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1.  Activated Protein C Inhibits Local Coagulation after Intrapulmonary Delivery of Endotoxin in Humans 
Rationale: Acute lung injury and pneumonia are associated with pulmonary activation of coagulation and suppression of fibrinolysis, resulting in fibrin deposition in the lung. Activated protein C (APC) has systemic anticoagulant effects in patients with sepsis. Objective: To determine the effect of systemic administration of recombinant human APC on endotoxin-induced hemostatic alterations in the bronchoalveolar space in humans. Methods: Healthy humans received intravenous APC (24 μg/kg/hour; n = 8) or vehicle (n = 7); all subjects were administered saline in one lung subsegment and endotoxin (4 ng/kg) into the contralateral lung. Bronchoalveolar lavage was performed 16 hours after saline and endotoxin administration. Measurements and Main Results: Endotoxin induced local activation of coagulation, as reflected by elevated levels of thrombin–antithrombin complexes (1.9 ± 0.1 ng/ml) and soluble tissue factor (15.0 ± 0.6 pg/ml) in bronchoalveolar lavage fluid, which was inhibited by APC (1.4 ± 0.1 ng/ml and 12.3 ± 0.4 pg/ml, respectively; both p < 0.01). Concurrently, endotoxin suppressed fibrinolysis, as indicated by reduced bronchoalveolar levels of plasminogen activator activity accompanied by elevated levels of plasminogen activator inhibitor type I activity. APC diminished the rise in plasminogen activator inhibitor type I activity (from 3.9 ± 0.1 to 3.0 ± 0.2 ng/ml, p = 0.002), while not significantly influencing plasminogen activator activity levels. Endotoxin reduced bronchoalveolar protein C concentrations, which was prevented by APC. Protein C did not influence the endotoxin-induced rise in local soluble thrombomodulin levels. Conclusion: APC exerts an anticoagulant effect in the human lung challenged with endotoxin.
PMCID: PMC2718442  PMID: 15750041
fibrinolysis; lipopolysaccharide; lung
2.  Urokinase-Type Plasminogen Activator Inhibits Efferocytosis of Neutrophils 
Rationale: Phagocytosis of apoptotic cells, also called efferocytosis, plays an essential role in the resolution of inflammation. Urokinase-type plasminogen activator (uPA) is a multifunctional protein that has been implicated in inflammatory conditions, including pneumonia and severe infection, which are often accompanied by the development of acute lung injury. However, the role of uPA in modulating efferocytosis of apoptotic neutrophils has not been defined.
Objectives: To characterize the role of uPA in regulation of efferocytosis and to delineate the underlying mechanisms involved in this process.
Methods: In vitro and in vivo phagocytosis, immunoprecipitation, and Western blotting assays.
Measurements and Main Results: The phagocytosis of apoptotic neutrophils by macrophages was significantly inhibited by uPA. Mutant uPA lacking the growth factor domain and catalytically inactive uPA had similar inhibitory effects on efferocytosis, as did wild-type uPA. In contrast, absence of the kringle domain abrogated the ability of uPA to diminish efferocytosis. Both the αVβ3 integrin and vitronectin seemed to be involved in the inhibition of efferocytosis by uPA. Incubation of macrophages with uPA also diminished activation of the small GTPase Rac-1, which normally occurs during ingestion of apoptotic neutrophils. Under in vivo conditions in the lungs, uPA decreased the uptake of apoptotic neutrophils by alveolar macrophages.
Conclusions: Our data demonstrate a novel role for uPA in which it is able to diminish the uptake of apoptotic neutrophils by macrophages under both in vitro and in vivo conditions.
PMCID: PMC3029937  PMID: 20656938
phagocytosis; integrin αvβ3; inflammation; acute lung injury
3.  Induction of Tissue Factor by Urokinase in Lung Epithelial Cells and in the Lungs 
Rationale: Urokinase-type plasminogen activator (uPA) regulates extracellular proteolysis in lung injury and repair. Although alveolar expression of uPA increases, procoagulant activity predominates.
Objectives: This study was designed to investigate whether uPA alters the expression of tissue factor (TF), the major initiator of the coagulation cascade, in lung epithelial cells (ECs).
Methods: Bronchial, primary airway ECs and C57B6 wild-type, uPA-deficient (uPA−/−) mice were exposed to phosphate-buffered saline, uPA, or LPS. Immunohistochemistry, protein, cellular, and molecular techniques were used to assess TF expression and activity.
