A novel role for matrix metalloproteinase-8 in sepsis.

OBJECTIVES: Matrix metalloproteinase-8 messenger RNA expression was previously found to be increased in whole blood of children with septic shock. The impact of this finding on the severity and inflammatory response to sepsis is unknown. Here, we investigate the relationship between matrix metalloproteinase-8 and disease severity in children with septic shock. We further corroborate the role of matrix metalloproteinase-8 in sepsis in a murine model. DESIGN: Retrospective observational clinical study and randomized controlled laboratory experiments. SETTING: Pediatric intensive care units and an animal research facility at an academic children's hospital. PATIENTS AND SUBJECTS: Patients age ≤10 yrs admitted to the intensive care unit with a diagnosis of septic shock. For laboratory studies, we utilized male mice deficient for matrix metalloproteinase-8 and male wild-type C57BL/6J mice. INTERVENTIONS: Blood from children with septic shock was analyzed for matrix metalloproteinase-8 messenger RNA expression and matrix metalloproteinase-8 activity, and correlated with disease severity based on mortality and degree of organ failure. A murine model of sepsis was used to explore the effect of genetic and pharmacologic inhibition of matrix metalloproteinase-8 on the inflammatory response to sepsis. Finally, activation of nuclear factor-κB was assessed both in vitro and in vivo. MEASUREMENTS AND MAIN RESULTS: Increased matrix metalloproteinase-8 mRNA expression and activity in septic shock correlates with decreased survival and increased organ failure in pediatric patients. Genetic and pharmacologic inhibition of matrix metalloproteinase-8 leads to improved survival and a blunted inflammatory profile in a murine model of sepsis. We also identify matrix metalloproteinase-8 as a direct in vitro activator of the proinflammatory transcription factor, nuclear factor-κB. CONCLUSIONS: Matrix metalloproteinase-8 is a novel modulator of inflammation during sepsis and a potential therapeutic target.

The immunomodulatory effects of albumin in vitro and in vivo.

Albumin appears to have proinflammatory effects in vitro. We hypothesized that albumin would induce a state of tolerance to subsequent administration of lipopolysaccharide (LPS) in vitro and in vivo. RAW264.7 and primary peritoneal macrophages were treated with increasing doses of bovine serum albumin (BSA) and harvested for NF-κB luciferase reporter assay or TNF-α ELISA. In separate experiments, RAW264.7 cells were preconditioned with 1 mg/mL BSA for 18 h prior to LPS (10 µg/mL) treatment and harvested for NF-κB luciferase reporter assay or TNF-α ELISA. Finally, C57Bl/6 mice were preconditioned with albumin via intraperitoneal administration 18 h prior to a lethal dose of LPS (60 mg/kg body wt). Blood was collected at 6 h after LPS administration for TNF-α ELISA. Albumin produced a dose-dependent and TLR-4-dependent increase in NF-κB activation and TNF-α gene expression in vitro. Albumin preconditioning abrogated the LPS-mediated increase in NF-κB activation and TNF-α gene expression in vitro and in vivo. The clinical significance of these findings remains to be elucidated.

C-peptide, a novel inhibitor of lung inflammation following hemorrhagic shock.

C-peptide is a 31-amino acid peptide cleaved from proinsulin during insulin synthesis. Initially thought to be inert, C-peptide may modulate the inflammatory response in the setting of endotoxemia and ischemia reperfusion. However, the spectrum of its biological effects is unclear. We hypothesized that exogenous administration of C-peptide would modulate pro- and anti-inflammatory signaling pathways and thereby attenuate lung inflammation in an in vivo model of hemorrhagic shock. Hemorrhagic shock was induced in male Wistar rats (aged 3-4 mo) by withdrawing blood to a mean arterial pressure of 50 mmHg. At 3 h after hemorrhage, rats were rapidly resuscitated by returning their shed blood. At the time of resuscitation and every hour thereafter, animals received C-peptide (280 nmol/kg) or vehicle parenterally. Animals were euthanized at 1 and 3 h after resuscitation. C-peptide administration at resuscitation following hemorrhagic shock ameliorated hypotension and blunted the systemic inflammatory response by reducing plasma levels of IL-1, IL-6, macrophage inflammatory protein-1α, and cytokine-induced neutrophil chemoattractant-1. This was associated with a reduction in lung neutrophil infiltration and plasma levels of receptor for advanced glycation end products. Mechanistically, C-peptide treatment was associated with reduced expression of proinflammatory transcription factors activator protein-1 and NF-κB and activation of the anti-inflammatory transcription factor peroxisome proliferator-activated receptor-γ. Our data suggest that C-peptide ameliorates the inflammatory response and lung inflammation following hemorrhagic shock. These effects may be modulated by altering the balance between pro- and anti-inflammatory signaling in the lung.

