Antimicrobial peptide LL-37 ameliorates a murine sepsis model via the induction of microvesicle release from neutrophils.

Sepsis is a life-threatening disease caused by systemic dys-regulated inflammatory response to infection. We previously revealed that LL-37, a human cathelicidin antimicrobial peptide, improves the survival of cecal ligation and puncture septic mice. Ectosomes, microvesicles released from neutrophils, are reported to be elevated in sepsis survivors; however, the functions of ectosomes in sepsis remain largely unknown. Therefore, we herein elucidated the protective action of LL-37 on sepsis, by focusing on LL-37-induced ectosome release in a cecal ligation and puncture model. The results demonstrated the enhancement of ectosome levels by LL-37 administration, accompanied by a reduction of bacterial load. Importantly, ectosomes isolated from LL-37-injected cecal ligation and puncture mice contained higher amounts of antimicrobial proteins/peptides and exhibited higher antibacterial activity, compared with those from PBS-injected cecal ligation and puncture mice, suggesting that LL-37 induces the release of ectosomes with antibacterial potential in vivo. Actually, LL-37 stimulated mouse bone-marrow neutrophils to release ectosomes ex vivo, and the LL-37-induced ectosomes possessed antibacterial potential. Furthermore, administration of LL-37-induced ectosomes reduced the bacterial load and improved the survival of cecal ligation and puncture mice. Together these observations suggest LL-37 induces the release of antimicrobial ectosomes in cecal ligation and puncture mice, thereby reducing the bacterial load and protecting mice from lethal septic conditions.

MrgX2­mediated internalization of LL­37 and degranulation of human LAD2 mast cells.

LL­37 is the sole antimicrobial peptide of human cathelicidin comprising 37 amino acids, which is expressed mainly in epithelial cells and neutrophils, and activates mast cells. In the present study, in order to elucidate the mechanism of mast cell activation by LL­37, the associations between the internalization of LL­37 and Mas­related gene X2 (MrgX2)­mediated mast cell activation (degranulation) was investigated using the human mast cell line, LAD2. LL­37 was rapidly internalized into the cells, and induced degranulation, as assessed by the extracellular release of ß­hexosaminidase. Pertussis toxin, a G­protein inhibitor, significantly suppressed the internalization of LL­37 and the degranulation of LAD2 cells. Furthermore, small interfering (si)­RNA­mediated knockdown of MrgX2, a putative G protein­coupled receptor for LL­37, inhibited the internalization of LL­37 and degranulation of LAD2 cells. Notably, LL­37 internalization was enhanced by the stable expression of MrgX2 in HMC­1 and 293 cells. In addition, the internalized LL­37 mainly colocalized with MrgX2 in the perinuclear region of LAD2 cells. Furthermore, neuraminidase treatment, which removes negatively charged sialic acid from the cell surface, markedly reduced the internalization of LL­37 and degranulation of LAD2 cells, and clathrin­mediated endocytosis inhibitors (dynasore and chlorpromazine) inhibited the internalization and degranulation of LAD2 cells. Taken together, these observations indicated that LL­37 may bind the negatively charged cell surface molecules, rapidly internalize into the cells via clathrin­mediated endocytosis and interact with MrgX2 to activate mast cells (LAD2 cells).

Protection of the endothelial glycocalyx by antithrombin in an endotoxin-induced rat model of sepsis.

INTRODUCTION: Injury and loss of the endothelial glycocalyx occur during the early phase of sepsis. We previously showed that antithrombin has a protective effect on this structure in vitro. Here, we investigated the possible protective effects of antithrombin in an animal model of sepsis. METHODS: Wistar rats were injected with endotoxin, and circulating levels of syndecan-1, hyaluronan, albumin, lactate and other biomarkers were measured in an antithrombin-treated group and an untreated control group (n = 6 in each group). Intravital microscopy was used to observe leukocyte adhesion, microcirculation, and syndecan-1 staining. RESULTS: The circulating levels of syndecan-1 and hyaluronan were significantly reduced in the antithrombin-treated group, compared with the untreated controls. Lactate levels and albumin reduction were significantly attenuated in the antithrombin-treated group. Intravital microscopic observation revealed that both leukocyte adhesion and blood flow were better maintained in the treatment group. The syndecan-1 lining was disrupted after endotoxin treatment, and this derangement was attenuated by treatment with antithrombin. CONCLUSION: Antithrombin effectively maintained microcirculation and vascular integrity by protecting the glycocalyx in a rat sepsis model.