Measurements and Main Results: uPA enhanced TF mRNA and protein expression, and TF-dependent coagulation in lung ECs. uPA-induced expression of TF involves both increased synthesis and enhanced stabilization of TF mRNA. uPA catalytic activity had little effect on induction of TF. By contrast, deletion of the uPA receptor binding growth factor domain from uPA markedly attenuated the induction of TF, suggesting that uPA receptor binding is sufficient for TF induction. Lung tissues of uPA-deficient mice expressed less TF protein and mRNA compared with wild-type mice. In addition, intratracheal instillation of mouse uPA increased TF mRNA and protein expression and accelerated coagulation in lung tissues. uPA−/− mice exposed to LPS failed to induce TF.
Conclusions: uPA increased TF expression and TF-dependent coagulation in the lungs of mice. We hypothesize that uPA-mediated induction of TF occurs in lung ECs to promote increased fibrin deposition in the airways during acute lung injury.
PMCID: PMC2894411  PMID: 20194819
urokinase; tissue factor; lung epithelial cells; idiopathic pulmonary fibrosis
4.  Antiinflammatory Effects of Hydrogen Peroxide in Neutrophil Activation and Acute Lung Injury 
Rationale: Although reactive oxygen species (ROS) are generally considered to be proinflammatory and to contribute to cellular and organ dysfunction when present in excessive amounts, there is evidence that specific ROS, particularly hydrogen peroxide (H2O2), may have antiinflammatory properties.
Objectives: To address the role that increases in intracellular H2O2 may play in acute inflammatory processes, we examined the effects of catalase inhibition or the absence of catalase on LPS-induced inflammatory responses.
Methods: Neutrophils from control or acatalasemic mice, or control neutrophils incubated with the catalase inhibitor aminotriazole, were treated with LPS, and levels of reactive oxygen species, proteasomal activity, NF-κB activation, and proinflammatory cytokine expression were measured. Acute lung injury (ALI) was produced by intratracheal injection of LPS into control, acatalasemic-, or aminotriazole-treated mice.
Measurements and Main Results: Intracellular levels of H2O2 were increased in acatalasemic neutrophils and in neutrophils exposed to aminotriazole. Compared with LPS-stimulated neutrophils from control mice, neutrophils from acatalasemic mice or neutrophils treated with aminotriazole demonstrated reduced 20S and 26S proteasomal activity, IκB-α degradation, NF-κB nuclear accumulation, and production of the proinflammatory cytokines TNF-α and macrophage inhibitory protein (MIP)-2. The severity of LPS-induced ALI was less in acatalasemic mice and in mice treated with aminotriazole as compared with that found in control mice.
Conclusions: These results indicate that H2O2 has antiinflammatory effects on neutrophil activation and inflammatory processes, such as ALI, in which activated neutrophils play a major role.
PMCID: PMC2668804  PMID: 19151196
reactive oxygen species; neutrophils catalase; LPS; NF-κB; acute lung injury
5.  Post-Transcriptional Regulation of Urokinase-type Plasminogen Activator Receptor Expression in Lipopolysaccharide-induced Acute Lung Injury 
Rationale: Urokinase-type plasminogen activator (uPA) receptor (uPAR) is required for the recruitment of neutrophils in response to infection. uPA induces its own expression in lung epithelial cells, which involves its interaction with cell surface uPAR. Regulation of uPAR expression is therefore crucial for uPA-mediated signaling in infectious acute lung injury (ALI).
Objectives: To determine the role of uPA in uPAR expression during ALI caused by sepsis.
Methods: We used Western blot, Northern blot, Northwestern assay, and immunohistochemistry. Phosphate-buffered saline– and lipopolysaccharide (LPS)-treated wild-type and uPA−/− mice were used.