C-peptide ameliorates kidney injury following hemorrhagic shock.

Reperfusion injury following hemorrhagic shock is accompanied by the development of a systemic inflammatory state that may lead to organ failure. Insulin connecting peptide (C-peptide) has been shown to exert anti-inflammatory effects in sepsis and myocardial ischemia-reperfusion injury and to ameliorate renal dysfunction in diabetic animals. Hence, we investigated the effect of C-peptide on kidney injury after hemorrhagic shock. We hypothesized that C-peptide would exert renoprotective effects by blunting inflammation. Hemorrhagic shock was induced in male rats (3-4 months old) by withdrawing blood from the femoral artery to a mean arterial pressure of 50 mmHg. Animals were kept in shock for 3 h, at which time they were rapidly resuscitated by returning their shed blood. At the time of resuscitation and every hour thereafter, one group of animals received C-peptide (280 nmol/kg), whereas another group received vehicle. Hemorrhagic shock resulted in significant rise in plasma levels of creatinine and elevated kidney neutrophil infiltration as evaluated by myeloperoxidase activity in vehicle-treated rats in comparison with sham rats, thus suggesting kidney injury. Treatment with C-peptide significantly attenuated the rise in creatinine and kidney myeloperoxidase activity when compared with vehicle group. At a molecular level, these effects of C-peptide were associated with reduced expression of the c-Fos subunit and reduced activation of the proinflammatory kinases, extracellular signal-regulated kinase 1/2 (ERK 1/2), and c-Jun N-terminal kinase and subsequently reduced DNA binding of activator protein 1 in the kidney. Thus, our data suggest that C-peptide may exert renoprotective effects after hemorrhagic shock by modulating activator protein 1 signaling.

Liver X receptor α activation with the synthetic ligand T0901317 reduces lung injury and inflammation after hemorrhage and resuscitation via inhibition of the nuclear factor κB pathway.

Liver X receptor α (LXRα) is a nuclear transcription factor that regulates lipid metabolism. Recently, it has been shown that activation of LXRα with synthetic ligands has anti-inflammatory effects in atherosclerosis and chemical-induced dermatitis. We investigated the effect of the LXRα agonist, T0901317, on lung inflammation in a rodent model of hemorrhagic shock. Hemorrhagic shock was induced in male rats by withdrawing blood to a goal mean arterial blood pressure of 50 mmHg. Blood pressure was maintained at this level for 3 h, at which point rats were rapidly resuscitated with shed blood. Animals were then treated with T0901317 (50 mg · kg) or vehicle i.p. and sacrificed at 1, 2, and 3 h after resuscitation. Treatment with T0901317 significantly improved the cardiac and stroke volume indices as well as the heart rate of rats during the resuscitation period as compared with vehicle-treated rats. The T0901317-treated animals showed significant improvement in the plasma level of lactate, whereas base deficit and bicarbonate levels both trended toward improvement. The T0901317-treated animals also showed lower levels of plasma cytokines and chemokines monocyte chemoattractant protein 1, macrophage inflammatory protein 1α, TNF-α, KC, and IL-6. Lung injury and neutrophil infiltration were reduced by treatment with T0901317, as evaluated by histology and myeloperoxidase assay. At molecular analysis, treatment with T0901317 increased nuclear LXRα expression and DNA binding while also inhibiting activation of nuclear factor κB, a proinflammatory transcription factor, in the lung. Thus, our data suggest that LXRα is an important modulator of the inflammatory response and lung injury after severe hemorrhagic shock, likely through the inhibition of the nuclear factor κB pathway.

Biological activity of truncated C-terminus human heat shock protein 72.

