Lung Injury In Humans Research Articles

Relationship between neutrophil elastase and acute lung injury in humans

We conducted clinical trials in patients with acute lung injury (ALI) associated with systemic inflammatory response syndrome using a selective neutrophil elastase inhibitor, sivelestat sodium hydrate (Sivelestat), to investigate the involvement of neutrophil elastase in ALI. In the phase III double-blind study (Study 1) in 230 patients, the efficacy of Sivelestat was evaluated with the pulmonary function improvement (PFI) rating as the primary endpoint, and the weaning rate from mechanical ventilator, the discharge rate from intensive care unit (ICU), and the survival rate as secondary endpoints. Afterwards, an unblinded study (Study 2) in 20 patients was conducted using procedures for weaning from mechanical ventilation to reevaluate its efficacy with ventilator-free days (VFD) value, the primary endpoint, and to compare with that of Study 1 subgroup, which met the selection criteria used in Study 2. Sivelestat increased PFI rating, reduced duration of mechanical ventilation, and shortened stay in ICU in Study 1, although there was no significant efficacy on the survival rate. VFD value in Study 2 was comparable to that in the optimal-dose group of Study 1 subgroup, and increase in VFD value correlated with PFI rating and increase in ICU free days. It was concluded that neutrophil elastase may be involved in the pathogenesis of ALI in humans.

Roles of iNOS and nNOS in sepsis-induced pulmonary apoptosis.

Apoptosis(programmed cell death) is induced in pulmonary cells and contributes to the pathogenesis of acute lung injury in septic humans. Previous studies have shown that nitric oxide (NO) is an important modulator of apoptosis; however, the functional role of NO derived from inducible NO synthase (iNOS) in sepsis-induced pulmonary apoptosis remains unknown. We measured pulmonary apoptosis in a rat model of Escherichia coli lipopolysaccharide (LPS)-induced sepsis in the absence and presence of the selective iNOS inhibitor 1400W. Four groups were studied 24 h after saline (control) or LPS injection in the absence and presence of 1400W pretreatment. Apoptosis was evaluated using DNA fragmentation, terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling staining, and caspase activation. LPS administration significantly augmented pulmonary cell apoptosis and caspase-3 activity in airway and alveolar epithelial cells. Pretreatment with 1400W significantly enhanced LPS-induced pulmonary apoptosis and increased caspase-3 and -7 activation. The antiapoptotic effect of iNOS was confirmed in iNOS-/- mice, which developed a greater degree of pulmonary apoptosis both under control conditions and in response to LPS compared with wild-type mice. By comparison, genetic deletion of the neuronal NOS had no effect on LPS-induced pulmonary apoptosis. We conclude that NO derived from iNOS plays an important protective role against sepsis-induced pulmonary apoptosis.

Clara cell protein as a biomarker for ozone-induced lung injury in humans.

Exposure to ozone (O3) impairs lung function, induces airway inflammation and alters epithelial permeability. Whilst impaired lung function and neutrophilia have been observed at relatively low concentrations, altered lung epithelial permeability is only seen after high-dose challenges. The appearance of Clara cell protein (CC16) in serum has been proposed as a sensitive marker of lung epithelial injury. Here, the use of CC16 as an injury biomarker was evaluated under a controlled exposure to O3 and the relationship between this marker of lung injury and early lung function decrements was investigated. Subjects (n=22) were exposed on two separate occasions to 0.2 parts per million O3 and filtered air for 2 h. Blood samples were drawn and lung function assessed at 2 h pre-exposure, immediately before and immediately after exposure as well as 2 and 4 h postexposure. O3 increased CC16 serum concentrations at 2 h (12.0+/-4.5 versus 8.4+/-3.1 microg x L(-1)) and 4 h postexposure (11.7+/-5.0 versus 7.9+/-2.6 microg x L(-1)) compared with air concentrations. Archived samples from O3 studies utilising the same design indicated that this increase was sustained for up to 6 h postexposure (9.1+/-2.6 versus 7.1+/-1.7 microg x L(-1)) with concentrations returning to baseline by 18 h (7.7+/-2.9 versus 6.6+/-1.7 microg x L(-1)). In these studies, the increased plasma CC16 concentration was noted in the absence of increases in traditional markers of epithelial permeability. No association was observed between increased CC16 concentrations and lung function changes. To conclude, Clara cell protein represents a sensitive and noninvasive biomarker for ozone-induced lung epithelial damage that may have important uses in assessing the health effects of air pollutants in future epidemiological and field studies.

