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The Clara cell secretory protein (CCSP) imparts a protective effect to the lung during oxidant injury. However, exposure to supplemental oxygen, a common therapeutic modality for lung disease, represses the expression of CCSP in the adult mouse lung. We investigated the mechanisms of hyperoxia-induced repression of the mouse CCSP promoter. Deletion experiments in vivo and in vitro indicated that the hyperoxia-responsive elements are localized to the proximal -166 bp of the CCSP promoter. Electrophoretic mobility shift and supershift analyses demonstrated increased binding of c-Jun at the activator protein-1 site, increased binding of CCAAT/enhancer binding protein (C/EBP) beta at the C/EBP sites, and decreased binding at the Nkx2.1 sites. Western analyses revealed that hyperoxia exposure induced an increase in the expression of the C/EBPbeta isoform liver-inhibiting protein (LIP) and an increase in cytoplasmic Nkx2.1. Cotransfection of LIP or c-Jun expression plasmids decreased the transcriptional activity of the proximal -166-bp CCSP promoter. These observations suggest that hyperoxia-induced repression of the CCSP gene is mediated, at least in part, at the level of transcription and that multiple mechanisms mediate this repression. Moreover, these novel observations may provide insights for generation of therapeutic interventions for the amelioration of oxidant-induced lung injury.

Interferon gamma (IFN-gamma), a potent cytokine inducing a wide range of immunologic activities, is increased in the airway secondary to viral infection or during an inflammatory response. This increase in IFN-gamma concentration may alter the expression of specific airway epithelial cell genes that regulate adaptation of airway inflammatory responses. One protein induced by IFN-gamma is Clara cell secretory protein (CCSP), which may contribute to the attenuation of airway inflammation. This study was done to investigate the molecular mechanism by which IFN-gamma stimulates the expression of the CCSP gene in mouse transformed Clara cells and transgenic mice. Deletion mapping and linker-scanning mutations demonstrated that IFN-gamma-induced expression of CCSP was regulated, in part, at the level of transcription. In vitro and in vivo studies verified that the minimal IFN-gamma-responsive segment was localized to the proximal 166 bp of the 5'-flanking region. Additionally, IFN-gamma-induced expression of CCSP was mediated indirectly through an interferon regulatory factor-1-mediated increase in hepatocyte nuclear factor-3beta.

Coenzyme A (CoASH) is compartmentalized preferentially in the mitochondria, and CoASH and its mixed disulfide with glutathione (CoASSG) undergo thiol/disulfide exchange reactions with glutathione (GSH) and glutathione disulfide (GSSG) in vitro. We measured CoASH and CoASSG in freeze-clamped lung tissues from Fischer-344 and Sprague-Dawley rats maintained in room air or exposed to >95% O(2) for 48 h to test the hypothesis that oxidant stresses on lung thiol status would be observed in the CoASH/CoASSG redox couple, suggesting oxidant stress responses in the mitochondria. Lung tissue concentrations of CoASSG in the Fischer-344 rats declined from 0.89 +/- 0.15 to 0.51 +/- 0.13 nmol/g of lung after 48 h of hyperoxia. CoASH levels declined from 6.40 +/- 0.84 to 3.0 +/- 0.65 nmol/g of lung, and acetyl CoA levels also were lower in the lungs of animals exposed to hyperoxia. CoASH/CoASSG ratios were lower in animals exposed to hyperoxia, satisfying our previously defined criteria for an oxidant stress on this thiol/disulfide redox couple, but absolute CoASSG levels were not increased, as would be expected for oxidant stresses driven simply by increases in reactive oxygen species or other oxidants. Pulmonary edema was observed in the hyperoxic rats and accounted for some of the declines in CoASH concentrations, but CoASH contents per total lung also declined. Lung mitochondrial succinate dehydrogenase activities were not diminished in rats exposed to hyperoxia, indicating that the decreases in CoASH concentrations are not attributable to general destruction of lung mitochondria. Lung GSSG contents were greater in the hyperoxia animals, but GSH/GSSG ratios, which are dominated by extramitochondrial pools, did not decrease in these animals. The mechanisms responsible for, and the possible pathophysiologic consequences of, the decreases in lung CoASH concentrations are not evident from the data available at the present time, but the loss of more than half the tissue contents of CoASH is likely to generate additional metabolic effects that could have significant pathophysiologic consequences.

