Natriuretic peptide C receptor in the developing sheep lung: role in perinatal transition.

Background: At birth, the release of surfactant from alveolar type II cells (ATIIs) is stimulated by increased activity of the beta-adrenergic/adenylyl cyclase/cyclic 3'-5' adenosine monophosphate-signaling cascade. Atrial natriuretic peptide (ANP) stimulates surfactant secretion through natriuretic peptide receptor A (NPR-A). ANP inhibits adenylyl cyclase activity through its binding to NPR-C. We wished to further understand the role of the NPR-C in perinatal transition. Methods: We studied ATII expression of NPR-C in fetal and newborn sheep using immunohistochemistry, and surfactant secretion in isolated ATIIs by measuring 3[H] choline release into the media. Results: ANP induced surfactant secretion, and, at higher doses, it inhibits the stimulatory effect of the secretagogue terbutaline. ATII NPR-C expression decreased significantly after birth. Premature delivery also markedly decreased ANP and NPR-C in ATIIs. Co-incubation of terbutaline (10-4 M) with ANP (10-6 M) significantly decreased 3[H] choline release from isolated newborn ATII cells when compared with terbutaline alone; this inhibitory effect was mimicked by the specific NPR-C agonist, C-ANP (10-10 M). Conclusion: ANP may act as an important epithelial-derived inhibitor of surfactant release in the fetal lung, and downregulation of ANP and NPR-C following birth may sensitize ATII cells to the effects of circulating catecholamines, thus facilitating surfactant secretion.

Late administration of antenatal vitamin A promotes pulmonary structural maturation and improves ventilation in the lamb model of congenital diaphragmatic hernia.

PURPOSE: The lungs in congenital diaphragmatic hernia (CDH) are hypoplastic and immature making respiratory support one of the most challenging aspects of caring for these neonates. Vitamin A is essential for normal lung growth and development. It also promotes alveolarization. The aim of this study is to investigate the effects of antenatal vitamin A on lung growth and alveolarization and ventilation in the lamb model of CDH. METHODS: This study was approved by the Animal Care Committee of the State University of New York at Buffalo, and conforms to the National Institute of Health guidelines. Diaphragmatic defects were created at 79-81 days gestation. Group 1 lambs (CDH, n = 5) were untreated. In group 2 (CDH + vitamin A, n = 6) and group 3 lambs (control + vitamin A, n = 3) right jugular venous catheters were inserted at 118-120 days and retinyl palmitate (vitamin A) was administered until 135 days. The control group (n = 5) consisted of twin littermates. Lambs were delivered at 136-139 days and ventilated for 2 h according to a set protocol. The left lungs were harvested and fixed for histology. RESULTS: Lung compliance was significantly higher in CDH + vitamin A (median 0.27, range 0.1-0.48 ml/cmH(2)O/kg) versus CDH lambs (median 0.07, range 0.07-0.18 ml/cmH(2)O/kg), P < 0.05. At 1 h CDH + vitamin A lambs experienced significantly lower PaCO(2) (median 115, range 35-194 mmHg vs. median 192, range 168-234 mmHg) and higher arterial pH (median 7.0, range 6.74-7.35 vs. median 6.73, range 6.5-6.81) than CDH lambs, P < 0.05. The lung weight to body weight ratio of CDH + vitamin A lambs was significantly less than that of CDH lambs (P < 0.05). Histology showed small thick walled air-spaces and no true alveoli in CDH lambs. In contrast, true alveoli and thinning of the inter-alveolar septums were seen in CDH + vitamin A lambs. CONCLUSION: This is the first study to demonstrate an improvement in lung function and structural maturation when antenatal vitamin A is given in a surgical model of CDH.

Postnatal lung development: immediate-early gene responses post ozone and LPS exposure.

