and found that mtALDH significantly reduced hyperoxia-induced cell death. Compared with control cells (Neo-A549), the necrotic cell death in mtALDH-overexpressing cells (mtALDH-A549) decreased from 25.3 to 6.5%, 50.5 to 9.1%, and 52.4 to 15.1% after 24-, 48-, and 72-h hyperoxic exposure, respectively. The levels of intracellular and mitochondria-derived reactive oxygen species (ROS) in mtALDH-A549 cells after hyperoxic exposure were significantly lowered compared with Neo-A549 cells. mtALDH overexpression significantly stimulated extracellular signal-regulated kinase (ERK) phosphorylation under normoxic and hyperoxic conditions. Inhibition of ERK phosphorylation partially eliminated the protective effect of mtALDH in hyperoxia-induced cell death, suggesting ERK activation by mtALDH conferred cellular resistance to hyperoxia. mtALDH overexpression augmented Akt phosphorylation and maintained the total Akt level in mtALDH-A549 cells under normoxic and hyperoxic conditions. Inhibition of phosphatidylinositol 3-kinase (PI3K) activation by LY294002 in mtALDH-A549 cells significantly increased necrotic cell death after hyperoxic exposure, indicating that PI3K-Akt activation by mtALDH played an important role in cell survival after hyperoxia. Taken together, these data demonstrate that mtALDH overexpression attenuates hyperoxia-induced cell death in lung epithelial cells through reduction of ROS, activation of ERK/MAPK, and PI3K-Akt cell survival signaling pathways.     

Deletion of ASK1 Protects against Hyperoxia-Induced Acute Lung Injury

Apoptosis signal-regulating kinase 1 (ASK1), a member of the MAPK kinase kinase kinase (MAP3K) family, is activated by various stimuli, which include oxidative stress, endoplasmic reticulum (ER) stress, calcium influx, DNA damage-inducing agents and receptor-mediated signaling through tumor necrosis factor receptor (TNFR). Inspiration of a high concentration of oxygen is a palliative therapy which counteracts hypoxemia caused by acute lung injury (ALI)-induced pulmonary edema. However, animal experiments so far have shown that hyperoxia itself could exacerbate ALI through reactive oxygen species (ROS). Our previous data indicates that ASK1 plays a pivotal role in hyperoxia-induced acute lung injury (HALI). However, it is unclear whether or not deletion of ASK1 in vivo protects against HALI. In this study, we investigated whether ASK1 deletion would lead to attenuation of HALI. Our results show that ASK1 deletion in vivo significantly suppresses hyperoxia-induced elevation of inflammatory cytokines (i.e. IL-1β and TNF-α), cell apoptosis in the lung, and recruitment of immune cells. In summary, the results from the study suggest that deletion of ASK1 in mice significantly inhibits hyperoxic lung injury.     

Identification of ectonucleotidases CD39 and CD73 in innate protection during acute lung injury

Acute lung injury (ALI), such as that which occurs with mechanical ventilation, contributes to morbidity and mortality of critical illness. Nonetheless, in many instances, ALI resolves spontaneously through unknown mechanisms. Therefore, we hypothesized the presence of innate adaptive pathways to protect the lungs during mechanical ventilation. In this study, we used ventilator-induced lung injury as a model to identify endogenous mechanisms of lung protection. Initial in vitro studies revealed that supernatants from stretch-induced injury contained a stable factor which diminished endothelial leakage. This factor was subsequently identified as adenosine. Additional studies in vivo revealed prominent increases in pulmonary adenosine levels with mechanical ventilation. Because ectoapyrase (CD39) and ecto-5'-nucleotidase (CD73) are rate limiting for extracellular adenosine generation, we examined their contribution to ALI. In fact, both pulmonary CD39 and CD73 are induced by mechanical ventilation. Moreover, we observed pressure- and time-dependent increases in pulmonary edema and inflammation in ventilated cd39(-/-) mice. Similarly, pharmacological inhibition or targeted gene deletion of cd73 was associated with increased symptom severity of ventilator-induced ALI. Reconstitution of cd39(-/-) or cd73(-/-) mice with soluble apyrase or 5'-nucleotidase, respectively, reversed such increases. In addition, ALI was significantly attenuated and survival improved after i.p. treatment of wild-type mice with soluble apyrase or 5'-nucleotidase. Taken together, these data reveal a previously unrecognized role for CD39 and CD73 in lung protection and suggest treatment with their soluble compounds as a therapeutic strategy for noninfectious ALI.     

Severe Vitamin E deficiency exacerbates acute hyperoxic lung injury associated with increased oxidative stress and inflammation

Hyperoxia causes acute lung injury along with an increase of oxidative stress and inflammation. It was hypothesized that vitamin E deficiency might exacerbate acute hyperoxic lung injury. This study used alpha-tocopherol transfer protein knockout (alpha-TTP KO) mice fed a vitamin E-deficient diet (KO E(-) mice) as a model of severe vitamin E deficiency. Compared with wild-type (WT) mice, KO E(-) mice showed a significantly lower survival rate during hyperoxia. After 72 h of hyperoxia, KO E(-) mice had more severe histologic lung damage and higher values of the total cell count and the protein content of bronchoalveolar lavage fluid (BALF) than WT mice. IL-6 mRNA expression in lung tissue and the levels of 8-iso-prostaglandin F(2alpha) (8-iso-PGF(2alpha)) in both lungs and BALF were higher in KO E(-) mice than in WT mice. It was concluded that severe vitamin E deficiency exacerbates acute hyperoxic lung injury associated with increased oxidative stress or inflammation.     

Cytochrome b5 and cytokeratin 17 are biomarkers in bronchoalveolar fluid signifying onset of acute lung injury

Acute lung injury (ALI) is characterized by pulmonary edema and acute inflammation leading to pulmonary dysfunction and potentially death. Early medical intervention may ameliorate the severity of ALI, but unfortunately, there are no reliable biomarkers for early diagnosis. We screened for biomarkers in a mouse model of ALI. In this model, inhalation of S. aureus enterotoxin A causes increased capillary permeability, cell damage, and increase protein and cytokine concentration in the lungs. We set out to find predictive biomarkers of ALI in bronchoalveolar lavage (BAL) fluid before the onset of clinical manifestations. A cutting edge proteomic approach was used to compare BAL fluid harvested 16 h post S. aureus enterotoxin A inhalation versus BAL fluid from vehicle alone treated mice. The proteomic PF 2D platform permitted comparative analysis of proteomic maps and mass spectrometry identified cytochrome b5 and cytokeratin 17 in BAL fluid of mice challenged with S. aureus enterotoxin A. Validation of cytochrome b5 showed tropic expression in epithelial cells of the bronchioles. Importantly, S. aureus enterotoxin A inhalation significantly decreased cytochrome b5 during the onset of lung injury. Validation of cytokeratin 17 showed ubiquitous expression in lung tissue and increased presence in BAL fluid after S. aureus enterotoxin A inhalation. Therefore, these new biomarkers may be predictive of ALI onset in patients and could provide insight regarding the basis of lung injury and inflammation.