Evaluation of the effect of recombinant thrombomodulin on a lipopolysaccharide-induced murine sepsis model.

To evaluate the effect of recombinant human thrombomodulin (rTM) on sepsis, the levels of nucleosome as well as high-mobility group box 1 (HMGB1) and cytokines in sera and peritoneal fluids were measured in a mouse model of lipopolysaccharide (LPS)-induced sepsis after administration of rTM. C57BL/6 mice were intraperitoneally injected with LPS (15 mg/kg; Escherichia coli O111:B4) with or without the intravenous administration of rTM (3 mg/kg; 30 min prior to or 2 h after LPS injection). The survival rates were evaluated and levels of tumor necrosis factor (TNF)-α, interleukin (IL)-6, monocyte chemoattractant protein (MCP)-1, HMGB1 and nucleosome in sera and peritoneal fluids were analyzed by ELISA. Administration of rTM prior to or after LPS improved the survival rate of septic mice. In addition, rTM administered prior to or after LPS suppressed the level of pro-inflammatory cytokine TNF-α in sera at 1-3 h after LPS injection, whereas only the administration of rTM after LPS suppressed the levels of HMGB1 and nucleosome (late-phase mediators of sepsis) (9-12 h) in sera after the LPS injection. Furthermore, administration of rTM prior to or after LPS suppressed the level of TNF-α in the peritoneal fluids at 1-3 h after LPS injection, whereas only the administration of rTM after LPS suppressed the levels of IL-6 and MCP-1 in the peritoneal fluids at 6-9 h after LPS injection. These observations indicated that administration of rTM significantly improves the survival rate and suppresses the increased levels of TNF-α, IL-6, MCP-1, HMGB1 and nucleosome in the LPS-induced septic shock model. Thus, rTM may exert a protective action on sepsis and reduce mortality, possibly by reducing not only the levels of cytokines and chemokine but also the levels of late-phase mediators of sepsis.

Ketamine suppresses the substance P-induced production of IL-6 and IL-8 by human U373MG glioblastoma/astrocytoma cells.

The neuropeptide substance P (SP) is an important mediator of neurogenic inflammation within the central and peripheral nervous systems. SP has been shown to induce the expression of pro-inflammatory cytokines implicated in the pathogenesis of several disorders of the human brain via the neurokinin-1 receptor (NK-1R). Ketamine, an intravenous anesthetic agent, functions as a competitive antagonist of the excitatory neurotransmission N-methyl-D­aspartate (NMDA) receptor, and also antagonizes the NK-1R by interfering with the binding of SP. In the present study, we investigated the anti-inflammatory effects of ketamine on the SP-induced activation of a human astrocytoma cell line, U373MG, which expresses high levels of NK-1R. The results from our experiments indicated that ketamine suppressed the production of interleukin (IL)-6 and IL-8 by the U373MG cells. Furthermore, ketamine inhibited the SP-induced activation of extracellular signal­regulated kinase (ERK)1/2, p38 mitogen-activated protein kinase (MAPK) and nuclear factor-κB (NF-κB). Taken together, these observations suggest that ketamine may suppress the SP-induced activation (IL-6 and IL-8 production) of U373MG cells by inhibiting the phosphorylation of signaling molecules (namely ERK1/2, p38 MAPK and NF-κB), thereby exerting anti­inflammatory effects. Thus, ketamine may modulate SP-induced inflammatory responses by NK-1R­expressing cells through the suppression of signaling molecules (such as ERK1/2, p38 MAPK and NF-κB).

Neutrophil extracellular traps induce IL-1ß production by macrophages in combination with lipopolysaccharide.