Exposure to environmental pollutants may severely affect lung growth and development. The present study was designed to test the hypothesis that lung damage caused either by ozone or lipopolysaccharide (LPS) occurs through distinct early responses, which are age dependent in the postnatal lung. C57Bl/6 mice ages 4, 10, and 56 days were exposed to inhalation of LPS with an estimated deposited dose of 26 EU and examined 0.5, 1, or 4 h post inhalation exposure; or to 1 or 2.5 ppm ozone for 4 h or sequential exposures of LPS followed by ozone. Abundance of c-fos, c-jun, interleukin (IL)-1beta, Toll-like receptor (TLR) 2, TLR 4, and tumor necrosis factor (TNF) alpha message levels were measured by RNase protection assay. Exposure to ozone for 4 h induced a c-fos and c-jun response in 4-; 10-; and 56-day-old mice in a dose-dependent manner, was localized to conducting and terminal airways, and also induced TLR 4 message abundance in 10- and 56-day-old mice. Exposure to LPS induced c-fos and c-jun 30 and 60 min postinhalation in 10- and 56-day-old mice only. TLR 2 and 4 message abundance was increased at 10 and 56 days, but was undetectable at 4 days of age, and correlated with proinflamatory message induction. Exposure to LPS followed by ozone increased message abundance of IL-1beta, TNFalpha, TLR 2, TLR 4, and c-jun/c-fos at 10 and 56 days, suggesting that combined exposures that induce cellular stresses can regulate gene expression by activating signaling pathways that operate through both transcription factors activator protein (AP)-1 and nuclear factor (NF)-kappaB. However, only c-jun/c-fos and TNFalpha were elevated in 4-day-old mice after sequential exposures, suggesting that the early activation of the inflammatory response after sequential exposures may occur through a TLR-independent pathway. These results suggest that sequential exposures induce multiple signaling pathways that are age dependent.

Antenatal vitamin A decreases ventilation-induced lung injury in the lamb model of congenital diaphragmatic hernia.

OBJECTIVE: Infants with congenital diaphragmatic hernia (CDH) are susceptible to ventilation-induced lung injury. Vitamin A may protect the lung from injury during ventilation. The authors investigated the effects of antenatal vitamin A on ventilation-induced lung injury in CDH lambs using lung myeloperoxidase (MPO) activity as an indicator of lung injury. METHODS: Left-sided diaphragmatic defects were created in 10 lambs at 79-81 days' gestation. Six CDH lambs had right jugular venous catheters inserted at 120 days' gestation and were given vitamin A until 135 days' gestation. Four CDH lambs were not treated. Twin littermates (n = 3) served as controls. All lambs were delivered at 136-139 days of gestation and ventilated for 2 hours. Lambs were sacrificed following ventilation and samples of left lung were snap frozen. MPO was extracted from lung tissue and MPO activity was assayed. RESULTS: CDH lambs treated with antenatal vitamin A demonstrated significantly lower MPO activity than untreated CDH lambs (0.0477 +/- 0.0150 vs. 0.1106 +/- 0.0230 units/mg protein, p < 0.05). CONCLUSION: This is the first study to look at the effect of vitamin A on lung injury in CDH. In the lamb model of CDH, antenatal vitamin A decreases ventilation-induced lung injury.

Surface properties of sulfur- and ether-linked phosphonolipids with and without purified hydrophobic lung surfactant proteins.

Two novel C16:0 sulfur-linked phosphonolipids (S-lipid and SO(2)-lipid) and two ether-linked phosphonolipids (C16:0 DEPN-8 and C16:1 UnDEPN-8) were studied for surface behavior alone and in mixtures with purified bovine lung surfactant proteins (SP)-B and/or SP-C. Synthetic C16:0 phosphonolipids all had improved adsorption and film respreading compared to dipalmitoyl phosphatidylcholine, and SO(2)-lipid and DEPN-8 reached maximum surface pressures of 72mN/m (minimum surface tensions of <1mN/m) in compressed films on the Wilhelmy balance (23 degrees C). Dispersions of DEPN-8 (0.5mg/ml) and SO(2)-lipid (2.5mg/ml) also reached minimum surface tensions of <1mN/m on a pulsating bubble surfactometer (37 degrees C, 20cycles/min, 50% area compression). Synthetic lung surfactants containing DEPN-8 or SO(2)-lipid+0.75% SP-B+0.75% SP-C had dynamic surface activity on the bubble equal to that of calf lung surfactant extract (CLSE). Surfactants containing DEPN-8 or SO(2)-lipid plus 1.5% SP-B also had very high surface activity, but less than when both apoproteins were present together. Adding 10wt.% of UnDEPN-8 to synthetic lung surfactants did not improve dynamic surface activity. Surfactants containing DEPN-8 or SO(2)-lipid plus 0.75% SP-B/0.75% SP-C were chemically and biophysically resistant to phospholipase A(2) (PLA(2)), while CLSE was severely inhibited by PLA(2). The high activity and inhibition resistance of synthetic surfactants containing DEPN-8 or SO(2)-lipid plus SP-B/SP-C are promising for future applications in treating surfactant dysfunction in inflammatory lung injury.

