Overexpression of manganese superoxide dismutase protects lung epithelial cells against oxidant injury.

To determine whether overexpression of antioxidant enzymes in lung epithelial cells prevents damage from oxidant injury, stable cell lines were generated with complementary DNAs encoding manganese superoxide dismutase (MnSOD) and/or catalase (CAT). Cell lines overexpressing MnSOD, CAT, or MnSOD + CAT were assessed for tolerance to hyperoxia or paraquat. After exposure to 95% O(2) for 10 d, 44 to 57% of cells overexpressing both MnSOD and CAT and 37 to 47% of cells overexpressing MnSOD alone were viable compared with 7 to 12% of empty vector or parental cells (P < 0.05). To assess if viable cells were capable of cell division after hyperoxic exposures (up to 5 d), a clonogenicity assay was performed. The clonogenic potential of cells overexpressing MnSOD + CAT and MnSOD alone were significantly better than those expressing CAT alone or empty vector controls. In addition, 54 to 72% of cells overexpressing both MnSOD and CAT survived in 1 mM paraquat compared with 58 to 73% with MnSOD alone and 27% with control cells. Overexpression of CAT alone did not improve survival in hyperoxia or paraquat. The combination of MnSOD + CAT did not provide additional protection from paraquat. Data demonstrate that overexpression of MnSOD protects cells from oxidant injury and CAT offers additional protection from hyperoxic injury when co-expressed with MnSOD.

Hyperoxia inhibits oxidant-induced apoptosis in lung epithelial cells.

It has previously been shown that hyperoxia induces nonapoptotic cell death in cultured lung epithelial cells, whereas hydrogen peroxide (H(2)O(2)) and paraquat cause apoptosis. To test whether pathways leading to oxidative apoptosis in epithelial cells are sensitive to molecular O(2), A549 cells were exposed to 95% O(2) prior to exposure to lethal concentrations of H(2)O(2). The extent of H(2)O(2)-induced apoptosis was significantly reduced in cells preexposed to hyperoxia compared with room-air controls. Preexposure of the hyperoxia-resistant HeLa-80 cell line to 80% O(2) also inhibited oxidant-induced apoptosis, suggesting that this inhibition is not due to O(2) toxicity. Because hyperoxia generates reactive oxygen species and activates the redox-sensitive transcription factor nuclear factor kappa B (NF-kappa B), the role of antioxidant enzymes and NF-kappa B were examined in this inhibitory process. The onset of inhibition appeared to be directly related to the degradation of I kappa B and subsequent activation of NF-kappa B (either by hyperoxia or TNF-alpha), whereas no significant up-regulation of endogenous antioxidant enzyme activities was found. In addition, suppression of NF-kappa B activities by transfecting A549 cells with a dominant-negative mutant construct of I kappa B significantly augmented the extent of H(2)O(2)-induced apoptosis. These data suggest that hyperoxia inhibits oxidant-induced apoptosis and that this inhibition is mediated by NF-kappa B.

Differential patterns of apoptosis in resolving and nonresolving bacterial pneumonia.

Infection with either Streptococcus sanguis or Streptococcus pneumoniae type 25 causes acute pneumonitis in rats. Pneumonia caused by S. sanguis resolves over the course of 8 d, whereas pneumonia caused by S. pneumoniae type 25 progresses to fibrosis. To examine the role of apoptosis in these models, we performed assays with the terminal deoxynucleotidyltransferase-uridine nucleotide end-labeling technique on tissue sections from rat lungs at various times, and quantified the results with image analysis. Apoptosis was a feature of both the acute and resolving stages of pneumonia. The pattern and extent of apoptosis were similar in both models during the acute stage, and the number of apoptotic nuclei increased in both models through 4 d after infection. Although there were differences in the cellular pattern of apoptosis after 2 d and 4 d of infection, the extent of apoptosis was the same in both models. After 8 d, major differences were observed. In the resolving model, apoptosis was limited primarily to an abscess in the base of the lung. In the nonresolving model, apoptosis was persistent. We also found that cyclin-dependent kinase-5 expression is upregulated during apoptosis induced by bacterial infection. These data indicate that the location and timing of apoptosis may determine whether pneumonia resolves or progresses to fibrosis.

Hyperoxia in cell culture. A non-apoptotic programmed cell death.

