Endotoxin Lethality Research Articles

Enhanced susceptibility to lipopolysaccharide-induced arthritis and endotoxin shock in interleukin-32 alpha transgenic mice through induction of tumor necrosis factor alpha

IntroductionThe present study assessed the potential functions of interleukin (IL)-32α on inflammatory arthritis and endotoxin shock models using IL-32α transgenic (Tg) mice. The potential signaling pathway for the IL-32-tumor necrosis factor (TNF)α axis was analyzed in vitro.MethodsIL-32α Tg mice were generated under control of a ubiquitous promoter. Two disease models were used to examine in vivo effects of overexpressed IL-32α: Toll-like receptor (TLR) ligand-induced arthritis developed using a single injection of lipopolysaccharide (LPS) or zymosan into the knee joints; and endotoxin shock induced with intraperitoneal injection of LPS and D-galactosamine. TNFα antagonist etanercept was administered simultaneously with LPS in some mice. Using RAW264.7 cells, in vitro effects of exogenous IL-32α on TNFα, IL-6 or macrophage inflammatory protein 2 (MIP-2) production were assessed with or without inhibitors for nuclear factor kappa B (NFκB) or mitogen-activated protein kinase (MAPK).ResultsSingle injection of LPS, but not zymosan, resulted in development of severe synovitis with substantial articular cartilage degradation in knees of the Tg mice. The expression of TNFα mRNA in inflamed synovia was highly upregulated in the LPS-injected Tg mice. Moreover, the Tg mice were more susceptive to endotoxin-induced lethality than the wild-type control mice 48 hours after LPS challenge; but blockade of TNFα by etanercept protected from endotoxin lethality. In cultured bone marrow cells derived from the Tg mice, overexpressed IL-32α accelerated production of TNFα upon stimulation with LPS. Of note, exogenously added IL-32α alone stimulated RAW264.7 cells to express TNFα, IL-6, and MIP-2 mRNAs. Particularly, IL-32α -induced TNFα, but not IL-6 or MIP-2, was inhibited by dehydroxymethylepoxyquinomicin (DHMEQ) and U0126, which are specific inhibitors of nuclear factor kappa B (NFκB) and extracellular signal regulated kinase1/2 (ERK1/2), respectively.ConclusionsThese results show that IL-32α contributed to the development of inflammatory arthritis and endotoxin lethality. Stimulation of TLR signaling with LPS appeared indispensable for activating the IL-32α-TNFα axis in vivo. However, IL-32α alone induced TNFα production in RAW264.7 cells through phosphorylation of inhibitor kappa B (IκB) and ERK1/2 MAPK. Further studies on the potential involvement of IL-32α-TNFα axis will be beneficial in better understanding the pathology of autoimmune-related arthritis and infectious immunity.

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.

MTORC1-S6K Activation by Endotoxin Contributes to Cytokine Up-Regulation and Early Lethality in Animals

BackgroundmTORC1 (mammalian target of rapamycin complex 1) activation has been demonstrated in response to endotoxin challenge, but the mechanism and significance are unclear. We investigated the effect of mTORC1 suppression in an animal model of endotoxemia and in a cellular model of endotoxin signaling.Methodology/Principal FindingsMice were treated with the mTORC1 inhibitor rapamycin or vehicle prior to lethal endotoxin challenge. Mortality and cytokine levels were assessed. Cultured macrophage-like cells were challenged with endotoxin with or without inhibitors of various pathways known to be upstream of mTORC1. Activated pathways, including downstream S6K pathway, were assessed by immunoblots. We found that mTORC1-S6K suppression by rapamycin delayed mortality of mice challenged with lethal endotoxin, and was associated with dampened circulating levels of VEGF, IL-1β, IFN-γ and IL-5. Furthermore, in vitro cellular studies demonstrated that LPS (lipopolysaccharide) activation of mTORC1-S6K still occurs in the presence of PI3K-Akt inhibition alone, but can be suppressed by concurrent inhibition of PI3K-Akt and MEK-ERK pathways.Conclusions/SignificanceWe conclude that cellular activation of mTORC1-S6K contributes to cytokine up-regulation and mortality in response to endotoxin, and may occur via multiple pathways.