Ontogeny of atrial natriuretic peptide and its receptor in the lung: effects on perinatal surfactant release.

During the transition at birth to air breathing, regulation of surfactant release from alveolar type II (ATII) cells is critical. Atrial natriuretic peptide (ANP) stimulates natriuretic peptide receptor-A (NPR-A) and increases intracellular cGMP. We examined the changes in ANP and NPR-A in respiratory epithelium during the perinatal period using immunohistochemistry and studied the effect of ANP on surfactant release from ATII cells isolated from fetal and newborn lambs. NPR-A mRNA was detected in the fetal lung by Northern Blot and RT-PCR. At 100 d gestation (term 145 d), ANP staining was absent and NPR-A staining was weak in cuboidal epithelial cells. ANP and NPR-A staining was prominent in ATII cells at 136 d gestation and was undetectable postnatally. ANP stimulated (maximal effect at 10(-10)M) surfactant release from both late gestation fetal and neonatal ATII cells. Protein kinase G inhibition significantly blocked this release. We conclude that ANP stimulates surfactant release in isolated perinatal ATII cells by a cGMP-dependent mechanism. ANP and NPR-A expression in ATII cells is greatest in late gestation and declines sharply postnatally. We speculate that increased activity of the ANP/NPR-A pathway in late gestation may prime the surfactant system, preparing the lung for air breathing.

Capsule and O-antigen from an extraintestinal isolate of Escherichia coli modulate cytokine levels in rat macrophages in vitro and in a rat model of pneumonia.

Gram-negative pneumonia results in significant morbidity, mortality, and cost to the healthcare system. Previously the authors demonstrated that capsule and O-antigen, virulence factors of the extraintestinal Escherichia coli isolate CP9, modulate pulmonary neutrophil influx in a rat pneumonia model. In this report, the authors utilized CP9 and mutants deficient in O-antigen (CP921), capsule (CP9.137), or both (CP923) to test the hypothesis that modulation of cytokine levels by capsule and/or O-antigen may be a contributory mechanism. Effects of capsule and O-antigen on cytokine levels in rats in vivo and in isolated pulmonary macrophages in vitro were assessed. In vivo, capsule and O-antigen had no significant effect on tumor necrosis factor (TNF)-alpha levels in bronchoalveolar lavage fluid (BALF), but both were associated with significant increases in the levels of interleukin (IL)-1beta and Cytokine-induced neutrophil Chemoattractant-1 (CINC-1). However, potential difficulties in interpreting data occurred because challenge bacterial strains exhibited differential growth, and clearance characteristics and mixed cell populations were present. Therefore, added mechanistic studies investigated specific interactions of capsule and O-antigen with pulmonary macrophages purified from normal rats and exposed to CP9, CP921, CP9.137, or CP923 in vitro. Results indicated that the presence of capsule led to significantly increased levels of TNF-alpha, IL-1beta, and CINC-1, whereas O-antigen significantly decreased macrophage-associated levels of these mediators. These findings support the hypothesis that CP9 capsule is proinflammatory for macrophage-induced neutrophil recruitment, whereas O-antigen attenuates macrophage production of proinflammatory mediators in pneumonia. These results expand our understanding on the mechanisms by which these virulence traits may contribute to the inflammatory pathogenesis of pneumonia.

Surfactant dysfunction and lung injury due to the E. coli virulence factor hemolysin in a rat pneumonia model.