Upon exposure to invading microorganisms, neutrophils undergo NETosis, a recently identified type of programmed cell death, and release neutrophil extracellular traps (NETs). NETs are described as an antimicrobial mechanism, based on the fact that NETs can trap microorganisms and exhibit bactericidal activity through the action of NET­associated components. In contrast, the components of NETs have been recognized as damage­associated molecular pattern molecules (DAMPs), which trigger inflammatory signals to induce cell death, inflammation and organ failure. In the present study, to clarify the effect of NETs on cytokine production by macrophages, mouse macrophage­like J774 cells were treated with NETs in combination with lipopolysaccharide (LPS) as a constituent of pathogen­associated molecular patterns. The results revealed that NETs significantly induced the production of interleukin (IL)­1ß by J774 cells in the presence of LPS. Notably, the NET/LPS­induced IL­1ß production was inhibited by both caspase­1 and caspase­8 inhibitors. Furthermore, nucleases and serine protease inhibitors but not anti­histone antibodies significantly inhibited the NET/LPS­induced IL­1ß production. Moreover, we confirmed that caspase­1 and caspase­8 were activated by NETs/LPS, and the combination of LPS, DNA and neutrophil elastase induced IL­1ß production in reconstitution experiments. These observations indicate that NETs induce the production of IL­1ß by J774 macrophages in combination with LPS via the caspase­1 and caspase­8 pathways, and NET­associated DNA and serine proteases are involved in NET/LPS­induced IL­1ß production as essential components.

Release mechanism of high mobility group nucleosome binding domain 1 from lipopolysaccharide-stimulated macrophages.

Alarmins are identified as endogenous mediators that have potent immune-activating abilities. High mobility group nucleosome binding domain 1 (HMGN1), a highly conserved, non-histone chromosomal protein, which binds to the inner side of the nucleosomal DNA, regulates chromatin dynamics and transcription in cells. Furthermore, HMGN1 acts as a cytokine in the extracellular milieu by inducing the recruitment and maturation of antigen-presenting cells (dendritic cells) to enhance Th1-type antigen-specific immune responses. Thus, HMGN1 is expected to act as an alarmin, when released into the extracellular milieu. The present study investigated the release mechanism of HMGN1 from macrophages using mouse macrophage­like RAW264.7 cells. The results indicated that HMGN1 was released from lipopolysaccharide (LPS)­stimulated RAW264.7 cells, accompanied by cell death as assessed by the release of lactate dehydrogenase (LDH). Subsequently, the patterns of cell death involved in HMGN1 release from LPS­stimulated RAW264.7 cells were determined using a caspase­1 inhibitor, YVAD, and a necroptosis inhibitor, Nec­1. YVAD and Nec­1 did not alter LPS­induced HMGN1 and LDH release, suggesting that pyroptosis (caspase­1­activated cell death) and necroptosis are not involved in the release of HMGN1 from LPS­stimulated RAW264.7 cells. In addition, flow cytometric analysis indicated that LPS stimulation did not induce apoptosis but substantially augmented necrosis, as evidenced by staining with annexin V/propidium iodide. Together these findings suggest that HMGN1 is extracellularly released from LPS­stimulated RAW264.7 macrophage­like cells, accompanied by unprogrammed necrotic cell death but not pyroptosis, necroptosis or apoptosis.

Human Host Defense Cathelicidin Peptide LL-37 Enhances the Lipopolysaccharide Uptake by Liver Sinusoidal Endothelial Cells without Cell Activation.

The liver is a major organ that removes waste substances from the blood, and liver sinusoidal endothelial cells (LSECs) are professional scavenger cells, which incorporate and degrade various endogenous and exogenous molecules including pathogenic factor LPS. Mammalian cells express a number of peptide antibiotics that function as effectors in the innate host defense systems. LL-37, a human cathelicidin antimicrobial peptide, has a potent LPS-neutralizing activity and exhibits protective actions on various infection models. However, the effect of LL-37 on the LPS clearance has not been clarified. In this study, to further understand the host-protective mechanism of LL-37, we evaluated the effect of LL-37 on the LPS clearance in vitro. LL-37 enhanced the LPS uptake by human LSECs. Of interest, LL-37 was similarly incorporated into LSECs both in the presence and the absence of LPS, and the incorporated LPS and LL-37 were colocalized in LSECs. Importantly, the uptake of LPS and LL-37 was inhibited by endocytosis inhibitors, heparan sulfate proteoglycan analogs, and glycosaminoglycan lyase treatment of the cells. Moreover, the uptake of LL-37-LPS did not activate TLR4 signaling in both MyD88-dependent and -independent pathways. In addition, the incorporated LL-37-LPS was likely transported to the lysosomes in LSECs. Together these observations suggest that LL-37 enhances the LPS uptake by LSECs via endocytosis through the complex formation with LPS and the interaction with cell-surface heparan sulfate proteoglycans, thereby facilitating the intracellular incorporation and degradation of LPS without cell activation. In this article, we propose a novel function of LL-37 in enhancing LPS clearance.

