The alveolar epithelium is composed of alveolar type 1 (AT1) and alveolar type 2 (AT2) cells, which represent ≈95% and ≈5% of the alveolar surface area, respectively. Lung liquid clearance is driven by the osmotic gradient generated by the Na,K-ATPase. AT2 cells have been shown to express the α1 Na,K-ATPase. We postulated that AT1 cells, because of their larger surface area, should be important in the regulation of active Na + transport. By immunofluorescence and electron microscopy, we determined that AT1 cells express both the α1 and α2 Na,K-ATPase isoforms. In isolated, ouabain-perfused rat lungs, the α2 Na,K-ATPase in AT1 cells mediated 60% of the basal lung liquid clearance. The β-adrenergic agonist isoproterenol increased lung liquid clearance by preferentially upregulating the α2 Na,K-ATPase protein abundance in the plasma membrane and activity in alveolar epithelial cells (AECs). Rat AECs and human A549 cells were infected with an adenovirus containing the rat Na,K-ATPase α2 gene (Adα2), which resulted in the overexpression of the α2 Na,K-ATPase protein and caused a 2-fold increase in Na,K-ATPase activity. Spontaneously breathing rats were also infected with Adα2, which increased α2 protein abundance and resulted in a ≈250% increase in lung liquid clearance. These studies provide the first evidence that α2 Na,K-ATPase in AT1 cells contributes to most of the active Na + transport and lung liquid clearance, which can be further increased by stimulation of the β-adrenergic receptor or by adenovirus-mediated overexpression of the α2 Na,K-ATPase.

Inhaled nitric oxide (iNO) is used clinically to treat pulmonary hypertension in newborns, often in conjunction with hyperoxia (NO/O2). Prolonged exposure to NO/O2 causes synergistic lung injury and death of lung epithelial cells. To explore the mechanisms involved, oxygen-resistant HeLa-80 cells were exposed to NO +/- O2. Exposure to NO and O2 induced a synergistic cytotoxicity, accompanied with apoptotic characteristics, including elevated caspase-3-like activity, Annexin V incorporation, and nuclear condensation. This apoptosis was associated with a synergistic suppression of NF-kappaB activity. Cells lacking functional NF-kappaB p65 subunit were more sensitive to NO/O2 than their wild type counterparts. This injury was partially rescued by transfection with a p65 expression construct, suggesting an inverse relationship between NF-kappaB and susceptibility to the cytotoxicity of NO/O2. Despite the reduced NF-kappaB activity in cells exposed to NO +/- O2, IkappaBalpha was degraded, suggesting that pathways regulating the steady-state levels of IkappaB were not involved. However, exposure to NO/O2 caused a marked reduction in nuclear localization and an increase in protein carbonyl formation of NF-kappaB p65 subunit. These results suggest that NO/O2-induced apoptosis occurs by suppressing NF-kappaB activity.

To determine whether liquid ventilation (LV) causes less cell injury and improves lung function compared with conventional gas ventilation (GV), we analyzed pulmonary physiological profiles, lung histology, and cell death in 110- and 120-day preterm lambs, which were randomized to receive either ventilation modality on FIO2 = 1. LV lungs were well expanded with adequate pulmonary function, whereas GV animals exhibited marked atelectasis, poor pulmonary function, and increased mortality. Both ventilatory strategies induced marked lung cell apoptosis, but with distinct patterns of distribution. Although GV induced apoptosis of epithelium primarily in the lining and within the lumina of bronchioles, LV induced significant apoptosis much more homogeneously throughout lung parenchyma including alveoli and interstitial spaces. These studies suggest that although both forms of ventilation cause regional apoptosis, LV more effectively delivers oxygen and recruits the lung more homogeneously than GV.

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.

