DNA microarray analysis of gene expression in alveolar epithelial cells in response to TNFalpha, LPS, and cyclic stretch.

Recent evidence suggests that alveolar epithelial cells (AECs) may contribute to the development, propagation, and resolution of acute lung injury (ALI)/acute respiratory distress syndrome (ARDS). Proinflammatory cytokines, pathogen products, and injurious mechanical ventilation are important contributors of excessive inflammatory responses in the lung. In the present study, we used cDNA microarrays to define the gene expression patterns of A549 cells (an AEC line) in the early stages of three models of pulmonary parenchymal cell activation: cells treated with tumor necrosis factor-alpha (TNFalpha) (20 ng/ml), lipopolysaccharide (LPS, 1 microg/ml), or cyclic stretch (20% elongation) for either 1 h or 4 h. Differential gene expression profiles were determined by gene array analysis. TNFalpha induced an inflammatory response pattern, including induction of genes for chemokines, inflammatory mediators, and cell surface membrane proteins. TNFalpha also increased genes related to pro- and anti-apoptotic proteins, signal transduction proteins, and transcriptional factors. TNFalpha further induced a group of genes that may form a negative feedback loop to silence the NFkappaB pathway. Stimulation of AECs with mechanical stretch changed cell morphology and activated Src protein tyrosine kinase. The combination of TNFalpha plus stretch enhanced or attenuated expression of multiple genes. LPS decreased microfilament polymerization but had less impact on NFkappaB translocation and gene expression. Results from this study indicate that AECs can tailor their response to different stimuli or/and combination of stimuli and subsequently play an important role in acute inflammatory responses in the lung.

Direct and indirect bacterial killing functions of neutrophil defensins in lung explants.

Studies of the antimicrobial activity of neutrophil defensins have mostly been carried out in microbiological media, and their effects on the host defense in physiological conditions are unclear. We examined 1) the antibacterial activity of defensins in physiological media with and without lung tissue present, 2) the effect of defensins on hydrogen peroxide (H(2)O(2)) production by lung tissue that had been exposed to bacteria, and 3) the effect of diphenyleneiodonium (DPI), an inhibitor of reactive oxygen species formation, on the antibacterial activity of defensins in the presence of lung tissue. Defensins were incubated with Escherichia coli or Pseudomonas aeruginosa in the absence or presence of primary cultured mouse lung explants. Defensins reduced bacterial counts by approximately 65-fold and approximately 25-fold, respectively, at 48 h; bacterial counts were further decreased by approximately 600-fold and approximately 12,000-fold, respectively, in the presence of lung tissue. Defensins induced H(2)O(2) production by lung tissue, and the rate of killing of E. coli by defensins was reduced by approximately 2,500-fold in the presence of 10 microM DPI. We conclude that defensins exert a significant antimicrobial effect under physiological conditions and that this effect is enhanced in the presence of lung tissue by a mechanism that involves the production of reactive oxygen species.

Comparison of lung protection strategies using conventional and high-frequency oscillatory ventilation.

This study compared pathophysiological and biochemical indexes of acute lung injury in a saline-lavaged rabbit model with different ventilatory strategies: a control group consisting of moderate tidal volume (V(T)) (10-12 ml/kg) and low positive end-expiratory pressure (PEEP) (4-5 cmH(2)O); and three protective groups: 1) low V(T) (5-6 ml/kg) high PEEP, 2-3 cmH(2)O greater than the lower inflection point; 2) low V(T) (5-6 ml/kg), high PEEP (8-10 cmH(2)O); and 3) high-frequency oscillatory ventilation (HFOV). The strategy using PEEP > inflection point resulted in hypotension and barotrauma. HFOV attenuated the decrease in pulmonary compliance, the lung inflammation assessed by polymorphonuclear leukocyte infiltration and tumor necrosis factor-alpha concentration in the alveolar space, and pathological changes of the small airways and alveoli. Conventional mechanical ventilation using lung protection strategies (low V(T) high PEEP) only attenuated the decrease in oxygenation and pulmonary compliance. Therefore, HFOV may be a preferable option as a lung protection strategy.

Effects of mechanical ventilation of isolated mouse lungs on surfactant and inflammatory cytokines.