The role of apoptosis in acute lung injury.

To discuss current aspects of our understanding of the role of apoptosis in lung injury. Review of English language literature. Apoptosis is a process that produces timely death in senescent cells. Apoptosis is important in developmental biology and in remodeling of tissues during repair. Many apoptosis pathways converge in intracellular protease cascades that lead to DNA cleavage and cell death. Apoptosis pathways can be triggered by surface receptors, which interact with soluble proteins or membrane-bound proteins, such as Fas ligand. Fas ligand accumulates in soluble form at sites of tissue inflammation and has the potential to initiate apoptosis of leukocytes, epithelial cells, and other parenchymal cells. Dysregulation of apoptosis pathways could contribute to the epithelial injury that is characteristic of acute lung injury in humans. The effects of Fas ligand are modulated by factors in lung fluids, such as cytokines (e.g., transforming growth factor-beta, surfactant protein A, and angiotensin II) and a specific Fas ligand decoy receptor (DcR3). Strategies to block apoptosis pathways could be useful in limiting some forms of acute lung injury in humans.

Science review: Apoptosis in acute lung injury

Apoptosis is a process of controlled cellular death whereby the activation of specific death-signaling pathways leads to deletion of cells from tissue. The importance of apoptosis resides in the fact that several steps involved in the modulation of apoptosis are susceptible to therapeutic intervention. In the present review we examine two important hypotheses that link apoptosis with the pathogenesis of acute lung injury in humans. The first of these, namely the 'neutrophilic hypothesis', suggests that during acute inflammation the cytokines granulocyte colony-stimulating factor and granulocyte/macrophage colony-stimulating factor prolong the survival of neutrophils, and thus enhance neutrophilic inflammation. The second hypothesis, the 'epithelial hypothesis', suggests that epithelial injury in acute lung injury is associated with apoptotic death of alveolar epithelial cells triggered by soluble mediators such as soluble Fas ligand. We also review recent studies that suggest that the rate of clearance of apoptotic neutrophils may be associated with resolution of neutrophilic inflammation in the lungs, and data showing that phagocytosis of apoptotic neutrophils can induce an anti-inflammatory phenotype in activated alveolar macrophages.

Upregulation of phosphoinositide 3-kinase and protein kinase B in alveolar macrophages following ozone inhalation. Role of NF-kappaB and STAT-1 in ozone-induced nitric oxide production and toxicity.

Inhalation of toxic doses of ozone causes lung injury and inflammation in humans and experimental animals. Using a rodent model of ozone toxicity, we have previously demonstrated that macrophages recruited to the lung following exposure to this oxidant contribute to the pathogenesis of tissue injury. In the present studies we analyzed potential mechanisms regulating alveolar macrophage activity following ozone inhalation and the role of inflammatory mediators in toxicity. Treatment of mice with ozone (0.8 ppm, 3 h) resulted in increased expression of inducible nitric oxide synthase (iNOS) protein and production of nitric oxide (NO) and peroxynitrite by alveolar macrophages. In contrast, these effects were not observed in macrophages from transgenic mice with a targeted disruption of the gene for iNOS, or in mice overexpressing superoxide dismutase. Moreover, ozone toxicity, as measured by bronchoalveolar lavage protein levels and nitrotyrosine staining of the lung was prevented in both of these transgenic mouse strains. The promoter/enhancer region of the iNOS gene contains binding sites for the transcription factors NF-kappaB and STAT-1 which regulate the activity of the gene. Ozone inhalation resulted in a rapid and prolonged activation of NF-kappaB in alveolar macrophages. Phosphoinositide 3-kinase (PI 3-K) and its down stream target, protein kinase B (PKB), which are known to regulate NF-kappaB activity, also increased in alveolar macrophages following ozone inhalation. These data, together with our findings that inhibitors of PI 3-K block NO production, suggest that these proteins are important in controlling expression of iNOS. Furthermore, the fact that macrophages from NF-kappaB p50 knockout mice did not generate reactive nitrogen intermediates and that these mice were protected from ozone induced toxicity demonstrate the importance of the NF-kappaB signaling pathway in lung injury. We also found that STAT-1 nuclear binding activity and STAT-1 protein expression were upregulated in macrophages from ozone treated animals. Taken together, these data suggest that biochemical signaling pathways that control the expression of genes critical for the inflammatory process play a role in ozone toxicity.