Antimicrobial cathelicidin peptide LL-37 inhibits the pyroptosis of macrophages and improves the survival of polybacterial septic mice.

LL-37 is the only known member of the cathelicidin family of antimicrobial peptides in humans. In addition to its broad spectrum of antimicrobial activities, LL-37 can modulate various inflammatory reactions. We previously revealed that LL-37 suppresses the LPS/ATP-induced pyroptosis of macrophages in vitro by both neutralizing the action of LPS and inhibiting the response of P2X7 (a nucleotide receptor) to ATP. Thus, in this study, we further evaluated the effect of LL-37 on pyroptosis in vivo using a cecal ligation and puncture (CLP) sepsis model. As a result, the intravenous administration of LL-37 improved the survival of the CLP septic mice. Interestingly, LL-37 inhibited the CLP-induced caspase-1 activation and pyroptosis of peritoneal macrophages. Moreover, LL-37 modulated the levels of inflammatory cytokines (IL-1ß, IL-6 and TNF-α) in both peritoneal fluids and sera, and suppressed the activation of peritoneal macrophages (as evidenced by the increase in the intracellular levels of IL-1ß, IL-6 and TNF-α). Finally, LL-37 reduced the bacterial burdens in both peritoneal fluids and blood samples. Together, these observations suggest that LL-37 improves the survival of CLP septic mice by possibly suppressing the pyroptosis of macrophages, and inflammatory cytokine production by activated macrophages and bacterial growth. Thus, the present findings imply that LL-37 can be a promising candidate for sepsis because of its many functions, such as the inhibition of pyroptosis, modulation of inflammatory cytokine production and antimicrobial activity.

Effects of anesthesia with sevoflurane and propofol on the cytokine/chemokine production at the airway epithelium during esophagectomy.

Post-operative pulmonary complications such as pneumonia, acute lung injury (ALI) and acute respiratory distress syndrome (ARDS) are closely associated with morbidity and mortality after esophagectomy. One lung ventilation (OLV) is commonly used during esophagectomy. However, the effect of the anesthetic agents on the inflammatory response induced by OLV has yet to be evaluated, particularly during esophagectomy, which causes several complications in the lung. The aim of the present study was to determine the effects of anesthetic agents, such as sevoflurane or propofol, on the inflammatory reactions at the airway. Twenty patients undergoing esophagectomy were randomized to receive either sevoflurane (n=10) or propofol (n=10) as a main anesthetic agent. Epithelial lining fluid (ELF) was obtained from ventilated­dependent lung (DL) and collapsed non-dependent lung (NDL) by a bronchoscopic microsampling method. The levels of inflammatory cytokines and chemokine [tumor necrosis factor (TNF)-α, interleukin (IL)-1ß, IL-6, IL-8, IL-10 and IL-12p70] in the ELF were measured using multiplexed bead-based immunoassays before and after OLV. The results indicated that the levels of IL-6 in ELF were significantly increased in both the ventilated DL and collapsed NDL after OLV compared with the levels prior to OLV in the sevoflurane group. By contrast, there was no significant change in the IL-6 levels in the propofol group in the ventilated DL and collapsed NDL before and after OLV. Similarly, IL-8 levels were markedly increased in the ventilated DL and collapsed NDL after OLV compared with those before OLV in the sevoflurane group, whereas there was no significant change in IL-8 levels in the propofol group in the ventilated DL and collapsed NDL before and after OLV. In contrast to the changes in IL-6 and IL-8 levels, levels of IL-10, an anti-inflammatory cytokine, were not obviously changed in both the ventilated DL and collapsed NDL before and after OLV in the sevoflurane group. However, IL-10 levels in the propofol group were increased in the ventilated DL and collapsed NDL after OLV compared with those before OLV. Of note, the levels of TNF-α, IL-1ß and IL-12p70 in ELF were below the detection limits. These observations suggested that propofol anesthesia more potently suppresses the surgical stress-induced inflammatory perturbation at the local milieu of the airway during esophagectomy compared with sevoflurane anesthesia.