Measurements and Main Results: Biological activities of uPA, including proteolysis, cell adhesion, migration, proliferation, and differentiation, are dependent on its association with uPAR. Bacterial endotoxin (LPS) is a major cause of pulmonary dysfunction and infection-associated mortality. The present study shows that LPS induces uPAR expression both in vitro and in vivo, and that the mechanism involves post-transcriptional stabilization of uPAR mRNA by reciprocal interaction of phosphoglycerate kinase (PGK) and heterogeneous nuclear ribonucleoprotein C (hnRNPC) with uPAR mRNA coding region and 3′ untranslated region determinants, respectively. The process involves tyrosine phosphorylation of PGK and hnRNPC. uPA−/− mice failed to induce uPAR expression after LPS treatment. In these mice, LPS treatment failed to alter the binding of PGK and hnRNPC protein with uPAR mRNA due to lack of tyrosine phosphorylation.
Conclusions: Our study shows that induction of LPS-mediated uPAR expression is mediated through tyrosine phosphorylation of PGK and hnRNPC. This involves expression of uPA as an obligate intermediary.
PMCID: PMC2643078  PMID: 19029002
LPS; urokinase-type plasminogen activator; urokinase-type plasminogen activator receptor; tyrosine phosphorylation
6.  Extracellular Superoxide Dismutase Haplotypes Are Associated with Acute Lung Injury and Mortality 
Rationale: Extracellular superoxide dismutase (EC-SOD) is a potent antioxidant that plays an important role in controlling oxidant-mediated stress and inflammation. High levels of EC-SOD are found in the lung. Acute lung injury (ALI) frequently occurs in patients with infection, and levels of EC-SOD have been shown to modulate severity of lung injury in transgenic animal models of endotoxemia-induced ALI. An R213G single nucleotide polymorphism (SNP) has been shown to alter levels of EC-SOD and patient outcomes in chronic obstructive pulmonary disease (COPD) and ischemic heart disease.
Objectives: To determine genetic variation in the promoter and EC-SOD gene and to examine whether EC-SOD haplotype blocks are associated with clinical outcomes.
Methods: We sequenced the EC-SOD promoter and gene to determine genetic variation and linkage disequilibrium (LD) patterns in a European American population. Two separate patient populations with infection-associated ALI were also evaluated to determine whether EC-SOD haplotypes were associated with clinical outcomes.
Measurements and Main Results: Sequencing resulted in the identification of 28 SNPs with relatively strong LD and 1 block consisting of 4691-5321-5360-5955-5982. This specific block was shown to be protective in two separate patient populations with infection associated ALI. In particular, patients with a GCCT haplotype had a reduced risk of time on the ventilator and mortality.
Conclusions: These results indicate that a GCCT haplotype may reduce inflammation in the lung, thereby decreasing the severity of lung injury and ultimately protecting patients from mortality associated with infection-induced ALI.
PMCID: PMC2633057  PMID: 18948423
EC-SOD; haplotypes; acute lung injury; single nucleotide polymorphism
7.  Potential Role of High-Mobility Group Box 1 in Cystic Fibrosis Airway Disease 
Rationale: High-mobility group box 1 (HMGB1) is a potent inflammatory mediator elevated in sepsis and rheumatoid arthritis, although its role in cystic fibrosis (CF) lung disease is unknown.
Objectives: To determine whether HMGB1 contributes to CF lung inflammation, including neutrophil chemotaxis and lung matrix degradation.
Methods: We used sputum and serum from subjects with CF and a Scnn1b-transgenic (Scnn1b-Tg) mouse model that overexpresses β-epithelial Na+ channel in airways and mimics the CF phenotype, including lung inflammation. Human secretions and murine bronchoalveolar lavage fluid (BALF) was assayed for HMGB1 by Western blot and ELISA. Neutrophil chemotaxis was measured in vitro after incubation with human neutrophils. The collagen fragment proline-glycine-proline (PGP) was measured by tandem mass spectroscopy.
Measurements and Main Results: HMGB1 was detected in CF sputum at higher levels than secretions from normal individuals. Scnn1b-Tg mice had elevated levels of HMGB1 by Western blot and ELISA. We demonstrated that dose-dependent chemotaxis of human neutrophils stimulated by purified HMGB1 was partially dependent on CXC chemokine receptors and that this could be duplicated in CF sputum and BALF from Scnn1b-Tg mice. Neutralization by anti-HMGB1 antibody, in both the sputum and BALF-reduced chemotaxis, which suggested that HMGB1 contributed to the chemotactic properties of these samples. Intratracheal administration of purified HMGB1 induced neutrophil influx into the airways of mice and promoted the release of PGP. PGP was also elevated in Scnn1b-Tg mice and CF serum.