Oxygen Regulation of Gene Expression: A Study in Opposites

Oxygen is crucial to aerobic metabolism, but excesses of oxygen or reactive oxygen species (ROS) can injure cells. This minireview addresses two transcription factors that regulate several cellular responses to oxygen tension. Hypoxia inducible factor-1 (HIF-1) is a heterodimeric protein activated by hypoxia. Levels of HIF-1 are regulated by removal of the HIF-1α subunit through ubiquination and proteasomal destruction under normoxic conditions. Hypoxia inhibits the ubiquination of HIF-1α, preventing its destruction and allowing it to bind to hypoxia-responsive elements in gene promoter, enhancer, and intronic sequences. HIF-1 induces the expression of the hypoxia responsive genes vascular endothelial growth factor and erythropoietin. Its dysregulation has been implicated in von Hippel–Lindau disease. Nuclear factor κB (NFκB) is a family of pleotropic, dimeric transcription factors, and has a complex pattern of regulation. Under normoxic conditions, NFκB is bound to one of several inhibitory proteins (e.g., IκB) that prevent its nuclear translocation. Hyperoxia or elevations of ROS cause the ubiquination and destruction of the inhibitory proteins, freeing NFκB and allowing it to bind to target gene promoters. Hyperoxia in cell and animal models and acute lung injury in humans induce the expression of multiple proinflammatory cytokines through NFκB-dependent mechanisms. Although HIF-1 and NFκB respond to changes in pO2, the precise nature of the oxygen sensing and transduction pathways is unclear in both cases. Both heme-protein and redox-sensitive mechanisms have been proposed. Improved understanding of oxygen-sensitive gene regulation may suggest targeted therapies for human disease.

Platelet-activating factor plays a pivotal role in the induction of experimental lung injury.

We have previously described a model of acute lung injury in the mouse in which intravenous administration of lipopolysaccharide (LPS) results in a marked sequestration of neutrophils in the pulmonary microvasculature, although this by itself was not sufficient to induce injury. If the sequestered neutrophils were exposed to zymosan, then a striking increase in pulmonary vascular permeability to albumin was found, suggesting that sequestered neutrophils may produce one or more mediators capable of acting directly on the capillary endothelium. Because activated neutrophils are known to release platelet-activating factor (PAF), we hypothesized that PAF produced locally within the pulmonary capillaries may be the mediator involved. Treatment of mice with the PAF antagonist UK-74,505 prior to administration of zymosan alone or combined LPS and zymosan resulted in a substantial attenuation of lung injury, as measured by the accumulation of extravascular 125I-labeled human serum albumin. UK-74,505 had no effect on neutrophil sequestration as measured by myeloperoxidase activity in whole lung tissue and as assessed by light microscopy. Administration of UK-74,505 after LPS, but before zymosan, was also effective at inhibiting lung injury but again, neutrophil sequestration was unaffected. In contrast, UK-74,505 had no effect on cobra venom factor-induced lung injury and neutrophil sequestration. These data suggest that PAF production is involved in the increases in pulmonary vascular permeability, but not in the sequestration of neutrophils, induced by zymosan alone or by combined LPS and zymosan treatment. Early treatment with PAF antagonists may be beneficial in preventing the development of acute lung injury in humans.

Genetic variability in combustion particle-induced chronic lung injury.