Mechanical ventilation of the lung is an essential but potentially harmful therapeutic intervention for patients with acute respiratory distress syndrome. The objective of the current study was to establish and characterize an isolated mouse lung model to study the harmful effects of mechanical ventilation. Lungs were isolated from BalbC mice and randomized to either a nonventilated group, a conventionally ventilated group (tidal volume 7 mL x kg(-1), 4 cm positive endexpiratory pressure (PEEP)) or an injuriously ventilated group (20 mL x kg(-1), 0 cm PEEP). Lungs were subsequently analysed for lung compliance, morphology, surfactant composition and inflammatory cytokines. Injurious ventilation resulted in significant lung dysfunction, which was associated with a significant increase in pulmonary surfactant, and surfactant small aggregates compared to the other two groups. Injurious ventilation also led to a significantly increased concentration of interleukin-6 and tumour necrosis factor-a in the lavage. It was concluded that the injurious effects of mechanical ventilation can effectively be studied in isolated mouse lung, which offers the potential of studying genetically altered animals. It was also concluded that in this model, the lung injury is, in part, mediated by the surfactant system and the release of inflammatory mediators.

Heat stress attenuates ventilator-induced lung dysfunction in an ex vivo rat lung model.

Our laboratory has previously shown decreased mortality rates and the attenuation of lung injury in rats exposed to heat stress (H) 18 h prior to induction of sepsis. In the present study, we examined the hypothesis that heat stress would protect lungs against ventilator-induced lung injury. Male Sprague-Dawley rats were anesthetized and randomly allocated to receive either sham treatment or exposure to heat (rectal temperature 41 degrees C, for 15 min). The lungs were harvested 18 h later, a pressure-volume (P- V) curve was constructed, and the lungs were either lavaged for cytokine and surfactant analyses (preventilation data) or were mechanically ventilated with VT 40 ml/kg in a warmed, humidified chamber. After 2 h of mechanical ventilation, another P-V curve was constructed and the lungs were lavaged for cytokine and surfactant analyses (postventilation data). Mechanical ventilation in control lungs produced a 47% decrease in chord compliance, an increase in lung lavage levels of tumor necrosis factor (TNF)-alpha (722 +/- 306 pg/ml), interleukin (IL)-1beta (902 +/- 322 pg/ml), and macrophage inflammatory protein-2 (MIP-2) (363 +/- 104 pg/ml) as compared with low levels of cytokines detected in preventilation data, and no change in percentage of surfactant large aggregates (LA). In contrast, in mechanically ventilated lungs from animals that were exposed to heat stress we observed a smaller decrease in chord compliance (17%), a significant attenuation in cytokine levels (TNF-alpha 233 +/- 119 pg/ml; IL-1beta 124 +/- 53 pg/ml; MIP-2 73 +/- 52 pg/ml; p < 0.05) and a significant increase in percentage LA compared with control animals. We conclude that exposing animals to heat stress confers protection against the effects of an injurious form of mechanical ventilation, by a mechanism that may involve attenuation of cytokines and preservation of some surfactant properties.

Neutrophil defensins mediate acute inflammatory response and lung dysfunction in dose-related fashion.

High concentrations of neutrophil defensins from airway and blood have been reported in patients with inflammatory lung diseases, but their exact role is unclear. We investigated the direct effect of defensins on the lungs of mice. Intratracheal instillation of purified defensins (5-30 mg/kg) induced a progressive reduction in peripheral arterial O(2) saturation, increased lung permeability, and enhanced the lung cytochrome c content. These indexes of acute lung dysfunction were associated with an increased total cell number and a significant neutrophil influx into the lung [5.1 +/- 0.04% in control vs. 48.6 +/- 12.7% in the defensin (30 mg/kg) group, P < 0.05]. Elastase concentrations in the bronchoalveolar lavage (BAL) fluids increased from 38 +/- 11 ng/ml (control) to 80 +/- 4 ng/ml (defensins, P < 0.05). Five hours after defensin instillation, concentrations of tumor necrosis factor-alpha and macrophage inflammatory protein-2 in BAL fluid were significantly increased. High levels of monocyte chemoattractant protein-1 in BAL fluid and plasma were also found after defensin stimulation. We conclude that intratracheal instillation of defensins causes acute lung inflammation and dysfunction, suggesting that high concentrations of defensins in the airways may play an important role in the pathogenesis of inflammatory lung diseases.