Here we discuss the morphological features and our current understanding of the pathways involved in non-apoptotic cell death from O2 toxicity. Preliminary data on hyperoxic signaling indicate that NF-kappa B translocation (and presumptive activation) is not a result of the p42/p44 MAPK pathway, but a likely downstream consequence of activation of the JNK pathway. Our observations suggest the existence of multiple signal transduction pathways in hyperoxia-induced cell death: one involved in the stress response which appears to be NF-kappa B-dependent and another in cell death.

Hyperoxia-induced cell death in the lung--the correlation of apoptosis, necrosis, and inflammation.

Prolonged exposure to hyperoxia causes tissue damage in many organs and tissues. Since the entire surface area of lung epithelium is directly exposed to O2 and other inhaled agents, hyperoxia leads to the development of both acute and chronic lung injuries. These pathologic changes in the lung can also be seen in acute lung injury (ALI) in response to other agents. Simple strategies to mitigate hyperoxia-induced ALI might not be effective by virtue of merely reducing or augmenting the extent of apoptosis of pulmonary cells. Identification of the specific cell types undergoing apoptosis and further understanding of the precise timing of the onset of apoptosis may be necessary in order to gain a greater understanding of the connection between apoptosis and tolerance to hyperoxia and ALI. Attention should also be focused on other forms of non-apoptotic programmed cell death.

Synergistic cytotoxicity from nitric oxide and hyperoxia in cultured lung cells.

Exogenous nitric oxide (NO) is being tested clinically for the treatment of pulmonary hypertension in infants and children. In most cases, these patients receive simultaneous oxygen (O2) therapy. However, little is known about the combined toxicity of NO + hyperoxia. To test this potential toxicity, human alveolar epithelial cells (A549 cells) and human lung microvascular endothelial lung cells were cultured in room air (control), hyperoxia (95% O2), NO (derived from chemical donors), or combined hyperoxia + NO. Control cells grew normally over a 6-day study period. In contrast, cell death from hyperoxia was evident after 4-5 days, whereas cells neither died nor divided in NO alone. However, cells exposed to both NO and hyperoxia began to die on day 2 and died rapidly thereafter. This cytotoxic effect was clearly synergistic, and cell death did not occur via apoptosis. As an indicator of peroxynitrite formation, nitrotyrosine-containing proteins were assayed using anti-nitrotyrosine antibodies. Two protein bands, at molecular masses of 25 and 35 kDa, were found to be increased in A549 cells exposed to NO or NO + hyperoxia. These results indicate that combined NO + hyperoxia has a synergistic cytotoxic effect on alveolar epithelial and lung vascular endothelial cells in culture.

Nuclear factor-kappaB is activated by hyperoxia but does not protect from cell death.

Oxidative insults that are lethal to epithelial cells kill either via apoptosis or necrosis. Nuclear factor-kappaB (NF-kappaB) is a redox-sensitive transcription factor that is activated by oxidative insult, and NF-kappaB activation can protect cells from apoptosis. To test if NF-kappaB can protect from necrotic cell death caused by high levels of molecular O2 (hyperoxia), we exposed human alveolar epithelial (A549) cells to hyperoxia. NF-kappaB was shown to be activated and was translocated to the nucleus within minutes. Nuclear translocation persisted over the course of several days, and the levels of NF-kappaB protein and mRNA increased as well. In hyperoxia, NF-kappaB regulation was independent of mitogen-activated protein kinase (MAPK). In sharp contrast, there was neither nuclear translocation of NF-kappaB nor any increase in expression after exposure to H2O2 at a concentration where this oxidant induces both MAPK and widespread apoptosis. Despite the activation and increased expression of NF-kappaB in hyperoxia, this oxidant remained lethal to the cells. These observations confirm the notion that apoptosis occurs in the absence of NF-kappaB activation but indicate that protection from cell death by NF-kappaB is probably limited to apoptosis.

Unscheduled apoptosis during acute inflammatory lung injury.

Apoptosis is a mode of cell death currently thought to occur in the absence of inflammation. In contrast, inflammation follows unscheduled events such as acute tissue injury which results in necrosis, not apoptosis. We examined the relevance of this paradigm in three distinct models of acute lung injury; hyperoxia, oleic acid, and bacterial pneumonia. In every case, it was found that apoptosis is actually a prominent component of the acute and inflammatory phase of injury. Moreover, using strains of mice that are differentially sensitive to hyperoxic lung injury we observed that the percent of apoptotic cells was well correlated with the severity of lung injury. These observations suggest that apoptosis may be one of the biological consequences during acute injury and the failure to remove these apoptotic cells may also contribute to the inflammatory response.