Occupational exposure to anthropogenic particles is associated with lung injury in humans. We hypothesized that residual oil fly ash (ROFA), an emission source particulate, may induce acute lung injury and fibrosis in sensitive rat strains and that fibronectin (Fn) gene expression will correspond to the development of fibrosis. Male Sprague-Dawley (SD), Wistar (WIS), and Fischer 344 (F-344) rats (60 days old) were exposed to saline or ROFA (8.3 mg/kg) by intratracheal instillation and examined for up to 12 wk. Histology indicated focal areas of lung damage showing inflammatory cell infiltration as well as alveolar, airway, and interstitial thickening in all three rat strains during 1-7 days postexposure. Trichrome staining of the lung sections indicated a sporadic incidence of focal alveolar fibrosis at 1, 3, and 12 wk in SD rats, whereas WIS and F-344 rats showed only a modest increase in trichrome staining in the septal areas. Of all Fn mRNA isoforms examined by polymerase chain reaction, only EIIIA(+) was upregulated during 6 h-1 wk in ROFA-exposed SD and WIS rats but not in F-344 rats. In situ hybridization analysis in SD rats revealed Fn mRNA expression by macrophage and alveolar and airway epithelium and within fibrotic areas. Immunohistochemical analysis revealed increased presence of Fn EIIIA(+) protein in the areas of fibrotic injury and basally to the airway epithelium. In summary, Fn EIIIA(+) increases early in the course of particle-induced lung injury and remodeling, which may or may not result in discernible alveolar fibrosis. There is a rat strain variation in ROFA-induced fibrosis and associated Fn EIIIA(+) expression.

Bronchoalveolar lavage fluid phospholipase A2 activities are increased in human adult respiratory distress syndrome

The role of phospholipase A2 (PLA2) in lung injury in humans is unclear. Previous studies have failed to identify an increase in PLA2 activity in bronchoalveolar lavage fluids (BALF) of patients with the adult respiratory distress syndrome (ARDS). In this study, increased phospholipase A2 (PLA2) activity was detected in BALF from patients with ARDS. PLA2 levels in BALF correlated positively with lung injury score in patients with lung disease. BALF PLA2 activity in patients with ARDS was resolved into heparin binding and nonbinding activities. Both PLA2 activities were increased in BALF of ARDS patients. The PLA2 activity that bound to heparin was identified as a group II PLA2 by its chromatographic characteristics, its inhibition by dithiothreitol, its substrate specificity, and its approximate molecular mass of 14 kDa. The second PLA2 activity was further purified and found to require Ca2+ at a concentration > 2 x 10(-4) M for activity. This form of PLA2 exhibited a neutral and broad pH optimum (pH 6.0-8.0) and hydrolyzed both phosphatidylethanolamine and phosphatidylcholine effectively. Its apparent molecular mass was estimated to be 80-90 kDa. Neither anti-pancreatic PLA2 antiserum nor anti-pig spleen cytosolic 100-kDa PLA2 antiserum immunoprecipitated the enzymatic activity. Thus at least two forms of PLA2 are increased in activity in BALF of patients with ARDS, a group II PLA2 and a biochemically and immunochemically form distinct from group I, group II, and cytosolic PLA2. Increased lung PLA2 activity may be important for the pathophysiology of ARDS.

Taurine protects rat bronchioles from acute ozone-induced lung inflammation and hyperplasia.