Overview of high-frequency ventilation modes, clinical rationale, and gas transport mechanisms.

HFV has been demonstrated to be a safe and effective way to ventilate and oxygenate patients, both short- and long-term, when used by experienced practitioners. It has carved out a niche in the specific management of respiratory problems in children and neonates; however, as understanding of the variables that independently contribute to VILI evolves, it is becoming clear that this mode of ventilation may be suited to the goals of lung protection. In addition, it is accepted also that initial assessment of HFV as a lung-protective strategy has failed to take into consideration significant variables that have been shown to be important in animal studies. This may have caused this mode of ventilation to be over looked as a possible strategy in the management of patients with severe lung disease. A number of trials are underway using ventilatory approaches based on current concepts of VILI, including improved CMV strategies. It is hoped that the results of these studies will identify the future role for HFV in the clinical setting.

Transplant immunosuppression increases and prolongs transgene expression following adenoviral-mediated transfection of rat lungs.

BACKGROUND: Gene therapy provides the potential to modify donor organs to better withstand transplantation, but this has yet to be realized. In vivo gene transfer using adenoviral vectors has had limited success because of host immune response that induces inflammation and limits the amount and duration of transgene expression. We hypothesize that transplantation immunosuppression can attenuate the post-transfection host-immune response to allow for improved gene transfer following adenoviral-mediated transfection. METHODS: We intratracheally transfected with adenovirus containing the beta-galactosidase gene and randomized the rats to either the immunosuppression group, receiving daily cyclosporine, azathioprine, and methylprednisolone, or the control group, receiving no immunosuppression. We evaluated transgene expression and post-transfection inflammation at time points ranging from 1 day to 5 weeks. RESULTS: Following transfection, control rats showed relatively low levels of transgene expression, which rapidly decreased to non-detectable levels. In contrast, immunosuppressed rats demonstrated significantly higher levels of transgene expression overall (p < 0.00005), peaking at almost 3 times that of the control group (p < 0.02), and showing prolonged and elevated transgene expression at 5 weeks (p < 0.02). On histologic sections of the lungs, immunosuppressed rats exhibited overall lesser grades of post-transfection inflammation. CONCLUSIONS: Transplant immunosuppression provides the means to attenuate the severe immune response to adenoviral-mediated gene transfection and thereby increase and prolong transgene expression.

Invited review: mechanisms of ventilator-induced lung injury: a perspective.

Despite advances in critical care, the mortality rate in patients with acute lung injury remains high. Furthermore, most patients who die do so from multisystem organ failure. It has been postulated that ventilator-induced lung injury plays a key role in determining the negative clinical outcome of patients exposed to mechanical ventilation. How mechanical ventilation exerts its detrimental effect is as of yet unknown, but it appears that overdistension of lung units or shear forces generated during repetitive opening and closing of atelectatic lung units exacerbates, or even initiates, significant lung injury and inflammation. The term "biotrauma" has recently been elaborated to describe the process by which stress produced by mechanical ventilation leads to the upregulation of an inflammatory response. For mechanical ventilation to exert its deleterious effect, cells are required to sense mechanical forces and activate intracellular signaling pathways able to communicate the information to its interior. This information must then be integrated in the nucleus, and an appropriate response must be generated to implement and/or modulate its response and that of neighboring cells. In this review, we present a perspective on ventilator-induced lung injury with a focus on mechanisms and clinical implications. We highlight some of the most recent findings, which we believe contribute to the generation and propagation of ventilator-induced lung injury, placing a special emphasis on their implication for future research and clinical therapies.

Mechanical stretch stimulates macrophage inflammatory protein-2 secretion from fetal rat lung cells.