Bronchopulmonary dysplasia (BPD) is a chronic lung disease that affects prematurely born infants and appears to evolve in part from early inflammatory responses in the lung. The inflammatory responses have been associated with protein and lipid oxidation in tracheal aspirate fluids. The present study was designed to test the hypothesis that in the first week of life specific oxidations and/ or altered expressions of proteins would be observed in tracheal aspirate fluids in infants who would subsequently develop BPD. We obtained tracheal aspirate fluids on Days of life 1, 3, and 6 from infants born at < or = 29 wk gestation, incubated the fluids with 2,4-dinitrophenylhyrazine (DNPH), separated the proteins electrophoretically, and assessed DNPH reactivity by immunonblots. DNPH reactivity of a protein that was identified as Clara cell secretory protein (CCSP) was observed more consistently in tracheal aspirate fluids from infants who later developed BPD than from infants who did not develop BPD. Tracheal aspirate fluid levels of immunoreactive CCSP were also lower on Day of life 1 in infants who developed BPD than in those who did not develop BPD. Increased CCSP oxidation and decreased immunoreactive CCSP expression in infants who subsequently developed BPD suggest that Clara cell function and CCSP expression may be critical for normal bronchoalveolar fluid homeostasis and that maintaining CCSP expression and function may be useful goals for targeted therapies for inhibition of the development of BPD.

Objective and Design: To test the hypothesis that glucocorticoid administration would diminish the lung expression of P-selectin mRNA in hyperoxia-exposed rats., Animals: Adult male Sprague-Dawley rats were divided into 6 separate groups containing 10 to 13 animals per group., Treatment: Rats were dosed with 1 mg/kg of dexamethasone or vehicle only, ip. Immediately after dosing, animals were placed in > 95 % oxygen. Some animals were maintained in room air and are presented as 0 h of exposure to hyperoxia. Another group of animals was dosed with 10 mg/kg lipopolysaccharide (LPS) ip immediately after dosing with either dexamethasone or vehicle as above., Methods: At 24 or 48 h, lung samples were obtained, and lung weight to body weight ratios calculated. In the LPS studies, samples were obtained 4 h after LPS dosing. In a subset of animals, lung sections were hybridized for P-selectin mRNA. All data except for hybridizations were analyzed with three-way ANOVA, with subsequent post-hoc testing. P-selectin hybridizations were quantified by counting the number of positive vessels per high-powered field, and subsequently analyzed by unpaired Student's t-test. Immunohistochemical analyses for P-selectin expression were also performed to determine whether changes in P-selectin mRNA were associated with differences in protein expression. All data are expressed as means +/- SEM., Results: Rats dosed with dexamethasone had higher lung/body weight ratios after 24 and 48 h of exposure to hyperoxia than did similarly exposed rats dosed only with vehicle (at 48 h, 0.87 +/- 0.04 versus 0.65 +/- 0.06, respectively, P < 0.05). The higher ratios in hyperoxic animals dosed with dexamethasone were associated with much higher levels of lung expression for P-selectin mRNA than was observed in similarly exposed rats dosed with vehicle alone (at 48 h, 3.93 +/- 1.02, versus 0.20 +/- 0.06, respectively, P < 0.01). In contrast dexamethasone dosing lead to lower lung P-selectin mRNA expression in animals exposed to LPS (1.23 +/- 1.08 in dexamethasone dosed animals versus 6.80 +/- 0.92 in vehicle only dosed animals). Consistent with the mRNA data, P-selectin immunoreactivity increased as a function of hyperoxia-exposure time in animals dosed with dexamethasone, while immunoreactivity decreased as a function of hyperoxia-exposure time in animals dosed with vehicle only., Conclusions: Increased P-selectin mRNA combined with increased P-selectin protein expression in animals exposed to hyperoxia and dosed with dexamethasone suggests that enhanced expression of P-selectin may contribute to the greater lung injury and inflammation caused by hyperoxia in rats treated with dexamethasone.