This study tests the hypothesis that the virulence factor hemolysin (Hly) expressed by extraintestinal pathogenic Escherichia coli contributes to surfactant dysfunction and lung injury in a rat model of gram-negative pneumonia. Rats were instilled intratracheally with CP9 (wild type, Hly-positive), CP9hlyA (Hly-minus), CP9/pEK50 (supraphysiological Hly), or purified LPS. At 6 h postinfection, rats given CP9 had a decreased percentage content of large surfactant aggregates in cell-free bronchoalveolar lavage (BAL), decreased large aggregate surface activity, decreased Pa(O2)/FiO2) ratio, increased BAL albumin/protein levels, and increased histological evidence of lung injury compared with rats given CP9hlyA or LPS. In addition, rats given CP9/pEK50 or CP9 had decreased large aggregate surface activity, decreased Pa(O2)/FiO2) ratios, and increased BAL albumin/protein levels at 2 h postinfection compared with rats given CP9hlyA. The severity of permeability lung injury based on albumin/protein levels in BAL at 2 h was ordered as CP9/pEK50 > CP9 > CP9hlyA > normal saline controls. Total lung titers of bacteria were increased at 6 h in rats given CP9 vs. CP9hlyA, but bacterial titers were not significantly different at 2 h, indicating that increased surfactant dysfunction and lung injury were associated with Hly as opposed to bacterial numbers per se. Further studies in vitro showed that CP9 could directly lyse transformed pulmonary epithelial cells (H441 cells) but that indirect lysis of H441 cells secondary to Hly-induced neutrophil lysis did not occur. Together, these data demonstrate that Hly is an important direct mediator of surfactant dysfunction and lung injury in gram-negative pneumonia.

Acid aspiration-induced lung inflammation and injury are exacerbated in NADPH oxidase-deficient mice.

Increased reactive oxidant intermediates (ROIs) from primed leukocytes have been implicated in the pathogenesis of acid aspiration lung injury. To evaluate the specific role of the phagocyte NADPH oxidase-derived ROIs in acid lung injury, the p47phox-/- knockout mouse model of chronic granulomatous disease was used. p47phox-/- mice developed a significantly greater alveolar neutrophilic leukocytosis compared with wild-type mice at all time points after acid injury, with the difference between genotypes being most marked at 48 h. In contrast, the p47phox-/- mice had a decreased number of macrophages in bronchoalveolar lavage (BAL) compared with wild-type at 48 h after acid or saline aspiration. Albumin concentration in BAL reflecting capillary leak was also greater in p47phox-/- compared with wild-type mice. BAL concentrations of proinflammatory cytokines and chemokines were greater in p47phox-/- compared with wild-type mice. These findings suggest that NADPH oxidase, directly or indirectly, plays a role in attenuating the acute neutrophilic response after acid lung injury. We speculate that this downmodulating effect may be mediated by promoting the transition from production of cytokines and chemokines involved in neutrophilic infiltration to a less injurious, chronic inflammatory response.

C-type natriuretic peptide and its receptor are downregulated in pulmonary epithelium following birth.

C-type natriuretic peptide (CNP) is a member of the natriuretic peptide family and acts through the membrane bound guanylyl cyclase linked natriuretic peptide receptor B (NPR-B) to increase intracellular cGMP. Activation of the CNP/NPR-B pathway in pulmonary epithelium has been linked to the inhibition of amiloride-sensitive sodium absorption and to the stimulation of the cystic fibrosis transmembrane conductance regulator (CFTR). Given the importance of ion movement across the pulmonary epithelium of the fetal and newborn lung, we sought to examine the expression of CNP and NPR-B in pulmonary epithelium of the developing fetal lamb and following the transition to air breathing. Lambs were sacrificed at 100 and 136 days of gestation and at 3 days, and 4 weeks after full term delivery. Lung sections were immunostained for CNP and NPR-B. At 100 days of gestation, staining for CNP and NPR-B was absent within all pulmonary epithelium. At 136 days of gestation, prominent staining for both CNP and NPR-B was seen within alveolar type II cells, non-ciliated cells of the distal airways (Clara cells), and ciliated epithelium of the upper airways. At both 3 days and 4 weeks following birth, staining for CNP and NPR-B was absent in alveolar type II cells, ciliated bronchial epithelium and was markedly reduced in Clara cells. The presence of CNP and NPR-B within the pulmonary epithelium in the nearterm fetal period and its rapid downregulation following birth suggests that CNP may contribute to the maintenance of the fluid-filled lung through the regulation of trans-epithelial ion flux.

Statistical prediction of the type of gastric aspiration lung injury based on early cytokine/chemokine profiles.