Role of MKP-1 in Osteoclasts and Bone Homeostasis

Bone mass is maintained through the complementary activities of osteoblasts and osteoclasts; yet differentiation of either osteoblasts and osteoclasts engages the mitogen-activated protein kinase (MAPK) pathway. The MAPKs are negatively regulated by a family of dual-specificity phosphatases known as the MAPK phosphatases (MKPs). MKP-1 is a stress-responsive MKP that inactivates the MAPKs and plays a central role in macrophages; however, whether MKP-1 plays a role in the maintenance of bone mass has yet to be investigated. We show here, using a genetic approach, that mkp-1(-/-) female mice exhibited slightly reduced bone mass. We found that mkp-1(+/+) and mkp-1(-/-) mice had equivalent levels of bone loss after ovariectomy despite mkp-1(-/-) mice having fewer osteoclasts, suggesting that mkp-1(-/-) osteoclasts are hyperactive. Indeed, deletion of MKP1 led to a profound activation of osteoclasts in vivo in response to local lipopolysaccharide (LPS) injection. These results suggest a role for MKP-1 in osteoclasts, which originate from the fusion of macrophages. In support of these observations, receptor activator for nuclear factor-kappaB ligand induced the expression for MKP-1, and osteoclasts derived from mkp-1(-/-) mice had increased resorptive activity. Finally, receptor activator of nuclear factor-kappaB ligand-induced p38 MAPK and c-Jun NH2-terminal kinase activities were enhanced in osteoclasts derived from mkp-1(-/-) mice. Taken together, these results show that MKP-1 plays a role in the maintenance of bone mass and does so by negatively regulating MAPK-dependent osteoclast signaling.

CHOLINE OR CDP-CHOLINE ALTERS SERUM LIPID RESPONSES TO ENDOTOXIN IN DOGS AND RATS: INVOLVEMENT OF THE PERIPHERAL NICOTINIC ACETYLCHOLINE RECEPTORS

We showed previously that choline administration protects dogs from endotoxin-induced multiple organ injury and platelet dysfunctions. Because sepsis/endotoxemia is associated with alterations in lipid metabolism, we have investigated whether choline or cytidine-5'-diphosphate choline, a choline donor, alters serum lipid responses to endotoxin in dogs and rats. In response to endotoxin, serum concentrations of triglycerides, choline-containing phospholipids, total cholesterol, and high-density lipoprotein cholesterol increased in a dose- and time-related manner. Administration of choline (20 mg/kg i.v. in dogs or 90 mg/kg i.p. in rats) or cytidine-5'-diphosphate choline (70 mg/kg i.v. in dogs) 5 min before and 4 and 8 h after endotoxin blocked or attenuated the increases in serum triglycerides, total cholesterol, and nonesterified fatty acids. Endotoxin-induced elevations in serum phospholipid levels did not change in rats and were enhanced in dogs by choline. In rats, serum lipid response to endotoxin was accompanied by severalfold elevations in serum levels of hepatorenal injury markers; their elevations were also blocked by choline. Pretreatment with hexamethonium blocked choline's effects on serum lipids and hepatorenal injury markers. Pretreatment with atropine blocked endotoxin-induced elevations in serum lipid and hepatorenal injury markers, but failed to alter choline's actions on these parameters. Choline treatment improved survival rate of rats in lethal endotoxin shock. In conclusion, these data show that choline treatment alters serum lipid responses to endotoxin and prevents hepatorenal injury during endotoxemia through a nicotinic acetylcholine receptor-mediated mechanism. Hence, choline and choline-containing compounds may have a therapeutic potential in the treatment of endotoxemia/sepsis.

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.