Antimicrobial cathelicidin peptide LL-37 inhibits the LPS/ATP-induced pyroptosis of macrophages by dual mechanism.

Pyroptosis is a caspase-1 dependent cell death, associated with proinflammatory cytokine production, and is considered to play a crucial role in sepsis. Pyroptosis is induced by the two distinct stimuli, microbial PAMPs (pathogen associated molecular patterns) and endogenous DAMPs (damage associated molecular patterns). Importantly, cathelicidin-related AMPs (antimicrobial peptides) have a role in innate immune defense. Notably, human cathelicidin LL-37 exhibits the protective effect on the septic animal models. Thus, in this study, to elucidate the mechanism for the protective action of LL-37 on sepsis, we utilized LPS (lipopolysaccharide) and ATP (adenosine triphosphate) as a PAMP and a DAMP, respectively, and examined the effect of LL-37 on the LPS/ATP-induced pyroptosis of macrophage-like J774 cells. The data indicated that the stimulation of J774 cells with LPS and ATP induces the features of pyroptosis, including the expression of IL-1ß mRNA and protein, activation of caspase-1, inflammasome formation and cell death. Moreover, LL-37 inhibits the LPS/ATP-induced IL-1ß expression, caspase-1 activation, inflammasome formation, as well as cell death. Notably, LL-37 suppressed the LPS binding to target cells and ATP-induced/P2X7-mediated caspase-1 activation. Together these observations suggest that LL-37 potently inhibits the LPS/ATP-induced pyroptosis by both neutralizing the action of LPS and inhibiting the response of P2X7 to ATP. Thus, the present finding may provide a novel insight into the modulation of sepsis utilizing LL-37 with a dual action on the LPS binding and P2X7 activation.

Effects of nitrous oxide on the production of cytokines and chemokines by the airway epithelium during anesthesia with sevoflurane and propofol.

The aim of this study was to evaluate the effects of nitrous oxide (a gaseous anesthetic) on the in vivo production of inflammatory cytokines and chemokines by the airway epithelium, when combined with sevoflurane or propofol. Subjects undergoing simple or segmental mastectomy were randomly assigned to the sevoflurane and nitrous oxide, sevoflurane and air, propofol and nitrous oxide, or propofol and air group (all n=13). Epithelial lining fluid (ELF) was obtained using the bronchoscopic microsampling method prior to and following the mastectomy to enable measurement of the pre- and post-operative levels of certain inflammatory cytokines and chemokines using a cytometric bead array system. Notably, the levels of interleukin (IL)-1ß, IL-8 and monocyte chemotactic protein-1 (MCP-1) in the ELF were significantly increased following the operations which involved the inhalation of sevoflurane and nitrous oxide, although the levels of these molecules were not significantly changed by the inhalation of sevoflurane and air. Furthermore, the IL-12p70 levels were significantly reduced in the ELF following the operations that involved the inhalation of sevoflurane and air, although the IL-12p70 levels were not significantly changed by the inhalation of nitrous oxide and sevoflurane. These observations suggest that the combination of sevoflurane and nitrous oxide induces an inflammatory response (increased production of IL-1ß, IL-8 and MCP-1) and suppresses the anti-inflammatory response (reduced production of IL-12p70) in the local milieu of the airway. Thus, the combination of these compounds should be carefully administered for anesthesia.

Comparative analysis of hepatic CD14 expression between two different endotoxin shock model mice: relation between hepatic injury and CD14 expression.