BACKGROUND: Unwitnessed gastric aspiration can be a diagnostic dilemma, and early discrimination of different forms may help to identify individuals with increased risk of development of severe clinical acute lung injury or acute respiratory distress syndrome. The authors hypothesized that inflammatory mediator profiles could be used to help diagnose different types of gastric aspiration. METHODS: Diagnostic modeling using a newly modified receiver operator characteristic approach was applied to recently published data from our laboratory on lavaged inflammatory mediators from rodents given intratracheal normal saline, hydrochloric acid, small nonacidified gastric particles, or a combination of acid and small gastric particles. Multiple animal groups and postaspiration times of injury were analyzed to gauge the applicability of the predictive approach: rats (6 and 24 h), C57/BL6 wild-type mice (5 and 24 h), and transgenic mice on the same background deficient in the gene for monocyte chemoattractant protein 1 (MCP-1 [-/-] mice; 5 and 24 h). RESULTS: Overall, the four types of aspiration were correctly discriminated in 85 of 96 rats (89%), 72 of 78 wild-type mice (92%), and 59 of 73 MCP-1 (-/-) mice (81%) by models that used a maximum of only two mediators. The severe "two-hit" aspirate of the combination of acid and small gastric particles was correctly predicted in 21 of 24 rats, 23 of 23 wild-type mice, and 21 of 21 MCP-1 (-/-) mice. Specific best-fit mediators or mediator pairs varied with aspirate type, animal type, and time of injury. Cytokines and chemokines that best predicted the combination of acid and small gastric particles were cytokine-induced neutrophil chemoattractant 1 (6 h) and MCP-1 (24 h) in rats, tumor necrosis factor alpha/macrophage inflammatory protein 2 (5 h) and tumor necrosis factor alpha/MCP-1 (24 h) in wild-type mice, and tumor necrosis factor alpha/macrophage inflammatory protein 2 (5 h) and tumor necrosis factor alpha/keratinocyte-derived cytokine (24 h) in MCP-1 (-/-) mice. CONCLUSIONS: These results support the potential feasibility of developing predictive models that use focused measurements of inflammatory mediators to help diagnose severe clinical forms of unwitnessed gastric aspiration, such as the combination of acid and small gastric particles, that may have a high risk of progression to acute lung injury/acute respiratory distress syndrome.

Sequential exposures to ozone and lipopolysaccharide in postnatal lung enhance or inhibit cytokine responses.

Sequential exposures to inhaled environmental pollutants may result in injuries/responses not predicted by evaluating exposures to an individual toxicant. This may indicate that the lung is damaged or primed by earlier events, so exposure to a nontoxic dose of an environmental pollutant may be sufficient to trigger adverse responses. The present study was designed to test the hypothesis that stimulating lung epithelial damage or inflammatory cell activation followed by a second stimulus leads to responses not seen after individual exposures in the postnatal lung. C57Bl/6 mice ages 4, 10, and 56 days were exposed to either a 10-minute inhalation of lipopolysaccharide (LPS), with an estimated deposited dose of 26 EU, followed immediately by 2.5 PPM ozone for 4 hours, or to 2.5 PPM ozone for 4 hours followed immediately by a 10-minute inhalation of LPS and examined 2 hours post exposure. Abundance of proinflammatory cytokine messages was measured by RNase protection assay. Exposure to LPS followed by ozone induced an inflammatory response in 4-day-old mice, which was not detected after LPS or ozone exposure alone. This exposure sequence also generated a synergistic increase in interleukin (IL)-6 mRNA abundance in 10- and 56-day-old mice but not in 4-day-old mice. Exposure to ozone followed by LPS inhibited IL-1alpha and IL-1beta responses in 4-, 10-, and 56-day-old mice; furthermore, this inhibitory effect was observed after 1.0 and 0.5 PPM ozone exposures. These results demonstrate that preexposure to LPS, which primarily activates inflammatory cell recruitment, can cause sensitization to a secondary stimulus. However, preexposure to ozone, which primarily damages the epithelium, inhibited proinflammatory responses. Thus it was concluded that sequential exposures to ozone and LPS resulted in responses not predicted by evaluating individual exposures during postnatal lung development.

Serine antiproteinase administration preserves innate superoxide dismutase levels after acid aspiration and hyperoxia but does not decrease lung injury.