Ozone is a potent respiratory irritant known to induce lung injury in humans and experimental animals. The present studies determined if ozone-induced lung inflammation was modified by pretreatment of animals with taurine, a detoxifying antioxidant. Rats were pretreated for 10 days with 5% taurine in their drinking water (controls received water only) prior to exposure to 2 ppm ozone for 3 h. At 2, 6, 12, 24, 48, and 72 h after ozone exposure, rats were anesthetized and the lungs were perfusion-fixed for histopathologic evaluation. An additional group of rats was used to examine bronchoalveolar lavage cell counts and hydroxyproline levels. A count of bronchoalveolar lavage cells 48 h after ozone exposure showed significantly fewer total inflammatory cells and fewer polymorphonuclear leukocytes accompanied by a reduction in hydroxyproline in the lavage fluid of ozone-exposed rats pretreated with taurine compared to rats that did not receive taurine. Light microscopy revealed an inflammatory cell infiltrate in the lungs of rats exposed to ozone. This was followed by focal hyperplasia in the terminal and respiratory bronchioles. Rats pretreated with taurine and then exposed to ozone showed none of these alterations. In addition, although there was a significant reduction in cell proliferation as measured by DNA precursor incorporation in the lungs of rats pretreated with taurine prior to ozone exposure compared to unsupplemented rats, the distribution of labeled cells was the same in taurine supplemented and unsupplemented groups. Also, significantly higher levels of taurine were found in the plasma, whole blood, and lavage fluid of rats pretreated with dietary taurine compared to rats that received water only. The results suggest that supplemental taurine protects rat lung epithelium from acute ozone-induced lung inflammation and hyperplasia.

Natural surfactant and hyperoxic lung injury in primates. II. Morphometric analyses

Diffuse lung injury is accompanied by low compliance and hypoxemia with histological evidence of endothelial and alveolar epithelial cell disruption. The histological effects of treatment of an acute diffuse lung injury with a natural surfactant product were evaluated in a primate model because surfactant function and content have been shown to be abnormal in diffuse lung injury in both animals and humans. Ten baboons were ventilated with 100% O2 for 96 h, and 5 were given an aerosol of natural porcine surfactant. Physiological and biochemical measurements of the effects of hyperoxia and surfactant treatment are presented in a companion paper. After O2 exposure, lungs were fixed and processed for quantitative electron microscopy. The responses to O2 included epithelial and endothelial cell injuries, interstitial edema, and inflammation. The hyperoxic animals treated with surfactant were compared with the untreated animals; the treatments altered neutrophil distribution, fibroblast proliferation, and changes in the volumes of type I epithelial cells and endothelial cells. Surfactant-treated animals also had decreased lamellar body volume density in type II epithelial cells and preservation of endothelial cell integrity. These changes suggest complex effects of natural surfactant on the pulmonary response to hyperoxia, including protection against epithelial and endothelial cell destruction as well as significant interstitial inflammation and fibroblast proliferation. We conclude that natural surfactant treatment of hyperoxic lung injury in primates resulted in partial protection of epithelial and endothelial cells but also increased the accumulation of fibroblasts in the lung.

Calcium ionophores injure alveolar epithelial cells: relation to phospholipase activity

Phospholipases and certain of their hydrolytic products are toxic to alveolar epithelial cells. Since many intracellular phospholipases are Ca2+ dependent, we postulated that elevating cytosolic Ca2+ with ionophores might cause epithelial injury via phospholipase activation. Isolated perfused hamster lungs exposed to an Ca2+ ionophore A23187 develop functional evidence of severe epithelial injury. Ultrastructural studies show widespread lysis of type I epithelial cells, with only minimal abnormalities in other lung cells, including the microvascular endothelium. Analysis of whole lung lipid extracts reveals a modest elevation in free arachidonic acid but no changes in other putative products of phospholipase activity. Parallel studies were performed in cultured cells of pulmonary origin. As measured by 51Cr release, A23187 causes substantial cytotoxicity in 3-day-old cultures of rat type II alveolar epithelial cells (RAEC) but not in cultured bovine pulmonary artery endothelial cells (BPAEC). RAEC prelabeled with [14C]stearic acid [( 14C]SA) and [3H]arachidonic acid [( 3H]AA) release radiolabeled free fatty acids (FFA) in response to A23187 in a dose- and time-dependent manner that parallels the cytotoxicity index. Analyses of putative phospholipase products in cells radiolabeled with [14C]SA and [3H]AA, with [14C]choline, or with [14C]ethanolamine suggest that liberation of radiolabeled FFA may be due to several phospholipases but with principal activity being exhibited by a phospholipase C having specificity toward phosphatidylcholine and phosphatidylethanolamine. Prelabeled BPAEC release only minimal quantities of FFA in response to A23187 under the same conditions. These studies demonstrate that elevations of intracytoplasmic Ca2+ are capable of severely and selectively damaging alveolar epithelial cells and that the injury is associated with activation of intracellular phospholipases. These findings may have implications in regard to the pathogenesis of acute lung injury in humans.