It has previously been shown that hyperoxia induces nonapoptotic cell death in cultured lung epithelial cells, whereas hydrogen peroxide (H2O2) and paraquat cause apoptosis. To test whether pathways leading to oxidative apoptosis in epithelial cells are sensitive to molecular O2, A549 cells were exposed to 95% O2 prior to exposure to lethal concentrations of H2O2. The extent of H2O2-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% O2 also inhibited oxidant-induced apoptosis, suggesting that this inhibition is not due to O2toxicity. Because hyperoxia generates reactive oxygen species and activates the redox-sensitive transcription factor nuclear factor κB (NF-κB), the role of antioxidant enzymes and NF-κB were examined in this inhibitory process. The onset of inhibition appeared to be directly related to the degradation of IκB and subsequent activation of NF-κB (either by hyperoxia or TNF-α), whereas no significant up-regulation of endogenous antioxidant enzyme activities was found. In addition, suppression of NF-κB activities by transfecting A549 cells with a dominant-negative mutant construct of IκB significantly augmented the extent of H2O2-induced apoptosis. These data suggest that hyperoxia inhibits oxidant-induced apoptosis and that this inhibition is mediated by NF-κB.

Cellular fibronectin (cFN) expression is characteristic of injured tissues. Unlike plasma FN, cFN mRNA often contains the EIIIA or EIIIB domains. We examined the lung cell-specific expression of total cFN mRNA and the EIIIA and EIIIB splice variants in rabbits after acute oxygen injury. By in situ hybridization, control lung had low cFN mRNA. After exposure to > 95% oxygen, mRNAs for total cFN and EIIIA were noted primarily in alveolar macrophages and large-vessel endothelial cells. By 3-5 days recovery, cFN and EIIIA mRNA abundance was increased in alveolar septal cells (i.e., alveolar epithelial, interstitial, or endothelial cells) and in some large-vessel endothelial cells but was low in bronchial epithelial cells. During recovery, EIIIB mRNA was low in alveolar septal cells but was noted mainly in chondrocytes. Immunostaining for EIIIA increased during recovery, paralleling the in situ hybridizations. Because FN may modulate alveolar type II cell phenotype, we investigated type II cell cFN mRNA expression in vivo. During recovery, neither isolated type II cells nor cells with surfactant protein C mRNA in vivo contained FN mRNA. In summary, these data suggest that cFN with the EIIIA domain has a role in alveolar cell recovery from oxygen injury and that type II cells do not express cFN during recovery.

An important component of the pathophysiologic response to hyperoxia (O2) is pulmonary inflammation, although the roles of specific inflammatory mediators during pulmonary O2 toxicity are not completely known. Interleukin-1 (IL-1) is an early inflammatory mediator and is sufficient to elicit many of the responses associated with acute injury. The IL-1 family comprises two bioactive proteins, IL-1alpha and IL-1beta, and their natural antagonist IL-1ra. Here we report studies of IL-1 regulation during hyperoxic lung injury in the adult mouse. When assayed by Northern blot, increases in IL-1beta mRNA were seen after 2 days of hyperoxia. In contrast, IL-1alpha mRNA was barely detectable before 4 days of hyperoxia. To further understand the cellular origin of IL-1beta expression in lungs, in situ hybridization and immunohistochemical analyses were performed. IL-1beta mRNA or protein was not detected in the lungs of unexposed animals. At 3 days, we observed the accumulation of IL-1beta transcripts in pulmonary interstitial macrophages and in a subset of neutrophils, and immunodetectable IL-1beta protein was co-localized in adjacent sections. At 4 days of exposure, IL-1beta transcripts were widespread in lung tissue, but many areas rich in IL-1beta mRNA were devoid of immunodetectable IL-1beta. However, it is not known whether increased synthesis of IL-1beta or the uncoupling of IL-1beta protein and mRNA accumulation has a role in pathophysiology of pulmonary O2 toxicity.