Ventilation-induced lung injury has been related to cytokine production. Immaturity and barotrauma are important contributors to the development of bronchopulmonary dysplasia in infants. In the present study, stretch of organotypic cultured fetal rat lung cells was used to simulate ventilation of preterm newborns. Cells were stimulated with lipopolysaccharide (LPS; 100 ng/ml) and/or mechanical stretch. After 4 h, stretch enhanced LPS-induced macrophage inflammatory protein (MIP)-2 production in a force- and frequency-dependent manner. The maximal effect of stretch was seen with 5% elongation at 40 cycles/min. In contrast, after 1 h of stimulation, stretch alone significantly increased MIP-2 production, which was not blocked by cycloheximide, an inhibitor of protein synthesis. At both the 1- and 4-h time points, only LPS increased MIP-2 mRNA levels. Stretch-induced MIP-2 release was associated with cell injury as measured by lactate dehydrogenase release and was not inhibited by gadolinium, a stretch-activated ion channel blocker. Taken together, these results suggest that the major effect of stretch on MIP-2 production from fetal rat lung cells is to stimulate its secretion.

Adverse ventilatory strategy causes pulmonary-to-systemic translocation of endotoxin.

Accumulating evidence strongly suggests that ventilatory strategy has an important impact on development of lung injury and patient outcome. Adverse ventilatory strategies have been shown to cause release of pulmonary-derived cytokines and may permit bacterial translocation from the lung to the systemic circulation. Because endotoxin is a potent and clinically important stimulant of cytokine-mediated systemic inflammatory responses that can lead to multiorgan failure, we investigated the effects of ventilatory strategy on lung-to-systemic translocation of endotoxin. We studied the effects of protective (tidal volume [VT] 5 ml. kg(-)(1), positive end-expiratory pressure [PEEP] 10 to 12.5 cm H(2)O) versus nonprotective (VT 12 ml. kg(-)(1), PEEP zero) ventilatory strategy on translocation of endotracheally instilled endotoxin. Anesthetized New Zealand White rabbits were subjected to saline lung lavage, and 32 were randomized to one of four groups: PS (protective ventilation + instilled saline); PE (protective ventilation + instilled endotoxin); NS (nonprotective ventilation + instilled saline); NE (nonprotective ventilation + instilled endotoxin), and ventilated for 3 h. Plasma endotoxin levels increased significantly in the NE group, and remained low and unchanged in the other groups. Peak levels of plasma tumor necrosis factor-alpha (TNF-alpha) were higher in NE versus other groups. Pa(O(2)) and mean arterial pressure (Pa) were lowest, and requirement for pressor and bicarbonate support greatest, in the NE group. Finally, plasma endotoxin levels were significantly greater in eventual nonsurvivors than survivors. These data provide convincing evidence for pulmonary translocation of lung-derived endotoxin. This translocation depends on ventilatory strategy, and suggests a pathophysiologic link between ventilatory strategy and outcome.

Production of tumour necrosis factor alpha by primary cultured rat alveolar epithelial cells.

Tumour necrosis factor alpha(TNF-alpha) is one of the most important pro-inflammatory cytokines, which plays an important role in host defense and acute inflammation related to tissue injury. The major source of TNF-alpha has been shown to be immune cells such as macrophages and neutrophils. In the present study, we demonstrated that LPS-treatment on alveolar epithelial cells isolated from adult rat lungs also induced a dose- and time-dependent release of TNF-alpha. The purity and identity of these cells were examined by immunofluorescent staining and confocal microscopy with antibodies for cytokeratin and pro-surfactant protein C, markers for epithelial cells and type II pneumocytes respectively. Positive staining of TNF-alpha was observed throughout the cell layer and localized intracellularly. LPS-induced TNF-alpha production from alveolar epithelial cells was blocked not only by cycloheximide, an inhibitor of protein translation, but also by actinomycin D, an inhibitor of gene transcription. The mRNA of TNF-alpha rapidly increased within 1 h of LPS stimulation. These data suggest that LPS-induced TNF-alpha production from alveolar epithelial cells is primarily regulated at the transcriptional level, which is different from that of macrophages and neutrophils. TNF-alpha produced by alveolar epithelial cells may function as an alert signal in host defense to induce production of other inflammatory mediators.

Perfluorocarbon blocks tumor necrosis factor-alpha-induced interleukin-8 release from alveolar epithelial cells in vitro.