CD14 is a glycoprotein that recognizes gram-negative bacterial lipopolysaccharide (LPS) and exists in both membrane-bound and soluble forms. Infectious and/or inflammatory diseases induce CD14 expression, which may be involved in the pathology of endotoxin shock. We previously found that the expression of CD14 protein differs among the endotoxin shock models used, although the reasons for these differences are unclear. We hypothesized that the differences in CD14 expression might be due to liver injury, because the hepatic tissue produces CD14 protein. We investigated CD14 expression in the plasma and liver in the carrageenan (CAR)-primed and D-galN-primed mouse models of endotoxin shock. Our results showed that severe liver injury was not induced in CAR-primed endotoxin shock model mice. In this CAR-primed model, the higher mRNA and protein expression of CD14 was observed in the liver, especially in the interlobular bile duct in contrast to D-galN-primed-endotoxin shock model mice. Our findings indicated that the molecular mechanism(s) underlying septic shock in CAR-primed and D-galN-primed endotoxin shock models are quite different. Because CD14 expression is correlated with clinical observations, the CAR-primed endotoxin shock model might be useful for studying the functions of CD14 during septic shock in vivo.

Effects of sevoflurane and propofol on pulmonary inflammatory responses during lung resection.

PURPOSE: Pulmonary inflammatory reactions are affected by one-lung ventilation (OLV) and anesthetic agents. However, the effects of anesthetic agents on pulmonary inflammatory reactions may vary. Our previous investigations suggested that inflammatory reactions were more pronounced in the dependent lung during lung resection under general anesthesia with propofol and remifentanil. Therefore, in the present study we attempted to determine the difference in pulmonary inflammatory reaction using either sevoflurane or propofol in both dependent and nondependent lungs during OLV. METHODS: Forty adult patients undergoing elective lung resection were randomized to receive either propofol (n = 20) or sevoflurane (n = 20) as the main anesthetic agent. Intraoperative analgesia was provided by remifentanil in both groups. Epithelial lining fluid (ELF) was obtained from each lung using a bronchoscopic microsampling method. ELF and plasma levels of inflammatory cytokines were measured using multiplexed bead-based immunoassays before and after OLV. RESULTS: Epithelial lining fluid levels of interleukin (IL)-1ß, IL-6, and IL-8 were significantly increased in the dependent lung and the nondependent lung after OLV compared with baseline levels (P < 0.05). Moreover, IL-6 ELF level in the dependent lung was significantly higher in the propofol group than in the sevoflurane group after OLV (P < 0.001). CONCLUSION: One-lung ventilation induced inflammatory responses of the bronchial epithelia in the dependent lung and the nondependent lung during lung resection. Moreover, this inflammatory response was significantly suppressed by sevoflurane compared with propofol. Furthermore, the antiinflammatory effect of sevoflurane was more pronounced in the dependent lung than in the nondependent lung during OLV.

Human anti-microbial cathelicidin peptide LL-37 suppresses the LPS-induced apoptosis of endothelial cells.

Sepsis is a systemic disease resulting from harmful host response to bacterial infections. During the exacerbation of severe sepsis or septic shock, apoptosis of endothelial cells is induced in susceptible organs such as the lung and liver and triggers microcirculatory disorder and organ dysfunction. LPS, an outer membrane component of Gram-negative bacteria, is one of the major virulence factors for the pathogenesis. We previously reported that LL-37, a human anti-microbial cathelicidin peptide, potently neutralizes the biological activity of LPS and protects mice from lethal endotoxin shock. However, the effect of LL-37 on the LPS-induced endothelial cell apoptosis remains to be clarified. In this study, to further elucidate the action of LL-37 on severe sepsis/endotoxin shock, we investigated the effects of LL-37 on the LPS-induced endothelial cell apoptosis in vitro and in vivo using lung-derived normal human microvascular blood vessel endothelial cells (HMVEC-LBls) and D-galactosamine hydrochloride (D-GalN)-sensitized murine endotoxin shock model. LL-37 suppressed the LPS-induced apoptosis of HMVEC-LBls. In addition, LL-37 inhibited the binding of LPS possibly to the LPS receptors (CD14 and toll-like receptor 4) expressed on the cells. Thus, LL-37 can suppress the LPS-induced apoptosis of HMVEC-LBls via the inhibition of LPS binding to the cells. Furthermore, LL-37 drastically suppressed the apoptosis of hepatic endothelial cells as well as hepatocytes in the liver of murine endotoxin shock model. Together, these observations suggest that LL-37 could suppress the LPS-induced apoptosis of endothelial cells, thereby attenuating lethal sepsis/endotoxin shock.