Acute lung injury after acid aspiration and increased ambient oxygen result in significant oxidative damage to the lungs. Lung antioxidant levels are also reduced. Because levels of serine proteinases in the airspaces are also dramatically increased, we hypothesized that these enzymes play a role in degrading lung antioxidants. Rats were treated with a serine proteinase inhibitor, aprotinin, before pulmonary aspiration of acid in the presence of increased ambient oxygen (hyperoxia). Lung Cu/Zn and Mn superoxide dismutase (SOD) activity (by colorimetric assay) and Cu/Zn SOD immune reactive protein (enzyme-linked immunosorbent assay) were assayed. The effects of antiproteinase treatment on acute lung injury were also assessed. Total SOD, Cu/Zn SOD, and Cu/Zn SOD antigenic protein levels were decreased in animals after acid aspiration and hyperoxia. However, Mn SOD activity was unchanged. The decrease in Cu/Zn SOD was attenuated in animals, where serine proteinase activity was inhibited. However, antiproteinase treatment did not decrease acute pulmonary injury, as assessed by leakage of radiolabeled albumin into the lung (permeability index), arterial blood gases, and markers of acute inflammation (pulmonary myeloperoxidase activity, a surrogate neutrophilic marker, and inflammatory cytokine profiles). We conclude that production of serine proteinases play a major role in degrading Cu/Zn SOD, thereby decreasing pulmonary antioxidant capacity. However, the role this plays in the pathogenesis of the acute lung injury is not clear.

Surfactant alterations in acute inflammatory lung injury from aspiration of acid and gastric particulates.

This study examines surfactant dysfunction in rats with inflammatory lung injury from intratracheal instillation of hydrochloric acid (ACID, pH 1.25), small nonacidified gastric particles (SNAP), or combined acid and small gastric particles (CASP). Rats given CASP had the most severe lung injury at 6, 24, and 48 h based on decreases in arterial oxygenation and increases in erythrocytes, total leukocytes, neutrophils, total protein, and albumin in bronchoalveolar lavage (BAL). The content of large surfactant aggregates in BAL was reduced in all forms of aspiration injury, but decreases were greatest in rats given CASP. Large aggregates from aspiration-injured rats also had decreased levels of phosphatidylcholine (PC) and increased levels of lyso-PC and total protein compared with saline controls (abnormalities for CASP were greater than for SNAP or ACID alone). The surface tension-lowering ability of large surfactant aggregates on a bubble surfactometer was impaired in rats with aspiration injury at 6, 24, and 48 h, with the largest activity reductions found in animals given CASP. There were strong statistical correlations between surfactant dysfunction (increased minimum surface tension and reduced large aggregate content) and the severity of lung injury based on arterial oxygenation and levels of albumin, protein, and erythrocytes in BAL (P < 0.0001). Surfactant dysfunction also correlated strongly with reduced lung volumes during inflation and deflation (P = 0.0004-0.005). These results indicate that surfactant abnormalities are functionally important in gastric aspiration lung injury and contribute significantly to the increased severity of injury found in CASP compared with ACID or SNAP alone.

E. coli virulence factor hemolysin induces neutrophil apoptosis and necrosis/lysis in vitro and necrosis/lysis and lung injury in a rat pneumonia model.

Enteric gram-negative bacilli, such as Escherichia coli are the most common cause of nosocomial pneumonia. In this study a wild-type extraintestinal pathogenic strain of E. coli (ExPEC)(CP9) and isogenic derivatives deficient in hemolysin (Hly) and cytotoxic necrotizing factor (CNF) were assessed in vitro and in a rat model of gram-negative pneumonia to test the hypothesis that these virulence factors induce neutrophil apoptosis and/or necrosis/lysis. As ascertained by in vitro caspase-3/7 and LDH activities and neutrophil morphology, Hly mediated neutrophil apoptosis at lower E. coli titers (1 x 10(5-6) cfu) and necrosis/lysis at higher titers (> or =1 x 10(7) cfu). Data suggest that CNF promotes apoptosis but not necrosis or lysis. We also demonstrate that annexin V/7-amino-actinomycin D staining was an unreliable assessment of apoptosis using live E. coli. The use of caspase-3/7 and LDH activities and neutrophil morphology supported the notion that necrosis, not apoptosis, was the primary mechanism by which neutrophils were affected in our in vivo gram-negative pneumonia model using live E. coli. In addition, in vivo studies demonstrated that Hly mediates lung injury. Neutrophil necrosis was not observed when animals were challenged with purified lipopolysaccharide, demonstrating the importance of using live bacteria. These findings establish that Hly contributes to ExPEC virulence by mediating neutrophil toxicity, with necrosis/lysis being the dominant effect of Hly on neutrophils in vivo and by lung injury. Whether Hly-mediated lung injury is due to neutrophil necrosis, a direct effect of Hly, or both is unclear.