OBJECTIVE: To determine whether tumor necrosis factor (TNF)-alpha-induced interleukin (IL)-8 production by pulmonary alveolar epithelial cells is blocked by perfluorocarbon (PFC). DESIGN: Controlled, laboratory investigation of IL-8 production by pulmonary alveolar epithelial cells after exposure to PFC in vitro. SETTING: University research laboratory. SUBJECT: The human alveolar epithelial cell line with pulmonary type II (A549) cell properties. INTERVENTIONS: The A549 cells on a polycarbonate porous filter were stimulated either on the apical or the basolateral side with TNF-alpha. To determine TNF-alpha-induced IL-8 production, IL-8 was measured by using a human IL-8 kit in both control and experimental groups. MEASUREMENTS AND MAIN RESULTS: TNF-alpha stimulation induced a large increase in IL-8. When PFC was added to the medium immediately after TNF-alpha stimulation, PFC separated the medium from the cells and IL-8 production was markedly reduced (TNF-alpha alone, 8342+/-470 pg vs. TNF-alpha followed by PFC, 417+/-88 pg, p < .05). Preincubation of A549 cells with PFC for 24 hrs before stimulation with TNF-alpha followed by removal of PFC did not affect IL-8 production (8834+/-204 vs. 8342+/-470 pg; p = NS). When added to the lower chamber, TNF-alpha also induced IL-8 production unaffected by the addition of PFC to the upper chamber. The decrease in TNF-alpha-induced IL-8 production depended on the time of PFC administration after the initiation of TNF-alpha stimulation. The earlier PFC was added, the more pronounced the diminution was in IL-8. CONCLUSIONS: PFC appears to function as a physical barrier, thus reducing cytokines produced by alveolar epithelial cells in vitro. This mechanism may partially explain the decreased inflammatory response observed during liquid ventilation in models of acute lung injury.

Alterations of nitric oxide synthase expression and activity during rat lung transplantation.

Decreased nitric oxide (NO) production has been reported during lung transplantation in patients. To study the effects of ischemia and reperfusion on endogenous NO synthase (NOS) expression, both an ex vivo and an in vivo lung injury model for transplantation were used. Donor rat lungs were flushed with cold low-potassium dextran solution and subjected to either cold (4 degrees C for 12 h) or warm (21 degrees C for 4 h) ischemic preservation followed by reperfusion with an ex vivo model. A significant increase in inducible NOS and a decrease in endothelial NOS mRNA was found after reperfusion. These results were confirmed in a rat single-lung transplant model after warm preservation. Interestingly, protein contents of both inducible NOS and endothelial NOS increased in the transplanted lung after 2 h of reperfusion. However, the total activity of NOS in the transplanted lungs remained at very low levels. We conclude that ischemic lung preservation and reperfusion result in altered NOS gene and protein expression with inhibited NOS activity, which may contribute to the injury of lung transplants.

Partial liquid ventilation decreases serum tumor necrosis factor-alpha concentrations in a rat acid aspiration lung injury model.

OBJECTIVE: To examine the hypothesis that partial liquid ventilation (PLV) with perfluorocarbon would decrease serum tumor necrosis factor-alpha concentrations in a rat acid aspiration lung injury model. DESIGN: Prospective, controlled animal study. SETTINGS: Research laboratory in a university setting. SUBJECTS: Male Sprague-Dawley rats. INTERVENTIONS: Treatment with intratracheal perflubron or control mechanical ventilation beginning 30 mins after acid aspiration. MEASUREMENTS AND MAIN RESULTS: PLV with perfluorocarbon compared with control ventilation resulted in significantly greater mean arterial blood pressures at 3 and 4 hrs and greater arterial Po2 at all times. Serum tumor necrosis factor-alpha at 2, 3, and 4 hrs was significantly less than that observed in the control group (4-hr values: 80+/-64 pg/mL vs. 658+/-688 pg/mL; p<.05), although no significant difference in tracheal fluid tumor necrosis factor-alpha concentrations (1425+/-1347 pg/mL vs. 2219+/-1933 pg/mL) was found. CONCLUSION: We conclude that the effects of PLV with perfluorocarbon can extend beyond improvements in pulmonary physiology and that PLV may be beneficial in reducing systemic sequelae of acute lung injury and inflammation.

