Epithelial cell death is an important contributor to oxidant-mediated acute lung injury

Abstract

Rationale: Acute lung injury and the acute respiratory distress syndrome are characterized by increased lung oxidant stress and apoptotic cell death. The contribution of epithelial cell apoptosis to the development of lung injury is unknown.

Objectives: To determine whether oxidant-mediated activation of the intrinsic or extrinsic apoptotic pathway contributes to the development of acute lung injury.

Methods: Exposure of tissue-specific or global knockout mice or cells lacking critical components of the apoptotic pathway to hyperoxia, a well-established mouse model of oxidant-induced lung injury, for measurement of cell death, lung injury, and survival.

Measurements and main results: We found that the overexpression of SOD2 prevents hyperoxia-induced BAX activation and cell death in primary alveolar epithelial cells and prolongs the survival of mice exposed to hyperoxia. The conditional loss of BAX and BAK in the lung epithelium prevented hyperoxia-induced cell death in alveolar epithelial cells, ameliorated hyperoxia-induced lung injury, and prolonged survival in mice. By contrast, Cyclophilin D-deficient mice were not protected from hyperoxia, indicating that opening of the mitochondrial permeability transition pore is dispensable for hyperoxia-induced lung injury. Mice globally deficient in the BH3-only proteins BIM, BID, PUMA, or NOXA, which are proximal upstream regulators of BAX and BAK, were not protected against hyperoxia-induced lung injury suggesting redundancy of these proteins in the activation of BAX or BAK.

Conclusions: Mitochondrial oxidant generation initiates BAX- or BAK-dependent alveolar epithelial cell death, which contributes to hyperoxia-induced lung injury.

Figure 1.

The generation of mitochondrial superoxide during exposure to hyperoxia contributes to mortality. Mice were intratracheally transfected with 1 × 10⁹ pfu/animal of Ad-Null or Ad-SOD2 in a 50% surfactant vehicle or sham infected (Sham). (A) After 7 days the mice were exposed to sublethal hyperoxia (84 h, >95% O₂) or maintained at normoxia (21% O₂) after which the lungs were harvested and immunoblotted for SOD2, a representative immunoblot and densitometry (n = 3) is shown. (B) Mice transfected with Ad-Null or Ad-SOD2 7 days earlier were exposed to hyperoxia for measurement of survival (n = 6 sham; n = 5 null; n = 11 SOD2). (C) Mice heterozygous for Mclk, which is required for synthesis of the mitochondrial antioxidant ubiquinone, were exposed to hyperoxia for measurement of survival (n = 10 each group). The median survival (LD₅₀) is in parentheses. Mice infected with Ad-Null or Ad-SOD2 were exposed to sublethal hyperoxia or maintained in normoxia for assessment of lung injury. Hematoxylin and eosin stained lung sections (×100) (D), wet-to dry lung weight ratios (E), and the percentage of TUNEL-positive nuclei (F) (n = 3 animals per group). Mclk^+/− or wild-type mice were exposed to sublethal hyperoxia or maintained in normoxia for measurement of lung injury. Hematoxylin and eosin stained lung sections (original magnification ×100) (E) and the percentage of TUNEL-positive nuclei (H). *P < 0.05 for comparison with normoxic controls. ^†P < 0.05 for comparison of difference between Ad-SOD2 and Ad-SOD null transfected animals or Mclk1^+/+ compared with Mclk1^+/− animals after exposure to hyperoxia.

Figure 2.

The mitochondrial permeability transition pore is not required for hyperoxia-induced mortality. (A) Mice lacking cyclophilin D, a critical component of the mitochondrial permeability transition pore, or wild-type mice on an identical background were exposed to hyperoxia for measurement of survival. The median survival (LD₅₀) is in parentheses (n = 18 for Cyclophilin D^+/+; n = 12 for Cyclophilin D^−/−). The difference between wild-type and knockout mice was not significant (P = 0.79). The same mouse strains were exposed to hyperoxia for 84 hours for measurement of lung injury. Hematoxylin and eosin stained lung sections (original magnification ×100) (B), wet-to-dry weight ratios of the lung (C), and the percentage of TUNEL positive nuclei (D) (n >4 animals per group). *P < 0.05 for comparison with nomoxic controls. No significant differences were observed between the Cyclophilin D^+/+ and Cyclophilin D^−/− animals.

Figure 3.

Mitochondrial superoxide production results in the activation of BAX and cell death. (A) Primary rat alveolar epithelial type II cells were infected with an adenovirus expressing no transgene (Ad-Null) or SOD2 (Ad-SOD2) and the relative abundance of SOD2 was measured by immunoblotting 48 hours later. (B) Cells were cotransfected with an adenovirus encoding an oxidant-sensitive GFP targeted to the mitochondrial matrix (mito-Ro-GFP) and either Ad-Null or Ad-SOD2 48 hours before exposure to hyperoxia or normoxia for 4 hours. The oxidation of mito-Ro-GFP was then assessed using flow cytometry. (C) Sham transfected cells or cells transfected with Ad-Null or Ad-SOD2 were exposed to hyperoxia for 16 hours after which the cells were immunostained with an antibody that recognizes an activated form of Bax (green, left column) and the mitochondrial marker MitoTracker Red (red, middle column). The right column is a merged image. Each column is a representative image sampled from three separate isolations. (D) Identically infected cells were exposed to hyperoxia for 48 hours and cell death was measured (LDH release). *P < 0.05 compared with normoxic controls. ^†P < 0.05 for comparison of difference between Ad-SOD2 and Ad-SOD null transfected cells.

