Although oxidative stress has been thought to play a general role in the activation of NF-κB, the involvement of reactive oxygen species (ROS) in facilitating nuclear translocation of NF-κB in neutrophils has not been described. In addition, the mechanisms by which ROS modulate the transcriptional activity of NF-κB in response to Toll-like receptor 4 (TLR4)-dependent signaling are not well characterized. To examine these issues, oxidant-dependent signaling events downstream of TLR4 were investigated in neutrophils stimulated with LPS. Pretreatment of neutrophils with the antioxidants N-acetylcysteine or α-tocopherol prevented LPS-induced nuclear translocation of NF-κB. Antioxidant treatment of LPS-stimulated neutrophils also inhibited the production of proinflammatory cytokines (TNF-α, macrophage inflammatory protein-2, and IL-1β), as well as activation of the kinases IκB kinase α, IκB kinase β, p38, Akt, and extracellular receptor-activated kinases 1 and 2. The decrease in cytoplasmic levels of IκBα produced by exposure of neutrophils to LPS was prevented by N-acetylcysteine or α-tocopherol. Activation of IL-1R-associated kinase-1 (IRAK-1) and IRAK-4 in response to LPS stimulation was inhibited by antioxidants. These results demonstrate that proximal events in TLR4 signaling, at or antecedent to IRAK-1 and IRAK-4 activation, are oxidant dependent and indicate that ROS can modulate NF-κB-dependent transcription through their involvement in early TLR4-mediated cellular responses.

Background Trauma induced coagulopathy (TIC) is associated with a four-fold increased risk of mortality. Hyperfibrinolysis is a component of TIC, but its mechanism is poorly understood. PAI-1 degradation by activated protein C has been proposed as mechanism for deregulation of the plasmin system in hemorrhagic shock, but in other settings of ischemia, tPA has been shown to be elevated. We hypothesized that the hyperfibrinolysis in TIC is not the result of PAI-1 degradation, but is driven by an increase in tPA, with resultant loss of PAI-1 activity through complexation with tPA. Methods 86 consecutive trauma activation patients had blood collected at the earliest time after injury, and were screened for hyperfibrinolysis using thrombelastography (TEG). Twenty-five hyperfibrinolytic patients were compared to 14 healthy controls using ELISAs for active tPA, active PAI-1 and PAI-1/tPA complex. Blood was also subjected to TEG with exogenous tPA-challenge as a functional assay for PAI-1 reserve. Results Total levels of PAI-1 (the sum of the active PAI-1 species and its covalent complex with tPA) are not significantly different between hyperfibrinolytic trauma patients and healthy controls: median 104 pM (IQR 48—201 pM) versus 115 pM (IQR 54—202 pM). The ratio of active to complexed PAI-1, however, was two orders of magnitude lower in hyperfibrinolysis than controls. Conversely, total tPA levels (active plus complex) were significantly higher in hyperfibrinolysis than controls: 139 pM (IQR 68—237 pM) versus 32 pM (IQR 16—37 pM). Hyperfibrinolytic trauma patients displayed increased sensitivity to exogenous challenge with tPA: median LY30 of 66.8% compared to 9.6% for controls. Conclusions Depletion of PAI-1 in TIC is driven by an increase in tPA, not PAI-1 degradation. The tPA-challenged TEG, based on this principle, is a functional test for PAI-1 reserves. Exploration of the mechanism of upregulation of tPA is critical to an understanding of hyperfibrinolysis in trauma.

High mobility group box 1 (HMGB1) is a novel late mediator of inflammatory responses that contributes to endotoxin-induced acute lung injury and sepsis-associated lethality. Although acute lung injury is a frequent complication of severe blood loss, the contribution of HMGB1 to organ system dysfunction in this setting has not been investigated. In this study, HMGB1 was detected in pulmonary endothelial cells and macrophages under baseline conditions. After hemorrhage, in addition to positively staining endothelial cells and macrophages, neutrophils expressing HMGB1 were present in the lungs. HMGB1 expression in the lung was found to be increased within 4 h of hemorrhage and then remained elevated for more than 72 h after blood loss. Neutrophils appeared to contribute to the increase in posthemorrhage pulmonary HMGB1 expression since no change in lung HMGB1 levels was found after hemorrhage in mice made neutropenic with cyclophosphamide. Plasma concentrations of HMGB1 also increased after hemorrhage. Blockade of HMGB1 by administration of anti-HMGB1 antibodies prevented hemorrhage-induced increases in nuclear translocation of NF-kappa B in the lungs and pulmonary levels of proinflammatory cytokines, including keratinocyte-derived chemokine, IL-6, and IL-1 beta. Similarly, both the accumulation of neutrophils in the lung as well as enhanced lung permeability were reduced when anti-HMGB1 antibodies were injected after hemorrhage. These results demonstrate that hemorrhage results in increased HMGB1 expression in the lungs, primarily through neutrophil sources, and that HMGB1 participates in hemorrhage-induced acute lung injury.