The cell biology and pathogenic role of pulmonary intravascular macrophages

Pulmonary intravascular macrophages (PIMs) are an extensive population of mature phagocytic cells adherent to the pulmonary capillary endothelium in selected species. They are not prevalent in lungs of commonly studied laboratory animals, such as rodents, and thus have only been recently appreciated. However, their potential role in host defense and acute lung injury has attracted interest, since a number of studies have demonstrated pulmonary localization of circulating particles, microbes, and endotoxin by PIMs. Those animal species, such as ruminants, that provide useful models of pathogen (or endotoxin)-induced acute lung injury demonstrate rapid pulmonary uptake of bacteria by PIMs. Inflammatory mediators released by activated PIMs may initiate the process and provoke accumulation of neutrophils and platelets. This review summarizes the morphological characteristics of PIMs and their species distribution. The role of these members of the mononuclear phagocyte system, both beneficial and potentially pathogenic, is reviewed. The question of whether PIMs have a role in acute lung injury in humans is also discussed.

Inhibition of lung damage caused by paraquat with lymphokines or cytokines

Gramoxone, containing paraquat as an active ingredient can cause severe lung injury in both humans and experimental animals. Biologically active fibroblast-stimulating factors produced by lymphocytes and macrophages may be of importance in the development of interstitial fibrosis. In our present study we have attempted to inhibit the process of paraquat induced lung fibrosis by lymphokine-enriched supernatants from concanavalin A stimulated spleen cell cultures. It was found that adequate supernatant treatment significantly reduced PQ-induced lung injury and its associated inflammatory response.

Rat alveolar macrophage production of chemoattractants for neutrophils: response to Escherichia coli endotoxin.

Endotoxemia in rats is associated with the accumulation of neutrophils (polymorphonuclear leukocytes) within the airspaces of the lung. Polymorphonuclear leukocyte influx appears to be regulated by the intrapulmonary accumulation of chemotactic activity. Since alveolar macrophages (AMS) are prevalent cells in the airspace and are known to release a variety of chemotactic factors, we investigated the effect of endotoxin exposure on AM production of chemotactic activity. We tested the hypothesis that endotoxin-exposed AMs have an augmented ability to produce chemoattractants. We recovered AMs by bronchoalveolar lavage from control rats and from rats treated in vivo with a "low dose" (2.5 mg/kg) or a "high dose" (5.0 mg/kg) of Escherichia coli endotoxin. These AMs were then cultured in vitro for 15 h in the absence or the presence of endotoxin (15 and 30 micrograms/ml) to stimulate the cells to produce chemoattractants. We found that in vitro endotoxin stimulated normal AMs to secrete chemoattractants in a dose-dependent fashion. AMs from rats treated with endotoxin in vivo spontaneously secreted more chemoattractants than AMs from control rats. Exposure to in vivo endotoxin followed by in vitro stimulation with endotoxin resulted in an even greater production of chemoattractants by AMs. We found a significant association between the percent polymorphonuclear leukocytes recovered by bronchoalveolar lavage from the airspaces and the production of chemoattractants by AMs from the same specimen. The level of chemotactic activity spontaneously produced by AMs predicted the degree of stimulated production of chemotactic activity. Partial purification indicated that this chemotactic activity has two molecular weight peaks, one near 1,000 and the other near 50,000. The activity was stable at 100 degrees C for at least 30 min and was degradable by trypsinization. We conclude that endotoxin can induce AM production of chemoattractants and that prior exposure to endotoxin in vivo affects the response of AM to in vitro endotoxin exposure. By inference, it is possible that this endotoxin-macrophage interaction may serve as a biologic amplifier of the effects of endotoxin and may have a role in the pathogenesis of septic lung injury in humans.