Heat shock protein 72 (Hsp72), a canonical intracellular molecular chaperone, may also function as an extracellular danger signal for the innate immune system. To further delineate the biological role of Hsp72 in the innate immune system, we generated two truncated versions of the full length human Hsp72 (N-terminus Hsp72, amino acids 1-430; and C-terminus Hsp72 amino acids 420-641) and directly compared their ability to activate cells from the macrophage/monocyte lineage. In RAW 264.7 macrophages transfected with a NF-κB-dependent luciferase reporter plasmid, C-terminus Hsp72 was a more potent inducer of NF-κB activity than N-terminus Hsp72, and this effect did not seem to be secondary to endotoxin contamination. C-terminus Hsp72-mediated activation of the NF-κB pathway was corroborated by increased activation of IκB kinase, degradation of IκBα, and increased NF-κB-DNA binding. C-terminus Hsp72 was a more potent inducer of tumor necrosis factor-α (TNFα) expression in RAW 264.7 macrophages and in primary murine peritoneal macrophages from wild-type mice. C-terminus Hsp72 did not induce TNFα expression in primary murine peritoneal macrophages from Toll-like receptor (TLR4) mutant mice, indicating a role for TLR4. In human THP-1 mononuclear cells, C-terminus Hsp72 induced tolerance to subsequent LPS stimulation, whereas N-terminus Hsp72 did not induce tolerance. Finally, control experiments using equimolar amounts of N-terminus or C-terminus Hsp72 demonstrated a higher biological potency for C-terminus Hsp72. These data demonstrate that the ability of human Hsp72 to serve as an activator for cells of the macrophage/monocyte lineage primarily lies in the C-terminus region spanning amino acids 420-641.

Phosphorylation of extracellular signal-regulated kinase (ERK)-1/2 Is associated with the downregulation of peroxisome proliferator-activated receptor (PPAR)-γ during polymicrobial sepsis.

Peroxisome proliferator-activated receptor (PPAR)-γ is a ligand-activated transcription factor and regulates inflammation. Posttranslational modifications regulate the function of PPARγ, potentially affecting inflammation. PPARγ contains a mitogen-activated protein kinase (MAPK) site, and phosphorylation by extracellular signal-regulated kinase (ERK)-1/2 leads to inhibition of PPARγ. This study investigated the kinetics of PPARγ expression and activation in parenchymal and immune cells in sepsis using the MAPK/ERK kinase (MEK)-1 inhibitor, an upstream kinase of ERK1/2. Adult male Sprague Dawley rats were subjected to polymicrobial sepsis by cecal ligation and puncture. Rats received intraperitoneal injection of vehicle or the MEK1 inhibitor PD98059 (5 mg/kg) 30 min before cecal ligation and puncture. Rats were euthanized at 0, 1, 3, 6 and 18 h after cecal ligation and puncture. Control animals used were animals at time 0 h. Lung, plasma and peripheral blood mononuclear cells (PBMCs) were collected for biochemical assays. In vehicle-treated rats, polymicrobial sepsis resulted in significant lung injury. In the lung and PBMCs, nuclear levels of PPARγ were decreased and associated with an increase in phosphorylated PPARγ and phosphorylated ERK1/2 levels. Treatment with the MEK1 inhibitor increased the antiinflammatory plasma adipokine adiponectin, restored PPARγ expression in PBMCs and lung, and decreased lung injury. The inflammatory effects of sepsis cause changes in PPARγ expression and activation, in part, because of phosphorylation of PPARγ by ERK1/2. This phosphorylation can be reversed by ERK1/2 inhibition, thereby improving lung injury.

Peroxisome proliferator-activated receptor {delta} regulates inflammation via NF-{kappa}B signaling in polymicrobial sepsis.

The nuclear peroxisome proliferator-activated receptor δ (PPARδ) is an important regulator of lipid metabolism. In contrast to its known effects on energy homeostasis, its biological role on inflammation is not well understood. We investigated the role of PPARδ in the modulation of the nuclear factor-κB (NF-κB)-driven inflammatory response to polymicrobial sepsis in vivo and in macrophages in vitro. We demonstrated that administration of GW0742, a specific PPARδ ligand, provided beneficial effects to rats subjected to cecal ligation and puncture, as shown by reduced systemic release of pro-inflammatory cytokines and neutrophil infiltration in lung, liver, and cecum, when compared with vehicle treatment. Molecular analysis revealed that treatment with GW0742 reduced NF-κB binding to DNA in lung and liver. In parallel experiments, heterozygous PPARδ-deficient mice suffered exaggerated lethality when subjected to cecal ligation and puncture and exhibited severe lung injury and higher levels of circulating tumor necrosis factor-α (TNFα) and keratinocyte-derived chemokine than wild-type mice. Furthermore, in lipopolysaccharide-stimulated J774.A1 macrophages, GW0742 reduced TNFα production by inhibiting NF-κB activation. RNA silencing of PPARδ abrogated the inhibitory effects of GW0742 on TNFα production. Chromatin immunoprecipitation assays revealed that PPARδ displaced the NF-κB p65 subunit from the κB elements of the TNFα promoter, while recruiting the co-repressor BCL6. These data suggest that PPARδ is a crucial anti-inflammatory regulator, providing a basis for novel sepsis therapies.