Figure 4.

BAX or BAK are required for hyperoxia-induced cell death in primary mouse alveolar epithelial cells. (A) Mice deficient in Bak with loxP sites flanking the Bax gene Bax^fl/flBak^−/− were intratracheally infected with an adenovirus encoding no transgene (Ad-Null) or one encoding Cre recombinase (Ad-Cre) (1 × 10⁹ pfu/animal) and 7 days later Cre abundance in lung homogenates was measured by immunoblotting. (B) Thirty days after infection with Ad-Null or Ad-Cre, primary ATII cells were harvested from mice and immunoblotted for the abundance of BAX (each lane represents pooled samples from four animals, densitometry represents three replicates). (C) These cells were exposed to hyperoxia for 48 hours and cell death was measured using an ELISA that detects DNA fragmentation (n = 3). *P < 0.05 for comparison with normoxic controls. ^†P < 0.05 for comparison of difference between Ad-Null and Ad-Cre transfected cells after exposure to hyperoxia.

Figure 5.

BAX or BAK contribute to hyperoxia-induced mortality in mice. (A) Bax^fl/flBak^−/− mice were intratracheally infected with an adenovirus encoding no transgene (Ad-Null) or one encoding Cre recombinase (Ad-Cre) (1 × 10⁹ pfu/animal) 30 days before exposure to hyperoxia (≥ 95% O₂) for measurement of survival (n = 11 for Ad-Null; n = 13 for Ad-Cre; ^†indicates P < 0.001 for comparison between Ad-Null and Ad-Cre infected mice after exposure to hyperoxia). Identically treated Bax^fl/flBak^−/− mice were exposed to hyperoxia or room air for 84 hours for assessment of lung injury. (B) Hematoxylin and eosin stained lung sections (original magnification ×100). (C) Wet-to-dry weight ratios of the lung. (D) Percentage of TUNEL-positive nuclei. n ≥ 4 for all measures. *P < 0.05 for comparison with normoxic controls. ^†P < 0.05 for comparison of difference between Ad-Null and Ad-Cre transfected animals after exposure to hyperoxia.

Figure 6.

Resistance of inflammatory cells to apoptosis does not alter hyperoxia-induced mortality. (A) Alveolar macrophages were identified in bronchoalveolar lavage fluid from wild-type mice and transgenic mice overexpressing Bcl-2 in cells of hematopoietic origin (vav-Bcl-2) as CD11c (+), CD 11b (−) cells and then intracellularly stained using an antibody against Bcl-2 . (B) Wild-type and vav-Bcl-2 mice were exposed to hyperoxia for measurement of survival (n = 8 each group). P = 1.

Figure 7.

The extrinsic apoptotic pathway is not required for hyperoxia-induced mortality in mice. (A) FADD^fl/fl mice were intratracheally infected with an adenovirus encoding no transgene (Ad-Null) or one encoding Cre recombinase (Ad-Cre) (1 × 10⁹ pfu/animal), and 30 days later alveolar type II cell lysates from four mice in each group were pooled and immunoblotted for the presence of the GFP-labeled Fas-Associated Death Domain (FADD). (B) Identically treated mice were exposed to hyperoxia (95% O₂) for measurement of survival (n = 14 for Ad-Null; n = 9 for Ad-Cre; P = 0.28 for comparison between FADD^fl/fl mice treated with Ad-Null or Ad-Cre). (C) Mice globally deficient in the proapoptotic BH3 protein Bid were exposed to hyperoxia for measurement of survival (n = 12 for wild-type; n = 14 for Bid^−/−; P = 0.8).

Figure 8.

The loss of an individual BH3 protein is not sufficient to prevent hyperoxia-induced mortality. Mice globally deficient in the proapoptotic BH3 proteins (A) Bim, (B) Puma, or (C) Noxa were exposed to hyperoxia for measurement of survival. Bim: n = 5 for wild-type and Bim^−/−, P = 0.8. Puma: n = 5 for wild-type and Puma^−/−, P = 0.87. Noxa: n = 21 for wild-type, n = 23 for Noxa^−/−, P = 0.87.

Figure 9.

Neither the BH3 domain only proteins Bim, Bid, and Puma alone or in combination nor ER localized BAK are required for BAX or BAK dependent cell death. (A and B) murine embryonic fibroblasts (MEFs) from Bax^−/− Bak^−/− and Bim^−/−Puma^−/− mice or from their wild-type controls were exposed to hyperoxia for measurement of cell death (LDH release). (C) MEFs from Bim^−/− Puma^−/− double knockout mice were stably transfected with two shRNA constructs against Bid (33) and exposed to hyperoxia for measurement of cell death (LDH release). (D) MEFs from Bax^−/− Bak^−/−mice were stably transfected with a control retrovirus (GFP) or retroviruses encoding wild-type BAK, a mitochondrially localized BAK (BAK-ActA) or an ER localized BAK (BAK-cb5). n = 3 for all measures, * indicates P < 0.05 for comparison with wild-type air exposed cells, ^†P < 0.05 between wild-type and mutant cells exposed to hyperoxia.