Neutrophils are critical initiators and effectors of the innate immune system and express Toll-like receptor 2 (TLR2) and TLR4. Although signaling through pathways involving phosphoinositide 3-kinase (PI3-K) and the downstream kinase Akt (protein kinase B) plays a central role in modulating neutrophil chemotaxis and superoxide generation in response to engagement of G protein-coupled receptors, the importance of these kinases in affecting inflammatory responses of neutrophils stimulated through TLR2 has not been examined. In these experiments, we found activation of Akt in neutrophils stimulated with the TLR2-specific ligands peptidoglycan and the lipopeptide tri-palmitoyl-S-glyceryl-Cys-Ser-(Lys)4 that occurred earlier and was of greater magnitude than that present after exposure to the TLR4 agonist LPS. The release of the proinflammatory mediators TNF-α and macrophage inflammatory protein-2 was inhibited in a dose-dependent manner by PI3-K blockade. The IC50 for inhibition of peptidoglycan-stimulated Akt activation and macrophage inflammatory protein-2 release correlated closely, indicating linkage of these two events. PI3-K blockade did not inhibit nuclear translocation of NF-κB, but did prevent Ser536 phosphorylation of the p65 subunit of NF-κB, an event required for maximal transcriptional activity of NF-κB. Inhibition of PI3-K also prevented activation of p38 mitogen-activated protein kinase and extracellular receptor-activated kinase 1/2 in TLR2-stimulated neutrophils. These results demonstrate that the PI3-K-Akt axis occupies a central role in TLR2-induced activation of neutrophils.

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Reactive oxygen species (ROS) can participate in cellular signaling and have been shown to modulate activation of the transcriptional regulatory factor NF-kappaB. However, the effects of ROS can differ in various cell populations. To examine the role of superoxide in neutrophil activation, we exposed resting neutrophils and neutrophils stimulated with LPS to paraquat, an agent that specifically increases intracellular superoxide concentrations. Culture of resting neutrophils with paraquat resulted in increased production of the proinflammatory cytokines TNF-alpha and MIP-2, enhanced degradation of IkappaB-alpha, and increased nuclear accumulation of NF-kappaB. Such effects of paraquat were due to intracellular superoxide (O2-) since they were blocked by the non-specific antioxidant N-acetyl cysteine and the cell permeable superoxide scavenger Tiron, but not by catalase, which facilitates the conversion of H2O2 to H2O and O2. Similar potentiating effects of paraquat were found in LPS-stimulated neutrophils. Exposure of neutrophils to paraquat also enhanced phosphorylation of Ser536 in the p65 subunit of NF-kappaB an event associated with increased transcriptional activity. Examination of kinases critical for LPS-stimulated gene expression showed that addition of paraquat to resting or LPS exposed neutrophils enhanced activation of p38 MAPK, but not that of Akt or ERK1/2. The potentiation of NF-kappaB translocation and proinflammatory cytokine production, but not of Ser536 p65 phosphorylation, by paraquat was dependent on activation of p38 MAPK. These results demonstrate that increased intracellular superoxide concentrations are proinflammatory in neutrophils, acting through a p38 MAPK dependent mechanism that results in enhanced nuclear accumulation of NF-kappaB and increased expression of NF-kappaB dependent proinflammatory cytokines.

Urokinase plasminogen activator (uPA) is a serine protease that catalyzes the conversion of plasminogen to plasmin. In addition, uPA has been shown to have proinflammatory properties, particularly in potentiating lipopolysaccharide (LPS)-induced neutrophil responses. To explore the mechanisms by which uPA exerts these effects, we examined the ability of specific uPA domains to increase cytokine expression in murine and human neutrophils stimulated with LPS. Whereas the addition of intact uPA to neutrophils cultured with LPS increased mRNA and protein levels of interleukin-1beta, macrophage-inflammatory protein-2, and tumor necrosis factor alpha, deletion of the kringle domain (KD) from uPA resulted in loss of these potentiating effects. Addition of purified uPA KD to LPS-stimulated neutrophils increased cytokine expression to a degree comparable with that produced by single-chain uPA. Inclusion of the arginine-glycine-aspartic but not the arginine-glycine-glutamic peptide to neutrophil cultures blocked uPA kringle-induced potentiation of proinflammatory responses, demonstrating that interactions between the KD and integrins were involved. Antibodies to alpha(V) or beta(3) integrins or to the combination of alpha(V)beta(3) prevented uPA kringle-induced enhancement of expression of proinflammatory cytokines and also of adhesion of neutrophils to the uPA KD. These results demonstrate that the KD of uPA, through interaction with alpha(V)beta(3) integrins, potentiates neutrophil activation.

Hypertonic saline (HTS) has been used intravenously to reduce organ dysfunction following injury and as an inhaled therapy for cystic fibrosis lung disease. The role and mechanism of HTS inhibition was explored in the TNFα and IL-1β stimulation of pulmonary epithelial cells. Hyperosmolar (HOsm) media (400 mOsm) inhibited the production of select cytokines stimulated by TNFα and IL-1β at the level of mRNA translation, synthesis and release. In TNFα stimulated A549 cells, HOsm media inhibited I-κBα phosphorylation, NF-κB translocation into the nucleus and NF-κB nuclear binding. In IL-1β stimulated cells HOsm inhibited I-κBα phosphorylation without affecting NF-κB translocation or nuclear binding. Incubation in HOsm conditions inhibited both TNFα and IL-1β stimulated nuclear localization of interferon response factor 1 (IRF-1). Additional transcription factors such as AP-1, Erk-1/2, JNK and STAT-1 were unaffected by HOsm. HTS and sorbitol supplemented media produced comparable outcomes in all experiments, indicating that the effects of HTS were mediated by osmolarity, not by sodium. While not affecting MAPK modules discernibly in A549 cells, both HOsm conditions inhibit IRF-1 against TNFα or IL-1β, but inhibit p65 NF-kB translocation only against TNFα but not IL-1β. Thus, anti-inflammatory mechanisms of HTS/HOsm appear to disrupt cytokine signals at distinct intracellular steps.