Liver apoptosis is age dependent and is reduced by activation of peroxisome proliferator-activated receptor-gamma in hemorrhagic shock.

A clinical observation in pediatric and adult intensive care units is that the incidence of multiple organ failure in pediatric trauma victims is lower than in adult patients. However, the molecular mechanisms are not yet defined. Recent experimental studies have shown that the nuclear peroxisome proliferator-activated receptor-gamma (PPARgamma) modulates the inflammatory process. In this study, we hypothesized that severity of liver injury may be age dependent and PPARgamma activation may provide beneficial effects. Hemorrhagic shock was induced in anesthetized young (3-5 mo old) and mature male Wistar rats (11-13 mo old) by withdrawing blood to a mean arterial blood pressure of 50 mmHg. After 3 h, rats were rapidly resuscitated with shed blood. Animals were euthanized 3 h after resuscitation. In mature rats, liver injury appeared more pronounced compared with young rats and was characterized by marked hepatocyte apoptosis, extravasation of erythrocytes, and accumulation of neutrophils. The ratio between the antiapoptotic protein Bcl-2 and the proapoptotic protein BAX was lower, whereas activity of caspase-3, the executioner of apoptosis, was higher in liver of mature rats compared with young rats. Plasma alanine aminotransferase levels were not different between the two age groups. This heightened liver apoptosis was associated with a significant downregulation of PPARgamma DNA binding in mature rats compared with young rats. Treatment with the PPARgamma ligand ciglitazone significantly reduced liver apoptosis in mature rats. Our data suggest that liver injury after severe hemorrhage is age dependent and PPARgamma activation is a novel hepatoprotective mechanism.

Changes in peroxisome proliferator-activated receptor-gamma activity in children with septic shock.

PURPOSE: To assess changes in peroxisome proliferator-activated receptor-gamma (PPARgamma) in peripheral blood mononuclear cells (PBMC) from critically ill children with sepsis. Additionally, to investigate the effects of sepsis on the endogenous activator of PPARgamma, 15-deoxy-(12,14)-PGJ(2) (15d-PGJ(2)), and the downstream targets of PPARgamma activity, adiponectin and resistin. METHODS: Single-center, prospective case-control study in critically ill children with systemic inflammatory response syndrome, sepsis or septic shock. RESULTS: PPARgamma nuclear protein expression was decreased but PPARgamma activity was increased in PBMC from children with septic shock compared with controls. PPARgamma activity on day 1 was significantly higher in patients with higher pediatric risk of mortality (PRISM) score compared with controls [mean 0.22 optical density (OD) +/- standard error of the mean (SEM) 0.03 versus 0.12 OD +/- 0.02; p < 0.001]. Patients with resolved sepsis had increased levels of the endogenous PPARgamma ligand, 15d-PGJ(2), compared with patients with systemic inflammatory response syndrome (SIRS) and septic shock (77.7 +/- 21.7 versus 58 +/- 16.5 pg/ml; p = 0.03). Plasma high-molecular-weight adiponectin (HMWA) and resistin levels were increased in patients with septic shock on day 1 and were significantly higher in patients with higher PRISM scores. Nonsurvivors from sepsis had higher resistin levels on the first day of hospitalization compared with survivors from septic shock [660 ng/ml, interquartile range (IQR) 585-833 ng/ml versus 143 ng/ml, IQR 66-342 ng/ml; p < 0.05]. CONCLUSIONS: Sepsis is associated with altered PPARgamma expression and activity in PBMC. Plasma adipokines correlate with risk of mortality scores in sepsis and may be useful biomarkers. Further studies are needed to understand the mechanisms underlying changes in PPARgamma in sepsis.

Ciglitazone ameliorates lung inflammation by modulating the inhibitor kappaB protein kinase/nuclear factor-kappaB pathway after hemorrhagic shock.