Lung toxicity of paraquat.

Paraquat (1, 1'-dimethyl-4, 4'-bipyridylium), a widely used and effective herbicide, can cause a severe lung injury in both humans and experimental animals. The injury is characterized by initial damage to the alveolar epithelium and capillary endothelium with the development of hemorrhagic pulmonary edema, followed by development of intra-alveolar and interstitial fibrosis. Although the lung is thought to be a primary target organ for paraquat toxicity, the mechanism of action of this herbicide remains to be uncertain. A possible mechanism of selective toxicity to the lung is the accumulation of paraquat in the lung. Data from experimental animals in vivo and in vitro indicate the accumulation of paraquat in the lung by an energy-dependent transport system. However, paraquat does not appear to be selectively concentrated by the lung in human paraquat poisoning. A widely accepted theory regards lipid peroxidation and NADPH depletion as the basic mechanism of paraquat toxicity. According to this theory, paraquat can be reduced by microsomal enzymes and undergoes a cyclic reduction and reoxidation, giving rise to a reactive oxygen radical. This continuous generation of oxygen radicals within the cells of the lung then in turn destroy this tissue through various mechanisms of oxidative damage such as lipid peroxidation and NADPH depletion. However, there are data in the literatures that contradict the active oxygen species theory of paraquat toxicity. Most of these findings show a lack of extensive oxidative damage to the lung. Extent of lipid peroxidation and NADPH depletion is not always correlated with that of lung tissue damage. Moreover, the mechanism of paraquat toxicity leading to the development of lung fibrosis have not been fully elucidated. Recent studies suggest that inflammatory cells such as polymorphonuclear leukocytes and alveolar macrophage, and chemical mediators released by these cells may play an important role in lung fibrosis caused by paraquat. To sum up, the mechanism of selective lung toxicity in paraquat poisoning is not clearly explained by the theory mentioned above. Study on the secondary pulmonary response following cell injury directly caused by paraquat may become a clue to elucidate this mechanism.

Local abnormalities of coagulation and fibrinolysis and alveolar fibrin deposition in sheep with oleic acid-induced lung injury.

Extravascular, primarily intra-alveolar, fibrin deposition is a histologic hallmark of acute lung injury in humans and experimental animals, but the mechanisms leading to this finding are poorly understood. To determine whether local abnormalities in the fibrinolytic-procoagulant balance contribute to alveolar fibrin deposition in acute lung injury, we studied bronchoalveolar lavage (BAL) fluids of anesthetized sheep that received intravenous oleic acid. Prominent alveolar fibrin deposition was observed within 2 h after oleic acid-induced lung injury. Procoagulant and fibrinolytic activities were determined in BAL samples of anesthetized, mechanically ventilated sheep before and 2 h after intravenous oleic acid or saline. BAL procoagulant activity was found to be due mainly to tissue factor associated with Factor VII. In baseline BAL samples, we found relatively low levels of procoagulant activity and relatively high levels of fibrinolytic activity. After induction of oleic acid-induced lung injury, the procoagulant activity of BAL was markedly increased, whereas fibrinolytic activity was either depressed or undetectable. Antiplasmin activity was detectable in BAL of sheep after oleic acid-induced lung injury, which contributed at least in part to the depressed fibrinolytic activity observed. These perturbations occurred with the appearance of extensive alveolar fibrin deposition. In control sheep, BAL fibrinolytic activity was decreased, and antiplasmin activity increased modestly after 2 h of mechanical ventilation, but procoagulant activity was unchanged and alveolar fibrin was not observed. Procoagulant activity in lung lymph and plasma after lung injury did not differ from baseline values, and fibrinolytic activity was undetectable in lymph or plasma samples. These data indicate that increased procoagulant activity and concurrent disruption of the balance of coagulation and fibrinolysis establish local conditions that promote acute fibrin deposition in the alveoli of mechanically ventilated, oleic acid-injured sheep.

Neutrophil-mediated pulmonary vascular injury. Synergistic effect of trace amounts of lipopolysaccharide and neutrophil stimuli on vascular permeability and neutrophil sequestration in the lung.