Acid and particulate-induced aspiration lung injury in mice: importance of MCP-1.

A model of aspiration lung injury was developed in WT C57BL/6 mice to exploit genetically modified animals on this background, i.e., MCP-1(-/-) mice. Mice were given intratracheal hydrochloric acid (ACID, pH 1.25), small nonacidified gastric particles (SNAP), or combined acid plus small gastric particles (CASP). As reported previously in rats, lung injury in WT mice was most severe for "two-hit" aspiration from CASP (40 mg/ml particulates) based on the levels of albumin, leukocytes, TNF-alpha, IL-1beta, IL-6, MCP-1, KC, and MIP-2 in bronchoalveolar lavage (BAL) at 5, 24, and 48 h. MCP-1(-/-) mice given 40 mg/ml CASP had significantly decreased survival compared with WT mice (32% vs. 80% survival at 24 h and 0% vs. 72% survival at 48 h). MCP-1(-/-) mice also had decreased survival compared with WT mice for CASP aspirates containing reduced particulate doses of 10-20 mg/ml. MCP-1(-/-) mice given 5 mg/ml CASP had survival similar to WT mice given 40 mg/ml CASP. MCP-1(-/-) mice also had differing responses from WT mice for several inflammatory mediators in BAL (KC or IL-6 depending on the particle dose of CASP and time of injury). Histopathology of WT mice with CASP (40 mg particles/ml) showed microscopic areas of compartmentalization with prominent granuloma formation by 24 h, whereas lung tissue from MCP-1(-/-) mice had severe diffuse pneumonia without granulomas. These results indicate that MCP-1 is important for survival in murine aspiration pneumonitis and appears to act partly to protect uninjured lung regions by promoting isolation and compartmentalization of tissue with active inflammation.

Differential proinflammatory cytokine responses of the lung to ozone and lipopolysaccharide exposure during postnatal development.

Age appears to be a critical variable in the ability of the lung to cope with external stress. Alterations in cellular responses associated with environmental toxicants are likely to modify the developmental processes. This would suggest that the timing and interaction between exposure and developmental events appears to play an important role as susceptible targets for environmental perturbation. C57BL/6 mice ages 2, 4, 7, 10, 14, 28, and 56 days were exposed to 2.5 PPM ozone for 4 hours or to a 10-minute inhalation of lipopolysaccharide (LPS) with an estimated deposited dose of 26 EU and examined 2 hours post exposure. Abundance of proinflammatory cytokine and chemokine mRNA were measured by RNase protection assay. After ozone exposure interleukin (IL)-6 was not detected in 2-, 4-, and 7-day-old mice; however, increases of 18- to 20-fold were measured in 10-, 14-, 28-, and 56-day-old mice. Macrophage inhibitory protein (MIP)-2 and cytokine-induced neutrophil chenocettractant (KC) were elevated slightly, with no differences between 2- and 56-day-old mice. After LPS exposure, IL-6 was not detected in 2- and 4-day-old mice; however, 8- to 10-fold increases were measured in 7-, 14-, and 28-day-old mice and approximately 20-fold in 56-day-old mice. IL-1beta was elevated approximately 4-fold at 2 and 4 days of age but was elevated 25- to 30-fold in 7-, 14-, 28-, and 56-day-old mice. MIP-2 and KC mRNA abundance was elevated 25- to 30-fold, with no differences between 2- and 56-day-old mice. These results demonstrate that critical time points exist during lung development to inhaled environmental pollutants and that differences exist in the maturation of inflammatory and epithelial defense mechanisms.

Bulk shear viscosities of endogenous and exogenous lung surfactants.