Conclusions: HMGB1 expression contributes to pulmonary inflammation and lung matrix degradation in CF airway disease and deserves further investigation as a biomarker and potential therapeutic target.
PMCID: PMC2566793  PMID: 18658107
cystic fibrosis; HMGB1; inflammation; collagen;; fragmentation; proline-glycine-proline
8.  Mitochondrial Respiratory Complex I Regulates Neutrophil Activation and Severity of Lung Injury 
Rationale: Mitochondria have important roles in intracellular energy generation, modulation of apoptosis, and redox-dependent intracellular signaling. Although reactive oxygen species (ROS) participate in the regulation of intracellular signaling pathways, including activation of nuclear factor (NF)-κB, there is only limited information concerning the role of mitochondrially derived ROS in modulating cellular activation and tissue injury associated with acute inflammatory processes.
Objectives: To examine involvement of the mitochondrial electron transport chain complex I on LPS-mediated NF-κB activation in neutrophils and neutrophil-dependent acute lung injury.
Methods: Neutrophils incubated with rotenone or metformin were treated with bacterial lipopolysaccharide (LPS) to determine the effects of mitochondrial complex I inhibition on intracellular concentrations of reactive oxygen species, NF-κB activation, and proinflammatory cytokine expression. Acute lung injury was produced by intratracheal injection of LPS into control, metformin, or rotenone-treated mice.
Measurements and Main Results: Inhibition of complex I with either rotenone or the antihyperglycemic agent metformin was associated with increased intracellular levels of both superoxide and hydrogen peroxide, as well as inhibition of LPS-induced IκB-α degradation, NF-κB nuclear accumulation, and proinflammatory cytokine production. Treatment of LPS-exposed mice with rotenone or metformin resulted in inhibition of complex I in the lungs, as well as diminished severity of lung injury.
Conclusions: These results demonstrate that mitochondrial complex I plays an important role in modulating Toll-like receptor 4–mediated neutrophil activation and suggest that metformin, as well as other agents that inhibit mitochondrial complex I, may be useful in the prevention or treatment of acute inflammatory processes in which activated neutrophils play a major role, such as acute lung injury.
PMCID: PMC2453511  PMID: 18436790
acute lung injury; neutrophil; mitochondria; reactive oxygen species; nuclear factor-κB
9.  Variant IRAK-1 Haplotype Is Associated with Increased Nuclear Factor–κB Activation and Worse Outcomes in Sepsis 
Rationale: The IL-1 receptor–associated kinase (IRAK-1) plays a central role in TLR2- and TLR4-induced activation of nuclear factor (NF)-κB, a critical event in the transcriptional regulation of many sepsis-associated proinflammatory mediators. There are two haplotypes for the IRAK-1 gene in Caucasians, with the variant haplotype consisting of five intron single-nucleotide polymorphisms (SNPs) and three exon SNPs.
Objectives: To examine the functional significance of the IRAK-1 variant haplotype in modifying nuclear translocation of NF-κB and affecting outcomes from sepsis.
Measurements and Main Results: One hundred fifty-five Caucasian patients with sepsis were included. Twenty-one (14%) were homozygous for the IRAK-1 variant haplotype as determined by a SNP in which T is replaced with C at nucleotide 1,595 within exon 12 of the IRAK-1 gene. The IRAK-1 variant haplotype was associated with increased nuclear levels of NF-κB in LPS-stimulated peripheral blood neutrophils from patients with sepsis compared with that found in patients with wild-type IRAK-1 haplotype (p = 0.0009). There was an increased incidence of shock (p = 0.047) (odds ratio [OR], 2.9; 95% confidence interval [CI], 1.1–7.7), greater requirement for more prolonged mechanical ventilator support (p = 0.04) (OR, 2.7; 95% CI, 1.05–6.9), and higher 60-d mortality (p = 0.05) (OR, 2.7; 95% CI, 1.0–6.8) in patients with the IRAK-1 variant haplotype compared with wild type.
Conclusions: These results indicate that the IRAK-1 variant haplotype is functionally significant in patients with sepsis, being associated with increased nuclear translocation of NF-κB, more severe organ dysfunction, and higher mortality.
PMCID: PMC2662973  PMID: 16528020
acute lung injury; haplotype, inflammation; neutrophil; NF-κ; B; single nucleotide polymorphism

Results 1-9 (9)