OBJECTIVE: Peroxisome proliferator-activated receptor-gamma is a ligand-activated transcription factor. Ciglitazone, a peroxisome proliferator-activated receptor-gamma ligand, has been shown to provide beneficial effects in experimental models of sepsis and ischemia/reperfusion injury. We investigated the effects of ciglitazone on lung inflammation after severe hemorrhage. DESIGN: Prospective, laboratory study, rodent model of hemorrhagic shock. SETTING: University hospital laboratory. SUBJECTS: Male rats. INTERVENTIONS: Hemorrhagic shock was induced by withdrawing blood to a mean arterial pressure of 50 mm Hg. At 3 hrs after hemorrhage, rats were rapidly resuscitated by returning their shed blood. At the time of resuscitation and every hour thereafter, animals received ciglitazone (10 mg/kg) or vehicle intraperitoneally. Heart rate and mean arterial pressure were measured throughout the experiment. Plasma and lung tissue were collected for analysis up to 3 hrs after resuscitation. MEASUREMENTS AND MAIN RESULTS: Ciglitazone treatment ameliorated mean arterial pressure, reduced lung injury, significantly blunted lung neutrophil infiltration, and lowered plasma interleukin-6, interleukin-10, and monocyte chemoattractant protein-1 levels. In a time course analysis, vehicle-treated rats had a significant increase in nuclear factor-kappaB DNA binding, which was preceded by increased inhibitor kappaB protein kinase activity and inhibitor kappaB alpha degradation in the lung. Treatment with ciglitazone significantly reduced inhibitor kappaB protein kinase activity and inhibitor kappaB alpha degradation and completely inhibited nuclear factor-kappaB DNA binding. This reduction of inhibitor kappaB protein kinase activity afforded by ciglitazone appeared to be a consequence of a physical interaction between peroxisome proliferator-activated receptor-gamma and increased inhibitor kappaB protein kinase. CONCLUSION: Ciglitazone ameliorates the inflammatory response and may reduce lung injury after hemorrhagic shock. These protective effects appear to be mediated through inhibition of the inhibitor kappaB protein kinase/nuclear factor-kappaB pathway.

Induction of endotoxin tolerance enhances bacterial clearance and survival in murine polymicrobial sepsis.

The fundamental mechanisms that underlie endotoxin tolerance remain to be elucidated, and the clinical significance of endotoxin tolerance in the context of active systemic infection remains in question. We hypothesized that the endotoxin tolerance phenotype would result in decreased inflammation at the expense of altered bacterial clearance and, thus, higher mortality in a murine model of polymicrobial sepsis induced by cecal ligation and puncture (CLP). Endotoxin tolerance was induced in C57Bl/6 mice with 5 mg/kg LPS or vehicle 18 h before subsequent CLP. Lung tissue, peritoneal fluid, and blood were collected at 1, 3, 6, and 18 h after surgery for subsequent analysis. Peritoneal macrophages were isolated for ex vivo phagocytosis assay. In separate experiments, mice were allowed to recover, and survival was monitored for 7 days. Endotoxin tolerance attenuated plasma TNF-alpha and IL-6 at 6 h after CLP. Peritoneal fluid cytokines were significantly attenuated as well. Endotoxin tolerance significantly improved bacterial clearance in both blood and peritoneal fluid after CLP. Similarly, ex vivo phagocytosis by primary peritoneal macrophages and RAW264.7 murine peritoneal macrophages was significantly improved after induction of the endotoxin tolerance phenotype. Contrary to our original hypothesis, we conclude that endotoxin tolerance significantly attenuates the host inflammatory response, augments bacterial clearance, and improves survival in this murine model of polymicrobial sepsis.

PP2A regulates upstream members of the c-jun N-terminal kinase mitogen-activated protein kinase signaling pathway.

We have previously demonstrated that inhibition of the serine-threonine phosphatase PP2A resulted in increased c-jun N-terminal kinase (JNK) activity, and that the regulatory subunit, A/alpha of PP2A, was physically associated with the JNK. Because there exists additional examples of phosphatases serving as negative regulators of multiple members of mitogen-activated protein kinase (MAPK) pathways in Drosophila and yeast, we hypothesized that PP2A may serve a homologous function in mammalian cells affording the regulation of additional upstream kinases in the JNK pathway. In human monocytes, activation of JNK by LPS proceeds through the MAPK kinase kinase MEKK-1 and, subsequently, the MAPK kinases MKK4 and/or MKK7. Using the human monocyte cell line THP-1, we show that pharmacological manipulation of the activity of PP2A seemed to regulate not only JNK but also the upstream kinases MKK4 and MEKK-1. Using coimmunoprecipitation, overexpression of tagged recombinant JNK, and bacterial two-hybrid strategies, evidence for physical interactions between the structural subunit, PP2A-A/alpha and MEKK-1, MKK4, and JNK was observed. These studies suggest that the target of regulation by PP2A includes upstream kinases in the JNK MAPK pathway. Furthermore, PP2A-A/alpha seems to serve as a structural protein to foster protein-protein interactions affording specificity of the regulation among members of this MAP kinase pathway.