Bulk shear viscosities were measured with a cone and plate microviscometer as a function of concentration, shear rate, and temperature for lavaged calf lung surfactant (LS), Exosurf, Infasurf, Survanta, and synthetic lipid mixtures dispersed in normal saline. Viscosity increased with phospholipid concentration for all surfactants, but its magnitude and shear dependence varied widely among the different preparations. Saline dispersions of Exosurf and synthetic phospholipids had low viscosities of only a few centipoise (cp) and exhibited minimal shear dependence. LS, Infasurf, Survanta, and lipid mixtures containing palmitic acid and tripalmitin had larger non-Newtonian viscosities that increased as shear rate decreased. At 35 mg of phospholipid/ml and 37 degrees C, viscosity values were 52.3 cp (Survanta), 31.1 cp (LS), and 25 cp (Infasurf) at a shear rate of 77 s(-1) and 16.9 cp (Survanta), 10.1 cp (LS), and 6.6 cp (Infasurf) at 770 s(-1). At 25 mg of phospholipid/ml and 37 degrees C, viscosity values at 77 s(-1) were 28.8 cp (Survanta), 4.7 cp (LS), and 12.5 cp (Infasurf). At fixed shear rate, viscosity was substantially decreased at 23 degrees C compared with 37 degrees C for LS and Infasurf but was increased for Survanta. Calcium (5 mM) greatly reduced the viscosity of both Survanta and Infasurf at 37 degrees C. Studies on synthetic mixtures indicated that phospholipid/apoprotein interactions were important in the rheology of lung-derived surfactants and that palmitic acid and tripalmitin contributed to the increased viscosity of Survanta. The viscous behavior of clinical exogenous surfactants potentially influences their delivery and distribution in lungs and varies significantly with composition, concentration, temperature, ionic environment, and physical formulation.

Halothane potentiation of hydrogen peroxide-induced inhibition of surfactant synthesis: the role of type II cell energy status.

UNLABELLED: Small concentrations of inhaled anesthetics can affect Type II cell surfactant production and exacerbate oxidant-mediated lung injury. We hypothesized that inhaled anesthetics augment oxidant-induced Type II pneumocyte dysfunction related to their different effects on cellular adenosine triphosphate (ATP) status. Freshly isolated Type II cells were exposed to different concentrations of hydrogen peroxide (H2O2) in the presence or absence of an in vitro halothane exposure. Cells exposed to 100 microM H2O2 alone demonstrated a 23% decrease in ATP levels and a 32% decrease in phosphatidylcholine (PC) synthesis compared with controls. Halothane alone decreased PC synthesis by only 12% and reduced ATP levels by 20%. However, when exposed to both halothane and H2O2 together, ATP levels decreased by 40%, and PC synthesis rates decreased by 51%. Pretreatment of cells with nicotinamide, an inhibitor of poly adenosine diphosphate ribose polymerase, completely prevented the ATP loss and PC synthesis decline caused by H2O2 alone, but it had no effect on the halothane-augmented portion of the cell injury. These data suggest that the ability of halothane to enhance oxidative damage may be related to its own specific effects on cell energetics that may not be amenable to the same treatments used to mitigate other cellular mechanisms of oxidative stress. IMPLICATIONS: A mediator of inflammation (hydrogen peroxide) and an inhaled anesthetic (halothane) interact to decrease cell energy and secretion of a substance (surfactant) required for healthy lung function from cells that line gas-exchange compartments. This interaction represents a possible mechanism by which inflammatory lung disease may become more severe intraoperatively.

Parathyroid hormone-related protein response to hyperoxic lung injury.

Parathyroid hormone-related protein (PTHrP) is a growth inhibitor for alveolar type II cells. Type II cell proliferation after lung injury from 85% oxygen is regulated, in part, by a fall in lung PTHrP. In this study, we investigated lung PTHrP after injury induced by >95% oxygen in rats and rabbits. In adult rats, lung PTHrP rose 10-fold over controls to 6,356 +/- 710 pg/ml (mean +/- SE) at 48 h of hyperoxia. Levels fell to 299 +/- 78 pg/ml, and staining for PTHrP mRNA was greatly reduced at 60 h (P < 0.05), the point of most severe injury and greatest pneumocyte proliferation. In adult rabbits, lung PTHrP peaked at 3,289 +/- 230 pg/ml after 64 h of hyperoxia with 24 h of normoxic recovery and then dropped to 1,629 +/- 153 pg/ml at 48 h of recovery (P < 0.05). Type II cell proliferation peaked shortly after the fall in PTHrP. In newborn rabbits, lavage PTHrP increased by 50% during the first 8 days of hyperoxia, whereas type II cell growth decreased. PTHrP declined at the LD(50), concurrent with increased type II cell division. In summary, lung PTHrP initially rises after injury with >95% hyperoxia and then falls near the peak of injury. Changes in PTHrP are temporally related to type II cell proliferation and may regulate repair of lung injury.