Mice exposed to 100% O2 die after 3 or 4 d with diffuse alveolar damage and alveolar edema. Extensive cell death is evident by electron microscopy in the alveolar septa, affecting both endothelial and epithelial cells. The damaged cells show features of both apoptosis (condensation and margination of chromatin) and necrosis (disruption of the plasma membrane). The electrophoretic pattern of lung DNA indicates both internucleosomal fragmentation, characteristic of apoptosis, and overall degradation, characteristic of necrosis. Hyperoxia induces a marked increase in RNA or protein levels of p53, bax, bcl-x, and Fas, which are known to be expressed in certain types of apoptosis. However, we did not detect an increased activity of proteases belonging to the apoptosis "executioner" machinery, such as CPP32 (caspase 3), ICE (caspase 1), or cathepsin D. Furthermore, administration of an ICE-like protease inhibitor did not significantly enhance the resistance to oxygen. Additionally, neither p53-deficient mice nor lpr mice (Fas null) manifested an increased resistance to hyperoxia-induced lung damage. These results show that both necrosis and apoptosis contribute to cell death during hyperoxia. Multiple apoptotic pathways seem to be involved in this, and an antiapoptotic strategy does not attenuate alveolar damage.

Lung injury is a frequent consequence of oxygen (O2) therapy administered to newborns and adults with respiratory distress. Acute exposure to hyperoxia results in a well-described pathophysiologic response in the lungs. Because inflammation is an important component of pulmonary O2 toxicity, we have an interest in identifying the inflammatory mediators that increase during hyperoxia. Platelet-endothelial cell adhesion molecule-1 (PECAM-1), a member of the immunoglobulin superfamily that is expressed at the junctions between endothelial cells, is essential to the transendothelial migration of leukocytes. We hypothesized that increased expression of PECAM-1 occurs in pulmonary endothelial cells during hyperoxic lung injury. Adult mice were exposed to 100% O2 for up to 96 h. We analyzed PECAM-1 expression by RNA blot hybridization, in situ hybridization, and immunohistochemistry. A increase in PECAM-1 mRNA was seen as soon as 2 d of hyperoxia relative to unexposed control mice. PECAM-1 mRNA and protein were found in endothelial cells of both large and small arteries. The expression of PECAM-1 in capillary vessels was further confirmed using in situ hybridization at the electron microscope level. This increase in PECAM-1 expression coincided with the appearance of leukocytes in lung tissue. These observations suggest that PECAM-1 expression is a relatively early step in the inflammation cascade, and intervention at this phase may be critical to the prevention of further damage.

Therapy with supraphysiological concentrations of oxygen (O2) is required in a number of clinical situations, but this use of O2 may be accompanied by tissue damage (Northway et al, 1967; Horowitz and Davis, 1996). For example, hyperoxia plays a role in the etiology of bronchopulmonary dysplasia (Pappas et al, 1983; Roberts and Frank, 1984) a disorder resulting from ventilatory O2 therapy for the respiratory distress syndrome of premature infants. The general problem of O2 toxicity involves several organs, most notably the lungs, which receive direct exposure (Wispe and Roberts, 1987). The pathology of O2induced lung injury includes inflammation and permeability changes of the alveolar-capillary membrane, causing extensive pulmonary edema and severe decreases in respiratory function (Freeman et al, 1986). Lung pathology resulting from acute O2 toxicity is accompanied by, and may result from biochemical alterations in lung cells.