The effect of one-lung ventilation upon pulmonary inflammatory responses during lung resection.

PURPOSE: One-lung ventilation (OLV) is commonly used during thoracic surgery. Clinical studies using bronchoalveolar lavage fluid analysis have demonstrated that OLV induces pulmonary inflammatory reactions in the ventilated dependent lung. However, few clinical studies have investigated such inflammatory reactions in the dependent lung compared with the collapsed nondependent lung. Here we used a bronchoscopic microsampling method to obtain epithelial lining fluid (ELF) from each lung, and then compared the inflammatory reactions in the dependent lung and the nondependent lung during thoracic surgery. METHODS: Twenty adult patients were studied. All patients underwent thoracic surgery using OLV. Propofol and remifentanil were used for total intravenous anesthesia. A double-lumen endotracheal tube was used to perform OLV. ELF was obtained from each lung using the bronchoscopic microsampling method. ELF levels of inflammatory mediators, tumor necrosis factor α, interleukin (IL)-1ß, IL-6, IL-8, IL-10, and IL-12p70 were measured using ELISA before and after OLV. RESULTS: ELF levels of IL-1ß, IL-6, and IL-8 were significantly increased in the dependent lung and the nondependent lung at the end of surgery compared with their baseline levels (p < 0.05). ELF level of IL-6 was significantly higher in the dependent lung than in the nondependent lung at the end of surgery (p = 0.019). CONCLUSIONS: One-lung ventilation induced inflammatory responses of the bronchial epithelia in the dependent lung and the nondependent lung during thoracic surgery. In addition, these inflammatory responses were more augmented in the dependent lung than in the nondependent lung.

Suppressive action of resolvin D1 on the production and release of septic mediators in D-galactosamine-sensitized endotoxin shock mice.

Endotoxin/septic shock is a severe condition induced during serious infections with Gram-negative bacteria. To evaluate the therapeutic potential of resolvin D1 (RvD1), a novel pro-resolving molecule, on endotoxin/septic shock, we investigated the effect of RvD1 on the extracellular release of high mobility group box-1 (HMGB1), the production of inflammatory cytokines, the accumulation of peritoneal cells and hepatocyte apoptosis in vivo using a D-galactosamine (GalN)-sensitized mouse endotoxin shock model. Serum HMGB1 levels were markedly elevated after challenge with lipopolysaccharide (LPS)/D-GalN, and RvD1 administration significantly reduced HMGB1 levels. Furthermore, the serum levels of inflammatory cytokines, such as TNF-α, IL-6, IL-10 and macrophage chemotactic protein (MCP)-1 were elevated in the endotoxin shock model. Importantly, RvD1 administration slightly reduced the TNF-α, IL-6 and IL-10 levels, and further lowered MCP-1 levels. Moreover, RvD1 administration affected the peritoneal cell accumulation and decreased the neutrophil population. Finally, LPS/D-GalN injection induced apoptosis in the liver (mostly of hepatocytes), and RvD1 administration reduced the apoptosis of hepatocytes. These observations suggest that RvD1 may be a therapeutic agent for sepsis/endotoxin shock by exerting suppressive action on the release and production of septic mediators (HMGB1 and inflammatory cytokines), the accumulation of peritoneal cells and hepatic apoptosis.

Evaluation of the effect of α-defensin human neutrophil peptides on neutrophil apoptosis.

Peptide antibiotics possess potent antimicrobial activities against invading micro-organisms and contribute to the innate host defense. Antimicrobial α-defensin human neutrophil peptides (HNPs) not only exhibit potent bactericidal activities against Gram-negative and -positive bacteria but also function as immunomodulatory molecules by inducing cytokine and chemokine production, as well as inflammatory and immune cell activation. Neutrophil is a critical effector cell in host defense against microbial infection, and its lifespan is regulated by various pathogen- and host-derived substances. Here, in order to further evaluate the role of HNPs in innate immunity, we investigated the action of HNPs-1 to -3 on neutrophil apoptosis. Neutrophil apoptosis was assessed using human blood neutrophils based on the morphological changes. Of note, HNP-1 most potently suppressed neutrophil apoptosis among HNPs-1 to -3, accompanied by the down-regulation of truncated Bid (a pro-apoptotic protein), the up-regulation of Bcl-xL (an anti-apoptotic protein), and the inhibition of mitochondrial membrane potential change and caspase 3 activity. It should be noted that, a selective P2Y6 antagonist, MRS2578, abolished the suppression of neutrophil apoptosis elicited by HNP-1 as well as UDP (a P2Y6 ligand). Collectively, these observations suggest that HNPs, especially HNP-1, can not only destroy bacteria but also modulate (suppress) neutrophil apoptosis via the P2Y6 signaling pathway. The suppression of neutrophil apoptosis results in the prolongation of their lifespan and could be advantageous for the host defense against bacterial invasion.