Forced expression of tropomyosin 2 or 3 in v-Ki-ras-transformed fibroblasts results in distinct phenotypic effects.

Transformation of cells in tissue culture results in a variety of cellular changes including alterations in cell growth, adhesiveness, motility, morphology, and organization of the cytoskeleton. Morphological and cytoskeletal changes are perhaps the most readily apparent features of transformed cells. Although a number of studies have documented a decrease in the expression of specific tropomyosin (TM) isoforms in transformed cells, it remains to be determined if the suppression of TM synthesis is essential in the establishment and maintenance of the transformed pheno-type. To address the roles of different TM isoforms in transformed cells we have examined the effects of expressing specific TM isoforms in transformed cells using a Kirsten virus-transformed cell line (ATCC NRK1569) as a model system. In contrast to normal fibroblasts, the NRK 1569 cells contain reduced levels of TM-1 and undetectable levels of TM-2 and TM-3. These cells have a rounded morphology and are devoid of stress fibers. Employing expression plasmids for TM-2 and TM-3, stable cell lines were established from the NRK 1569 cells that express these isoforms individually. We demonstrate that expression of TM-2 or TM-3 leads to increased cell spreading accompanied by the formation of identifiable microfilament bundles, as well as significant restoration of well-defined vinculin-containing focal adhesion plaques, although expression of each isoform exhibited distinct properties. In addition, cells expressing TM-2, but not TM-3, exhibited contact-inhibited cell growth and a requirement for serum.

In vivo expression of intercellular adhesion molecule 1 in type II pneumocytes during hyperoxia.

Cell-to-cell communication is often disrupted when tissue damage occurs, triggering new signals to cope with the injury. The expression of intercellular adhesion molecule (ICAM-1), a protein involved in the migration, binding, and activation of leukocytes, is markedly increased in mouse lungs damaged by acute hyperoxic exposure. Type I alveolar epithelial cells are sensitive to hyperoxic lung injury, and must be removed from the air spaces following their destruction. In contrast, type II pneumocytes are relatively resistant to hyperoxia and may have a role in the removal process. Two reports demonstrate increased ICAM-1 in alveoli after hyperoxia (Welty et al., 1993, AJRCMB 9:393-400; and Kang et al., 1993, AJRCMB 9:350-355), but the cellular site(s) of ICAM-1 synthesis were not determined. We hypothesized that during in vivo exposure to 100% oxygen (O2), type II pneumocytes synthesize and secrete ICAM-1, an important step in attracting inflammatory cells to the site of injury. Adult mice were exposed to 100% O2 for up to 72 h. To determine whether type II cells express ICAM-1, tissue sections were studied by electron microscopy single-label in situ hybridization or light microscopy dual-label in situ hybridization, using radiolabeled and nonradiolabeled probes. In the lungs of unexposed animals, ICAM-1 mRNA was detected in many cells-including type I pneumocytes-but not in type II cells. After hyperoxia, ICAM-1 transcripts were detected in bona fide, surfactant protein C mRNA-containing, type II alveolar epithelial cells. This observation suggests that type II cells play an important and previously unrecognized role in pulmonary inflammation from O2 toxicity and emphasizes the importance of type II pneumocytes in alveolar repair after injury.

Cellular oxygen toxicity. Oxidant injury without apoptosis.

All forms of aerobic life are faced with the threat of oxidation from molecular oxygen (O2) and have evolved antioxidant defenses to cope with this potential problem. However, cellular antioxidants can become overwhelmed by oxidative insults, including supraphysiologic concentrations of O2 (hyperoxia). Oxidative cell injury involves the modification of cellular macromolecules by reactive oxygen intermediates (ROI), often leading to cell death. O2 therapy, which is a widely used component of life-saving intensive care, can cause lung injury. It is generally thought that hyperoxia injures cells by virtue of the accumulation of toxic levels of ROI, including H2O2 and the superoxide anion (O2-), which are not adequately scavenged by endogenous antioxidant defenses. These oxidants are cytotoxic and have been shown to kill cells via apoptosis, or programmed cell death. If hyperoxia-induced cell death is a result of increased ROI, then O2 toxicity should kill cells via apoptosis. We studied cultured epithelial cells in 95% O2 and assayed apoptosis using a DNA-binding fluorescent dye, in situ end-labeling of DNA, and electron microscopy. Using all approaches we found that hyperoxia kills cells via necrosis, not apoptosis. In contrast, lethal concentrations of either H2O2 or O2- cause apoptosis. Paradoxically, apoptosis is a prominent event in the lungs of animals injured by breathing 100% O2. These data indicate that O2 toxicity is somewhat distinct from other forms of oxidative injury and suggest that apoptosis in vivo is not a direct effect of O2.