Bronchopulmonary dysplasia (BPD) is recognized as an important cause of morbidity and mortality in preterm infants. Because the role of congenital infections in BPD has been debated, the purpose of this study was to test the hypothesis that detection of infectious agents in tracheal aspirate samples was associated with the development of BPD. Tracheal aspirate samples were obtained within the 1st week of life and screened by polymerase chain reaction for adenovirus, cytomegalovirus, parvovirus, enteroviruses, Ureaplasma urealyticum, Mycoplasma hominis, Mycoplasma pneumoniae, and Chlamydia species. BPD was defined as persistent oxygen dependence at 28 d of age and 36 wk postconceptional age (PCA). Infants that expired before these time points were excluded from statistical analysis. Out of 89 infants studied, at 28 d of life, 13 had expired, 45 had BPD, and 31 had no BPD (controls). At 36 wk PCA, 15 infants expired, 39 still had BPD, and 35 did not. A significant increase in the frequency of adenovirus genome was identified in BPD patients compared with controls, both at 28 d of life (12/45 = 27% versus 1/31 = 3%: p< or =0.01) and at 36 wk PCA (10/39 = 29% versus 2/35 = 6%: p = 0.01). Other microorganisms were rarely detected and not associated with the development of BPD. This is the first study reporting the frequency of detection of adenovirus DNA in tracheal aspirate samples obtained during the 1st week of life from infants with BPD and suggests that prenatal acquisition may be important in the development of BPD.

Objective: To test the hypothesis that in infants born at

Infants and adults on oxygen often are treated with glucocorticoids in an attempt to reduce lung inflammatory injury. However, glucocorticoids hasten the development of hyperoxic lung injury in some animal models. The purpose of this study was to test the hypothesis that dexamethasone alters the lung inflammatory responses to hyperoxia exposure. We studied male Sprague-Dawley rats, placing them in >95% oxygen immediately after administration of 0, 0.1, 1, or 10 mg/kg of dexamethasone. At 0, 24, or 48 hr of exposure to hyperoxia, extravascular lung water contents were measured, and lung inflammatory responses were assessed by lung myeloperoxidase activities, lung neutrophil counts, and lung expression of E-Selectin and intercellular adhesions molecule-1 (ICAM-1). Dexamethasone, independent of exposure to hyperoxia, led to marked increases in lung neutrophil counts, without increases in lung myeloperoxidase activities or increases in the expression of the adhesion molecules. Hyperoxia exposure also enhanced lung neutrophil accumulation, and extravascular lung water increased earlier in animals exposed to hyperoxia and dexamethasone than in those exposed to hyperoxia alone. In conclusion, the increase in lung neutrophils in dexamethasone-treated rats without enhanced expression of E-Selectin or intracellular adhesions molecule-1 suggests that dexamethasone leads to lung neutrophil accumulation by its effect on neutrophils. The more rapid development of hyperoxic lung injury associated with earlier lung neutrophil accumulation suggests that dexamethasone-induced lung neutrophil sequestration primes the lung for the development of hyperoxic lung injury and supports further the conclusion that lung inflammation contributes significantly to the development of hyperoxic lung injury.

The pulmonary damage caused by prolonged exposure to high oxygen concentrations is accompanied by lung inflammation, which may contribute to the expression of hyperoxic lung injury. In turn, adhesion molecules are crucial for initiating inflammatory responses. The goal of the present study was to investigate the association of contents of soluble adhesion molecules in plasma or alveolar fluids of hyperoxic rats with lung expression of adhesion molecules, lung inflammation and lung injury. We exposed adult Sprague-Dawley rats to > 95% oxygen for up to 60 h and measured the contents of intercellular adhesion molecule-I (ICAM-I) and E-Selectin in plasma and lung tissue expression of the same molecules, and we assessed lung myeloperoxidase (MPO) activties and lung water contents as indices of lung inflammation and injury, respectively. We also assessed ICAM-I content in lavage samples, because ICAM-I may be shed from the alveolar epithelium. Lung water was elevated at 60 h of hyperoxia-exposure, and this effect was preceded by increases in lung MPO activities. Lung ICAM-I expression was more than doubled at 48 h, although soluble ICAM-I contents were not elevated in plasma or lavage. Soluble E-Selectin was increased by more than 50% at 24 h of hyperoxia-exposure, while lung expressions of E-Selectin were not increased until 48 h. The sequence of the events observed in the present studies suggests that E-Selectin contributes to lung inflammation in hyperoxia and the acceleration of lung injury immediately following the inflammatory response suggests a pivotal role for inflammation in this injury.