Annals of the New York Academy of SciencesVolume 834, Issue 1 p. 651-652 Alpha-2 Na,K-ATPase Contributes to Lung Liquid Clearancea K. RIDGE, K. RIDGE Pulmonary and Critical Care Medicine Division Michael Reese Hospital and University of Illinois Chicago, Illinois 60680; Winthrop Hospital New York, New York 11501; Washington University St. Louis, Missouri 63110; Rush Presbyterian-St. Luke's Medical Center and Northeastern Illinois University Chicago, Illinois 60637Search for more papers by this authorW. OLIVERA, W. OLIVERA Pulmonary and Critical Care Medicine Division Michael Reese Hospital and University of Illinois Chicago, Illinois 60680; Winthrop Hospital New York, New York 11501; Washington University St. Louis, Missouri 63110; Rush Presbyterian-St. Luke's Medical Center and Northeastern Illinois University Chicago, Illinois 60637Search for more papers by this authorD. H. RUTSCHMAN, D. H. RUTSCHMAN Pulmonary and Critical Care Medicine Division Michael Reese Hospital and University of Illinois Chicago, Illinois 60680; Winthrop Hospital New York, New York 11501; Washington University St. Louis, Missouri 63110; Rush Presbyterian-St. Luke's Medical Center and Northeastern Illinois University Chicago, Illinois 60637Search for more papers by this authorR. W. MERCER, R. W. MERCER Pulmonary and Critical Care Medicine Division Michael Reese Hospital and University of Illinois Chicago, Illinois 60680; Winthrop Hospital New York, New York 11501; Washington University St. Louis, Missouri 63110; Rush Presbyterian-St. Luke's Medical Center and Northeastern Illinois University Chicago, Illinois 60637Search for more papers by this authorB. UHAL, B. UHAL Pulmonary and Critical Care Medicine Division Michael Reese Hospital and University of Illinois Chicago, Illinois 60680; Winthrop Hospital New York, New York 11501; Washington University St. Louis, Missouri 63110; Rush Presbyterian-St. Luke's Medical Center and Northeastern Illinois University Chicago, Illinois 60637Search for more papers by this authorS. HOROWITZ, S. HOROWITZ Pulmonary and Critical Care Medicine Division Michael Reese Hospital and University of Illinois Chicago, Illinois 60680; Winthrop Hospital New York, New York 11501; Washington University St. Louis, Missouri 63110; Rush Presbyterian-St. Luke's Medical Center and Northeastern Illinois University Chicago, Illinois 60637Search for more papers by this authorF. HUGHES, F. HUGHES Pulmonary and Critical Care Medicine Division Michael Reese Hospital and University of Illinois Chicago, Illinois 60680; Winthrop Hospital New York, New York 11501; Washington University St. Louis, Missouri 63110; Rush Presbyterian-St. Luke's Medical Center and Northeastern Illinois University Chicago, Illinois 60637Search for more papers by this authorP. FACTOR, P. FACTOR Pulmonary and Critical Care Medicine Division Michael Reese Hospital and University of Illinois Chicago, Illinois 60680; Winthrop Hospital New York, New York 11501; Washington University St. Louis, Missouri 63110; Rush Presbyterian-St. Luke's Medical Center and Northeastern Illinois University Chicago, Illinois 60637Search for more papers by this authorM. L. BARNARD, M. L. BARNARD Pulmonary and Critical Care Medicine Division Michael Reese Hospital and University of Illinois Chicago, Illinois 60680; Winthrop Hospital New York, New York 11501; Washington University St. Louis, Missouri 63110; Rush Presbyterian-St. Luke's Medical Center and Northeastern Illinois University Chicago, Illinois 60637Search for more papers by this authorJ. I. SZNAJDER, Corresponding Author J. I. SZNAJDER Pulmonary and Critical Care Medicine Division Michael Reese Hospital and University of Illinois Chicago, Illinois 60680; Winthrop Hospital New York, New York 11501; Washington University St. Louis, Missouri 63110; Rush Presbyterian-St. Luke's Medical Center and Northeastern Illinois University Chicago, Illinois 60637Address for correspondence: Jacob Iasha Sznajder, MD, Pulmonary and Critical Care Medicine, Michael Reese Hospital, 2929 S. Ellis, RC-216, Chicago, IL 60616 (tel: 312-791-5776; fax: 312-791-2349.Search for more papers by this author K. RIDGE, K. RIDGE Pulmonary and Critical Care Medicine Division Michael Reese Hospital and University of Illinois Chicago, Illinois 60680; Winthrop Hospital New York, New York 11501; Washington University St. Louis, Missouri 63110; Rush Presbyterian-St. Luke's Medical Center and Northeastern Illinois University Chicago, Illinois 60637Search for more papers by this authorW. OLIVERA, W. OLIVERA Pulmonary and Critical Care Medicine Division Michael Reese Hospital and University of Illinois Chicago, Illinois 60680; Winthrop Hospital New York, New York 11501; Washington University St. Louis, Missouri 63110; Rush Presbyterian-St. Luke's Medical Center and Northeastern Illinois University Chicago, Illinois 60637Search for more papers by this authorD. H. RUTSCHMAN, D. H. RUTSCHMAN Pulmonary and Critical Care Medicine Division Michael Reese Hospital and University of Illinois Chicago, Illinois 60680; Winthrop Hospital New York, New York 11501; Washington University St. Louis, Missouri 63110; Rush Presbyterian-St. Luke's Medical Center and Northeastern Illinois University