Diverse cardioprotective signaling mechanisms of peroxisome proliferator-activated receptor-gamma ligands, 15-deoxy-Delta12,14-prostaglandin J2 and ciglitazone, in reperfusion injury: role of nuclear factor-kappaB, heat shock factor 1, and Akt.

Peroxisome proliferator-activated receptor-gamma (PPAR-gamma) is a nuclear receptor that regulates diverse biological functions including inflammation. The PPARgamma ligands have been reported to exert cardioprotective effects and attenuate myocardial reperfusion injury. Here, we examined the molecular mechanisms of their anti-inflammatory effects. Male Wistar rats were subjected to myocardial ischemia and reperfusion and were treated with the PPAR-gamma ligands, 15-deoxy-Delta-prostaglandin J2 (15d-PGJ2) or ciglitazone, or with vehicle only, in the absence or presence of the selective PPAR-gamma antagonist GW-9662. In vehicle-treated rats, myocardial injury was associated with elevated tissue activity of myeloperoxidase, indicating infiltration of neutrophils, and elevated plasma levels of creatine kinase and tumor necrosis factor-alpha. These events were preceded by activation of the nuclear factor-kappaB pathway. The PPAR-gamma DNA binding was also increased in the heart after reperfusion. Treatment with ciglitazone or 15d-PGJ2 reduced myocardial damage and neutrophil infiltration and blunted creatine kinase levels and cytokine production. The beneficial effects of both ligands were associated with enhancement of PPAR-gamma DNA binding and reduction of nuclear factor-kappaB activation. Treatment with 15d-PGJ2, but not ciglitazone, enhanced DNA binding of heat shock factor 1 and upregulated the expression of the cardioprotective heat shock protein 70. Treatment with 15d-PGJ2, but not ciglitazone, also induced a significant increase in nuclear phosphorylation of the prosurvival kinase Akt. The cardioprotection afforded by ciglitazone was attenuated by the PPAR-gamma antagonist GW-9662. In contrast, GW-9662 did not affect the beneficial effects afforded by 15d-PGJ2. Thus, our data suggest that treatment with these chemically unrelated PPAR-gamma ligands results in diverse anti-inflammatory mechanisms.

The green tea polyphenol epigallocatechin-3-gallate improves systemic hemodynamics and survival in rodent models of polymicrobial sepsis.

Epigallocatechin-3-gallate (EGCG) is the main polyphenolic flavonoid found in green tea. Recent in vitro studies have suggested that EGCG inhibits activation of the nuclear factor-kappaB (NF-kappaB) pathway. The NF-kappaB is a transcriptional factor required for gene expression of many inflammatory mediators, including the inducible isoform of nitric oxide synthase (NOS2). Excessive NO production by NOS2 is directly linked to the vasoplegia, shock, and mortality associated with sepsis. Accordingly, we hypothesized that EGCG administration would inhibit NOS2 gene expression and thereby improve survival in a rodent model of polymicrobial sepsis. Polymicrobial sepsis was induced in male Sprague-Dawley rats (hemodynamic study) and C57BL6 mice (mortality study) via cecal ligation and double puncture (CL2P). Rodents were treated with either EGCG (10 mg/kg intraperitoneally) or vehicle at 1 and 6 h after CL2P and every 12 h thereafter. In the hemodynamic study, mean arterial blood pressure was monitored for 18 h, and rats were killed at 3, 6, and 18 h after CL2P. In the mortality study, survival was monitored for 72 h after CL2P in mice. In vehicle-treated rodents, CL2P was associated with profound hypotension and greater than 80% mortality rate. Epigallocatechin-3-gallate treatment significantly improved both the hypotension and survival. In vitro experiments further showed that EGCG inhibited activation of NF-kappaB and subsequent NOS2 gene expression in a primary culture of rat aortic smooth muscle cells. Epigallocatechin-3-gallate may therefore represent a potential nutritional supplement or pharmacologic agent in patients with sepsis.

Proinsulin c-peptide exerts beneficial effects in endotoxic shock in mice.