Tissue specific distribution of Drosophila sarcomeric myosin heavy-chain protein isoforms.

The sarcomeric myosin heavy-chain (sMHC) gene of Drosophila is single-copy and RNA transcription from this gene is developmentally regulated. Numerous sMHC mRNAs that differ in exon composition can be formed by alternate RNA processing. These transcriptional events result in the presence of multiple sMHC isoforms in the developing organism. We have developed and characterized two antibodies which are specific for different types of sarcomeric myosin heavy-chain protein isoforms in Drosophila and have begun to examine the tissue distribution and function of these various protein isoforms. One of the antibodies (anti-A) is capable of distinguishing between two classes of sMHC protein isoforms which differ in their carboxy terminal amino acid sequences. The second antibody (anti-MHC) recognizes a separate and distinct domain in sMHC protein isoforms. We demonstrate the specificity and the utility of these antibodies in examining the developmental and tissue-specific expression of sMHC protein isoforms in the developing fly.

Functional properties of non-muscle tropomyosin isoforms.

Tropomyosins are a family of actin filament binding proteins. They have been identified in many organisms, including yeast, nematodes, Drosophila, birds and mammals. In metazoans, different forms of tropomyosin are characteristic of specific cell types. Most non-muscle cells, such as fibroblasts, express five to eight isoforms of tropomyosins. The various isoforms exhibit distinct biochemical properties that appear to be required for specific cellular functions.

Structure and expression of a muscle specific gene which is adjacent to the Drosophila myosin heavy-chain gene and can encode a cytochrome b related protein.

We report the characterization of a transcription unit at the second chromosome locus 36B, designated TU-36B which is adjacent to the 3' end of the Drosophila myosin heavy-chain (Mhc) gene. We have isolated and sequenced a complementary DNA clone and the region of genomic DNA which represents this gene. The sequencing studies reveal that this gene contains one intron, the mRNA is 1480 nucleotides in length, the TU-36B mRNA is transcribed in an orientation opposite to the Mhc mRNAs, and the poly(A) addition site of this gene is 99 nucleotides downstream of poly(A) addition site A-2 of the Drosophila Mhc gene. The mRNA contains a continuous open reading frame which can encode a protein product of 47,000 daltons in molecular weight. The proposed protein shares homology with cytochrome b proteins. Comparison of in situ hybridization of Mhc specific and TU-36B specific probes to tissue sections demonstrates that both mRNAs are predominantly transcribed in the same muscle tissues of the developing fly.

Tissue-specific expression of the alternately processed Drosophila myosin heavy-chain messenger RNAs.

The myosin heavy-chain (Mhc) gene of Drosophila is a single-copy gene from which four messenger RNAs are transcribed. Two of these mRNAs, CA-1 and CA-2, are expressed in all stages of development when Mhc mRNA is detected. The 3' ends of these mRNAs differ by alternate choice of poly(A) addition sites. Two additional Mhc mRNAs, CBA-1 and CBA-2, are detected only in midpupal to adult stages of development. The 3' ends of these mRNAs are alternately polyadenylated as the above mRNAs; however, these mRNAs contain an additional alternately spliced exon. We have used in situ hybridization to tissue sections to determine the tissue-specific expression of the alternately processed Mhc mRNAs. Four probes were used in the in situ hybridization experiments: one that detects all Mhc mRNAs, one that is specific for mRNA molecules polyadenylated at the downstream site 2, one that is specific for alternately spliced mRNAs containing the B exon, and one that is specific for Mhc mRNAs Ca-1 and CA-2. This last probe is an oligodeoxynucleotide, while the others are single-stranded RNA molecules synthesized in vitro. Our results demonstrate that the alternate splicing of Mhc mRNAs is muscle-cell-type-specific during pupal development, while the polydenylation site usage at the downstream site 2 is not muscle-cell-type-specific during either embryonic or pupal development.