Chicago, Illinois 60637Search for more papers by this authorR. W. MERCER, R. W. MERCER Pulmonary and Critical Care Medicine Division Michael Reese Hospital and University of Illinois Chicago, Illinois 60680; Winthrop Hospital New York, New York 11501; Washington University St. Louis, Missouri 63110; Rush Presbyterian-St. Luke's Medical Center and Northeastern Illinois University Chicago, Illinois 60637Search for more papers by this authorB. UHAL, B. UHAL Pulmonary and Critical Care Medicine Division Michael Reese Hospital and University of Illinois Chicago, Illinois 60680; Winthrop Hospital New York, New York 11501; Washington University St. Louis, Missouri 63110; Rush Presbyterian-St. Luke's Medical Center and Northeastern Illinois University Chicago, Illinois 60637Search for more papers by this authorS. HOROWITZ, S. HOROWITZ Pulmonary and Critical Care Medicine Division Michael Reese Hospital and University of Illinois Chicago, Illinois 60680; Winthrop Hospital New York, New York 11501; Washington University St. Louis, Missouri 63110; Rush Presbyterian-St. Luke's Medical Center and Northeastern Illinois University Chicago, Illinois 60637Search for more papers by this authorF. HUGHES, F. HUGHES Pulmonary and Critical Care Medicine Division Michael Reese Hospital and University of Illinois Chicago, Illinois 60680; Winthrop Hospital New York, New York 11501; Washington University St. Louis, Missouri 63110; Rush Presbyterian-St. Luke's Medical Center and Northeastern Illinois University Chicago, Illinois 60637Search for more papers by this authorP. FACTOR, P. FACTOR Pulmonary and Critical Care Medicine Division Michael Reese Hospital and University of Illinois Chicago, Illinois 60680; Winthrop Hospital New York, New York 11501; Washington University St. Louis, Missouri 63110; Rush Presbyterian-St. Luke's Medical Center and Northeastern Illinois University Chicago, Illinois 60637Search for more papers by this authorM. L. BARNARD, M. L. BARNARD Pulmonary and Critical Care Medicine Division Michael Reese Hospital and University of Illinois Chicago, Illinois 60680; Winthrop Hospital New York, New York 11501; Washington University St. Louis, Missouri 63110; Rush Presbyterian-St. Luke's Medical Center and Northeastern Illinois University Chicago, Illinois 60637Search for more papers by this authorJ. I. SZNAJDER, Corresponding Author J. I. SZNAJDER Pulmonary and Critical Care Medicine Division Michael Reese Hospital and University of Illinois Chicago, Illinois 60680; Winthrop Hospital New York, New York 11501; Washington University St. Louis, Missouri 63110; Rush Presbyterian-St. Luke's Medical Center and Northeastern Illinois University Chicago, Illinois 60637Address for correspondence: Jacob Iasha Sznajder, MD, Pulmonary and Critical Care Medicine, Michael Reese Hospital, 2929 S. Ellis, RC-216, Chicago, IL 60616 (tel: 312-791-5776; fax: 312-791-2349.Search for more papers by this author First published: 17 December 2006 https://doi.org/10.1111/j.1749-6632.1997.tb52340.xCitations: 7 a This work was supported by HL-48129, the American Lung Association, and Michael Reese Hospital. Read the full textAboutPDF ToolsRequest permissionExport citationAdd to favoritesTrack citation ShareShare Give accessShare full text accessShare full-text accessPlease review our Terms and Conditions of Use and check box below to share full-text version of article.I have read and accept the Wiley Online Library Terms and Conditions of UseShareable LinkUse the link below to share a full-text version of this article with your friends and colleagues. Learn more.Copy URL Share a linkShare onEmailFacebookTwitterLinkedInRedditWechat No abstract is available for this article. REFERENCES 1 Olivera, W., K. Ridge, L. D. H. Wood & J. I. Sznajder. 1994. Active sodium transport and alveolar epithelial Na,K-ATPase increase during subacute hyperoxia in rats. Am. J. Physiol. 266: L577–L584. 2 Matthay, M. A. & J. P. Wiener-Kronish. 1990. Intact epithelial barrier function is critical for the resolution of alveolar edema in humans. Am. Rev. Respir. Dis. 142: 1250–1257. 3 Sznajder, J. I., W. G. Olivera, K. M. Ridge & D. H. Rutschman. 1995. Mechanisms of lung liquid clearance during hyperoxia in isolated rat lungs. Am. J. Respir. Crit. Med. 151: 1519–1525. 4 Schneeberger, E. E. & K. M. McCarthy. 1986. Cytochemical localization of Na+-K+-ATPase in rat type II pneumocytes. J. Appl. Physiol. 60: 1584–1589. 5 Suzuki, S., D. Zuege & Y. Berthiaume. 1995. Sodium-independent modulation of Na,K-ATPase activity by β-adrenergic agonist in alveolar type II cells. Am. J. Physiol. 268: L983–L990. 6 Shyjan, A. W. & R. Levenson. 1989. Antisera specific for the α1, α2, α3, and β subunits in rat tissue membranes. Biochemistry 28: 4531–4535. 7 Orlowski, J. & J. B. Lingrel. 1988. Tissue specific and developmental regulation of rat Na,K-ATPase catalytic α and β subunit mRNAs. J. Biol. Chem. 263: 10436–10442. 8 Adamson, I. Y. R. & D. H. Bowden. 1974. The type 2 cell as progenitor of alveolar epithelial regeneration. Lab. Invest. 30: 35–42. Citing Literature Volume834, Issue1Na/K‐ATPase and Related Transport ATPases: Structure, Mechanism, and RegulationNovember 1997Pages 651-652 ReferencesRelatedInformation