OBJECTIVE: Insulin connecting peptide (c-peptide) aids the folding of proinsulin and has been considered to have little biological activity. Recently, c-peptide has been shown to improve diabetic neuropathy and nephropathy as well as vascular inflammation. In vitro studies have reported that c-peptide may activate peroxisome proliferator-activated receptor-gamma, a nuclear transcription factor that plays a regulatory role in inflammation. This study was designed to investigate the biological effects of c-peptide during endotoxemia. DESIGN: Prospective, randomized laboratory investigation that used an established murine model of endotoxic shock. SETTING: University hospital laboratory. SUBJECTS: Mice were subjected to endotoxic shock by intraperitoneal administration of Escherichia coli lipopolysaccharide. INTERVENTIONS: Mice received vehicle or c-peptide (70-140 nmol/kg) intraperitoneally at 3 hrs and 6 hrs after lipopolysaccharide. Mortality was monitored for 96 hrs. In a separate experiment, mice were killed at 4, 7, and 18 hrs after lipopolysaccharide administration. Lungs and plasma were collected for biochemical assays. MEASUREMENTS AND MAIN RESULTS: In vehicle-treated mice, endotoxic shock resulted in lung injury and was associated with a 41% survival rate and elevation in plasma tumor necrosis factor-alpha, macrophage inflammatory protein-1alpha, monocyte chemoattractant protein-1, and keratinocyte-derived chemokine levels. Lung nuclear levels of phosphorylated extracellular signal-regulated kinases 1 and 2 were significantly increased in vehicle-treated mice. On the other hand, lung nuclear expression and DNA binding of proliferator-activated receptor-gamma were decreased in comparison to control animals. Treatment with c-peptide (140 nmol/kg) improved survival rate (68%) and reduced plasma levels of tumor necrosis factor-alpha, macrophage inflammatory protein-1alpha, and monocyte chemoattractant protein-1, but it did not exert hypoglycemic effects. Treatment with c-peptide also up-regulated lung nuclear expression and DNA binding of proliferator-activated receptor-gamma and reduced phosphorylation of extracellular signal-regulated kinases 1 and 2 in comparison to vehicle-treated mice. CONCLUSIONS: Our data show that c-peptide has beneficial effects in endotoxic shock, and this therapeutic effect is associated with activation of proliferator-activated receptor-gamma.

Hepatocyte NF-kappaB activation is hepatoprotective during ischemia-reperfusion injury and is augmented by ischemic hypothermia.

The present study examined the role of hepatocyte NF-kappaB activation during ischemia-reperfusion injury. Second, we evaluated the effects of ischemic hypothermia on NF-kappaB activation and liver injury. C57BL/6 mice underwent 90 min of partial hepatic ischemia and up to 8 h of reperfusion. Body temperature was regulated during the ischemic period between 35 and 37 degrees C, 33 and 35 degrees C, 29 and 33 degrees C or unregulated, where temperature fell to <29 degrees C. Liver injury, as measured by serum alanine aminotransferase as well as liver histopathology, was inversely proportional to regulated body temperature, with the unregulated group (<29 degrees C) being highly protected and the normothermic group (35-37 degrees C) displaying the greatest injury. Inflammation, as measured by production of TNF-alpha and liver recruitment of neutrophils, was greatest in the normothermic groups and lowest in the ischemic hypothermia groups. Interestingly, hepatocyte NF-kappaB activation was highest in the hypothermic group and least in the normothermic group. Paradoxically, degradation of IkappaB proteins, IkappaB-alpha and IkappaB-beta, was greatest in the normothermic group, suggesting an alternate NF-kappaB regulatory mechanism during ischemia-reperfusion injury. Subsequently, we found that NF-kappaB p65 protein was increasingly degraded in normothermic versus hypothermic groups, and this degradation was specific for hepatocytes and was associated with decreased expression of the peptidyl-prolyl isomerase Pin1. The data suggest that NF-kappaB activation in hepatocytes is a protective response during ischemia-reperfusion and can be augmented by ischemic hypothermia. Furthermore, it appears that Pin1 promotes NF-kappaB p65 protein stability such that decreased expression of Pin1 during ischemia-reperfusion results in p65 degradation, reduced nuclear translocation of NF-kappaB, and enhanced hepatocellular injury.

Induction of HSP70 is dispensable for anti-inflammatory action of heat shock or NSAIDs in mast cells.