Antimicrobial cathelicidin polypeptide CAP11 suppresses the production and release of septic mediators in D-galactosamine-sensitized endotoxin shock mice.

Endotoxin shock is a severe systemic inflammatory response that is caused by the augmented production and release of septic mediators. Among them, inflammatory cytokines such as tumor necrosis factor-alpha, IL-1beta and IL-6 play a pivotal role. In addition, anandamide, an endogenous cannabinoid and high-mobility group box-1 (HMGB1), a non-histone chromosomal protein has recently been recognized as members of septic mediators. We previously reported that cationic antibacterial polypeptide of 11-kDa (CAP11), an antimicrobial cathelicidin peptide (originally isolated from guinea pig neutrophils), potently neutralizes the biological activity of LPS and protects mice from lethal endotoxin shock. In this study, to clarify the protective mechanism of CAP11 against endotoxin shock, we evaluated the effects of CAP11 on the production and release of septic mediators in vitro and in vivo using a murine macrophage cell line RAW264.7 and a D-galactosamine-sensitized murine endotoxin shock model. LPS stimulation induced the production of inflammatory cytokines and anandamide and release of HMGB1 from RAW264.7 cells. Importantly, CAP11 suppressed the LPS-induced production and release of these mediators by RAW264.7 cells. Moreover, LPS administration enhanced the serum levels of HMGB1, anandamide and inflammatory cytokines in the endotoxin shock model. Of note, CAP11 suppressed the LPS-induced increase of these mediators in sera, and LPS binding to CD14-positive cells (peritoneal macrophages), accompanied with the increase of survival rates. Together these observations suggest that the protective action of CAP11 on endotoxin shock may be explained by its suppressive effect on the production and release of septic mediators by CD14-positive cells possibly via the inhibition of LPS binding to the targets.

Antimicrobial cathelicidin peptide CAP11 suppresses HMGB1 release from lipopolysaccharide-stimulated mononuclear phagocytes via the prevention of necrotic cell death.

High mobility group box-1 (HMGB1) is extracellularly released from mononuclear phagocytes by lipopolysaccharide (LPS)-stimulation accompanied with cell death, and plays an important role in septic/endotoxin shock as a late phase mediator. Notably, CAP11 (cationic antibacterial polypeptide of 11-kDa), a member of cathelicidin family of antimicrobial peptides, has a potential to bind with LPS and neutralize the biological activity of LPS. In this context, we previously revealed that CAP11 can suppress the elevation of serum HMGB1 level in mouse endotoxin shock model and protect mice from endotoxin lethality. In the present study, to clarify the inhibitory mechanism of CAP11 on HMGB1 release, we evaluated the effect of CAP11 on the LPS-induced HMGB1 release and apoptotic/necrotic cell death using a murine macrophage cell line RAW264.7. The results indicated that LPS-stimulation induced the release of HMGB1 from RAW264.7 cells, accompanied with both apoptotic and necrotic cell death. Of interest, CAP11 markedly inhibited the binding of LPS to target RAW264.7 cells, and suppressed HMGB1 release as well as necrotic cell death; however, CAP11 could not affect the LPS-induced apoptotic cell death. These observations clearly indicate that CAP11 can efficiently abolish necrotic cell death via the inhibition of LPS-binding to target cells, thereby suppressing the release of HMGB1. Thus, CAP11 could be a therapeutic agent for septic/endotoxin shock, with a potential to regulate the release of HMGB1 from LPS-stimulated mononuclear phagocytes via the suppression of LPS-binding to target cells and prevention of necrotic cell death due to its potent LPS-binding activity.