Oxidative insults that are lethal to epithelial cells kill either via apoptosis or necrosis. Nuclear factor-kappa B (NF-kappa B) is a redox-sensitive transcription factor that is activated by oxidative insult, and NF-kappa B activation can protect cells from apoptosis. To test if NF-kappa B can protect from necrotic cell death caused by high levels of molecular O-2 (hyperoxia), we exposed human alveolar epithelial (A549) cells to hyperoxia. NF-kappa B 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-kappa B protein and mRNA increased as well. In hyperoxia, NF-kappa B regulation was independent of mitogen-activated protein kinase (MAPK). In sharp contrast, there was neither nuclear translocation of NF-kappa B 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-kappa B in hyperoxia, this oxidant remained lethal to the cells. These observations confirm the notion that apoptosis occurs in the absence of NF-kappa B activation but indicate that protection from cell death by NF-kappa B is probably limited to apoptosis.

Oxygen therapy is an essential component of treatment for newborns with respiratory distress, but hyperoxia (>21% O2) is toxic to all cells and causes lung injury. Mechanisms of cytotoxicity from, or tolerance to hyperoxia are not completely understood. A stable mutant line of HeLa cells(called HeLa-80 cells) was selected to grow in 80% O2, yet these cells show no increases in any known antioxidants. Giant 2-D gel electrophoresis was used to compare the patterns of protein synthesis between HeLa-80 cells and the parental cell line (HeLa-20). Out of about 10,00 spots analyzed there were only 15 consistent differences. If any of the proteins are regulated at the mRNA level, it should facilitate the cloning of cDNAs that encode them. Toward that end, we have used the differential display polymerase chain reaction to isolate and identify partial cDNA fragments corresponding to mRNAs that are differentially expressed in HeLa-20 and -80 cells. To date, we have screened approximately 50% of the mRNA complexity of the cells. A total of 68 candidate fragments have been delineated. Northern blots of HeLa-20 and -80 cell mRNA were probed with radiolabeled fragments from 14 of the candidates; 6 of them correspond to differentially-expressed mRNAs. To date, complete DNA sequence analysis of two of these fragments indicates they are derived from novel genes. These genes are expressed at different levels in a wide variety of human tissues. Expression of their full-length cDNAs will be used to determine their potential role in resistance or sensitivity to O2 toxicity.(Funded by grants from the American Heart Association and Winthrop-University Hospital).

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.