The pathogenesis of acute lung injury in humans is obscure, but lipopolysaccharide (LPS), complement activation, and neutrophils have been implicated. We investigated in rabbits the interaction of small amounts of intravascularly administered LPS (100 ng) with neutrophil chemotactic factors, the synthetic chemotactic peptide formyl-norleucyl-leucyl-phenylalanine (FNLP), and the biologically relevant chemotactic fragments of C5 (C5f). These neutrophil stimuli produce neutropenia when injected intravascularly in rabbits, reflecting neutrophil adherence to vascular endothelium. When LPS was injected with FNLP, the duration of neutropenia was enhanced. Studies with radiolabeled neutrophils infused in vivo demonstrated prolonged neutrophil sequestration within the lung in rabbits that were given FNLP plus LPS, an effect that was visible for 4 h after injection. Morphometric analysis of tissue sections 4 h after infusion confirmed the presence of greater numbers of neutrophils in the lungs of animals receiving LPS and FNLP. When a combination of LPS and chemotactic factors was infused at both zero and 6 h, we found a marked enhancement of lung vascular permeability at 24 h (as assessed by radiolabeled albumin accumulation), an effect not seen with either LPS or chemotactic factor alone. Ultrastructural studies revealed neutrophil sequestration and alteration in endothelial cells in the animals that received the combination of LPS and chemotactic factors. Neutrophil depletion with nitrogen mustard completely abolished the increased vascular permeability seen in animals that received LPS and chemotactic factors. This study suggests that small amounts of intravascularly administered LPS enhance the sequestration of neutrophils within the lung and increase lung vascular permeability and endothelial injury caused by neutrophils stimulated by intravascularly administered chemotactic factors. This mechanism may be relevant to the production of acute lung injury in human beings.

Bronchoalveolar Lavage Procoagulant Activity in Bleomycin-Induced Lung Injury in Marmosets: Characterization and Relationship to Fibrin Deposition and Fibrosis

Although pulmonary fibrin deposition and coagulation abnormalities have been observed in acute lung injury in humans, their role in the pathogenesis of pulmonary disorders is unclear. In order to gain further insights into the role of the coagulation in lung injury, we examined the relationship between procoagulant activity in bronchoalveolar lavage (BAL) fluids and the evolution of bleomycin-induced lung injury in marmosets. The BAL procoagulant activity was increased at 1, 2, and 4 wk after bleomycin challenge compared with that in control subjects, and it was capable of shortening the recalcification times of plasmas deficient in factor VII and factor VIII but not in factor X. This profile suggested the presence in BAL of an activator of factor X. Activation of purified human factor X by BAL was demonstrated by measuring the amidolytic activity of the generated factor Xa on its N-benzoyl-L-isoleucyl L-glutamyl-glycyl-L-argenine-p-nitroanilide substrate. Factor X activating activity was increased in BAL at 2 wk after bleomycin challenge. Cleavage of 125I-labeled human factor X by BAL from bleomycin-challenged marmosets yielded a 55,500 Mr product that comigrated with factor Xa, the appearance of which correlated strongly with amidolytic evidence of factor Xa activity. Electron microscopy of the lungs of animals from all groups revealed pulmonary fibrin deposition at 2 wk after bleomycin challenge, at the time of increased BAL procoagulant and factor X activating activity. The BAL procoagulant activity was completely sedimentable by ultracentrifugation and was inhibited by concanavalin A and phospholipase C. Activation of purified factor X by BAL was inhibited by monospecific polyclonal goat and rabbit antibodies to human factor VII as well as antibody to bovine tissue factor, demonstrating that factor X activating activity in BAL was attributable to tissue factor associated with material similar to factors VII or VIIa. We conclude that procoagulant activity in BAL increases after bleomycin challenge in marmosets and is attributable to activation of factor X by tissue factor associated with factors VII or VIIa-like material. Increased BAL procoagulant activity is temporally associated with pulmonary fibrin deposition and pulmonary fibrosis during bleomycin-induced pulmonary injury in the marmoset.