OBJECTIVE: It is well known that nonsteroidal anti-inflammatory drugs (NSAIDs), such as acetylsalicylic acid, ibuprofen, and indomethacin, induce anti-inflammatory effects through inhibition of cyclooxygenase enzyme activity. However, it has also been established that a variety of their anti-inflammatory effects are independent of cyclooxygenase. In the search for alternative modes of action, it was found that NSAIDs share some cellular effects with heat shock treatment. This prompted us to investigate whether NSAIDs modulate production of proinflammatory cytokines by mast cells through the heat shock response. MATERIALS AND METHODS: In mouse mast cells, derived from a culture of bone marrow cells of male BALB/cBy and null HSF-1(-/-) mice, responsiveness to heat shock and NSAIDs was monitored by measuring tumor necrosis factor-alpha (TNF-alpha) and interleukin-6 (IL-6) production and signaling pathways. RESULTS: In bone marrow-derived mast cells (BMMC), we found that heat shock and a number of NSAIDs induced heat shock protein 70 (HSP70), which was closely paralleled with inhibition of IL-6 and TNF-alpha production. Surprisingly, in BMMC from HSF-1(-/-)mice, heat shock and selected NSAIDs were still able to suppress cytokine production in the absence of HSP70 induction. CONCLUSION: In this article, we provide evidence that inhibition of release of proinflammatory cytokines by NSAIDs and heat shock may be attributed to inhibition of the inhibitory nuclear factor kappaB (NF-kappaB) kinase activity, extracellular signal-regulated kinases 1/2, and p38 pathways, resulting in decreased transcriptional activity of the NF-kappaB pathway.

Immunoregulatory effects of regulated, lung-targeted expression of IL-10 in vivo.

Regulation of pulmonary inflammation involves an intricate balance of both pro- and anti-inflammatory mediators. Acute lung injury can result from direct pulmonary insults that activate alveolar macrophages to respond with increased cytokine expression. Such cytokine gene expression is mediated in part via NF-kappaB. IL-10 has been previously identified as an important endogenous anti-inflammatory cytokine in vivo on the basis of inhibiting NF-kappaB activation; however, the mechanism of this inhibition remains incompletely defined. We hypothesized that IL-10 regulated NF-kappaB activation in vivo via IkappaK inhibition. A bitransgenic mouse that allowed for externally regulated, lung-specific human IL-10 overexpression was generated. In the bitransgenic mice, introduction of doxycycline induced lung-specific, human IL-10 overexpression. Acute induction of IL-10 resulted in significant decreases in bronchoalveolar lavage fluid neutrophils (48%, P = 0.03) and TNF (62%, P < 0.01) following intratracheal LPS compared with bitransgenic negative mice. In vitro kinase assays showed this decrease to correlate to diminished lung IkappaK activity. Furthermore, we also examined the effect of chronic IL-10 overexpression in these transgenic mice. Results show that IL-10 overexpression in lungs of mature mice increased the number of intrapulmonary cells the phenotype of which was skewed toward increased B220+/CD45+ B cells and CD4+ T cells and was associated with increased CC chemokine expression. Thus regulated, lung-specific IL-10 overexpression may have a variety of complex immunologic effects depending on the timing and duration of expression.

Contribution of MKP-1 regulation of p38 to endotoxin tolerance.

Endotoxin tolerance has been characterized as diminished TNF-alpha expression after a second LPS stimulus and is dependent on new protein synthesis. LPS-induced expression of TNF-alpha is partly regulated by the p38 mitogen-activated protein (MAP) kinase, which post-transcriptionally stabilizes TNF-alpha mRNA. The dual-specific phosphatase, MKP-1, has been shown to negatively regulate p38 via dephosphorylation. We hypothesized that MKP-1 expression induced during tolerance regulates TNF-alpha expression by inhibiting p38 activity. To test this hypothesis, tolerance was induced in THP-1 cells, and naive or tolerized cells were rechallenged 18 h later with LPS (1 microg/mL) and TNF-alpha production was measured. Under similar conditions, nuclear proteins were isolated after LPS stimulation and were analyzed for phospho-p38 and MKP-1 by Western blot. Transient overexpression of MKP-1 was achieved using an adenoviral expression strategy and infected cells subsequently treated with LPS for TNF-alpha production and p38 activation. Results showed that LPS tolerance was induced as reflected by decreased TNF-alpha. Induction of LPS hyporesponsiveness could be mimicked by overexpression of MKP-1 but not beta-gal. MKP-1 expression was noted only in LPS-tolerized or Ad-MKP-1 infected cells. In the canonical and Ad-MKP-1-mediated tolerance models, decreased phospho-p38 activity was observed. MKP-1s role in mediating endotoxin tolerance was further confirmed by demonstrating the inability to fully tolerize peritoneal macrophages isolated from MKP-1 null mutant (vs. wild type) mice (24% vs. 72% reductions, respectively). These data demonstrate that the dual specific phosphatase MKP-1 is an important mediator of endotoxin tolerance via p38 regulation.