Transtracheal gene transfection of donor lungs prior to organ procurement increases transgene levels at reperfusion and following transplantation.

BACKGROUND: Gene therapy's potential to modify donor organs to better withstand the process of transplantation has yet to be realized. To determine whether gene transfection is feasible to treat the early post-transplant injury of ischemia-reperfusion, we compared transfection of lungs in the donor prior to organ procurement with transfection of harvested ex vivo lungs in a rat single lung transplant model. METHODS: Lewis rats (donor transfection [DT]; n = 4) underwent transtracheal adenoviral-mediated transfection with 10(9) plaque forming unit of the beta-galactosidase reporter gene. Donor lungs were harvested following 6 hours of in vivo post-transfection ventilation, and then preserved for 6 hours at 4 degrees C prior to left single-lung transplantation. Ex vivo transfection was performed following organ retrieval; lungs were then preserved at 4 degrees C for 6 hours (EVT6h; n = 6) and 12 hours (EVT12h; n = 6) prior to transplantation. Lung transgene expression was measured by chemiluminescence at reperfusion, and at 2 hours following lung transplantation. RESULTS: Donor transfection lungs showed significantly higher levels of transgene expression as compared with EVT lungs at the time of reperfusion (DT = 3,408+/-1,301 relative light units/mg protein; EVT6h = 218+/-7; EVT12h = 213+/-26; p < 0.02) and at 2 hours after lung transplantation (DT = 2900+/-870; EVT6h = 62+/-27; EVT12h = 123+/-21; p < 0.005). Transgene expression measured in the heart, liver, kidney, and serum from DT rats demonstrated virtually no evidence of collateral transfection at 12 hours post-transfection (all <5.0). CONCLUSIONS: Gene transfection of donor lungs produces significantly higher levels of transgene expression in lungs at the critical time of reperfusion and in the early period following lung transplantation as compared to ex vivo transfection of cold preserved lungs. Transtracheal donor-lung transfection does not appear to result in collateral transfection of other transplantable organs. Local adenoviral-mediated transfection of the lungs is possible in the multiorgan donor prior to organ procurement and may provide the optimal strategy for gene therapeutic manipulations to address post-transplant ischemia-reperfusion injury.

Effect of adrenoreceptors on endotoxin-induced cytokines and lipid peroxidation in lung explants.

Lung tissue may be an important source of systemic inflammation associated with sepsis and the acute respiratory distress syndrome (ARDS). An ex vivo model of freshly explanted lung tissue in culture was developed to evaluate the ability of lipopolysaccharide (LPS) to directly stimulate lung tissues under conditions where indirect mechanisms such as recruitment of blood-derived inflammatory cells could not be implicated. Under control conditions, lung explants produced a high level of macrophage inflammatory protein-2 (MIP-2). Eight hours after LPS challenge, there were marked increases in the production of tumor necrosis factor-alpha (TNF-alpha) from 0.18 +/- 0.04 to 4.13 +/- 0.23 ng/ml/g tissue (p < 0.05), MIP-2 from 60.0 +/- 7.4 to 165.6 +/- 10.3 ng/ml/g tissue (p < 0.05), and tissue lipid peroxidation (malonaldehyde from 27.6 +/- 2.5 to 48.4 +/- 17.5 microM/g tissue; and 4-hydroxyalkenal from 34.0 +/- 3.0 to 59.7 +/- 18.8 microM/g tissue, both p < 0.05) from lung explants. Treatment with the beta-adrenoreceptor agonist isoproterenol (1 ng/ml) attenuated LPS-induced release of TNF-alpha and lipid peroxidation in association with an increase in intracellular cAMP levels. The adenylate cyclase activator, forskolin, also inhibited LPS-induced changes in TNF-alpha and lipid peroxidation. In conclusion, increasing intracellular levels of cAMP through beta-adrenoreceptor activation can attenuate the acute inflammatory response induced in the lung by LPS. LPS did not significantly impair the beta-adrenoreceptor reactivity in lung explants. Lung explants allow for the quantitative assessment of pulmonary inflammatory responses independent of influences from the circulation, and thus may be a useful ex vivo model to investigate cellular and molecular mechanisms of lung injury.