Pretreatment with R(+)-verapamil significantly reduces mortality and cytokine expression in murine model of septic shock

It is well known that cytokines play an important role in the pathogenesis of sepsis and septic shock. There is evidence indicating that the membrane transporter, P-glycoprotein (P-gp), may be involved in the release of cytokines, such as IL-2, IL-4 or IFN-γ. The aim of this study was to assess the influence of P-gp inhibitor, R(+)-verapamil, on cytokine expression in serum and tissues as well as survival rate of mice with LPS-induced septic shock. These effects were compared with the response to treatment with pentoxifylline, lisofylline, and prednisolone administered alone or after pretreatment with R(+)-verapamil. When given as a single agent, R(+)-verapamil significantly decreased serum levels of TNF-α and IFN-γ and protected mice from endotoxin lethality. Moreover, it decreased up-regulated by LPS TNF-α gene expression in the liver and lungs. Given concomitantly with immunomodulatory compounds, it enhanced their beneficial impact on the survival of mice with septic shock. The highest increase in survival rate was observed in combination with pentoxifylline (7% vs. 67%). The most striking differences observed between saline and R(+)-verapamil pretreated animals on combination therapy included down-regulation of TNF-α, higher levels of IL-6, and decreased IFN-γ concentrations. These results suggest that P-gp may be involved in the release of IFN-γ, and possibly also TNF-α, in mice with septic shock. R(+)verapamil improves survival of mice receiving a lethal dose of LPS and significantly potentiates the protective effect of pentoxifylline and prednisolone against LPS-induced lethality, probably as a result of both P-gp inhibition and a synergistic interaction at the gene level.

Immune Complex/Ig Negatively Regulate TLR4-Triggered Inflammatory Response in Macrophages through FcγRIIb-Dependent PGE2 Production

Excessive activation of TLR may induce endotoxin shock and inflammatory diseases, so the negative regulation of TLR-triggered inflammatory response attracts much attention. Nonpathogenic immune complex (IC) and Ig (IC/Ig) have been shown to play important roles in the regulation of immune responses and to be therapeutic in some kinds of autoimmune diseases. However, the role of IC/Ig in the regulation of TLR-triggered inflammatory responses and the underlying mechanisms remain to be fully understood. In this study we demonstrate that IC/Ig can significantly inhibit LPS-induced secretion of TNF-alpha and IL-6 from macrophages by preferentially inducing PGE(2). Pretreatment of mice with IC can protect wild-type mice, but not Fc gammaRIIb(-/-) mice, from lethal endotoxin shock, and significantly reduce the levels of serum TNF-alpha and IL-6 in wild-type mice but not in Fc gammaR IIb(-/-) mice. Furthermore, blockade of PGE(2) by celecoxib restores LPS-induced production of TNF-alpha and IL-6 in the presence of IC both in vitro and in vivo. Accordingly, blockade of PGE(2) production in vivo results in the increased sensitivity of IC-pretreated mice to lethal endotoxin shock. Therefore, IC/Ig can negatively regulate TLR4-triggered inflammatory response in macrophages through Fc gammaRIIb-dependent PGE(2). In addition, our results suggest that down-regulation of NF-kappaB activation and TLR4 expression but activation of protein kinase A pathway in macrophages by IC/Ig contribute to the negative regulatory process. Thus we provide new manner for the immune regulation and mechanistic explanation for nonpathogenic IC/Ig in the treatment of inflammatory or autoimmune diseases.

Caffeic acid phenethyl ester protects mice from lethal endotoxin shock and inhibits lipopolysaccharide-induced cyclooxygenase-2 and inducible nitric oxide synthase expression in raw 264.7 macrophages via the P38/ERK and NF-KB pathways

Caffeic acid phenethyl ester protects mice from lethal endotoxin shock and inhibits lipopolysaccharide-induced cyclooxygenase-2 and inducible nitric oxide synthase expression in raw 264.7 macrophages via the P38/ERK and NF-KB pathways

Proteolytic Cleavage of High Mobility Group Box 1 Protein by Thrombin-Thrombomodulin Complexes

High mobility group box 1 protein (HMGB1) was identified as a mediator of endotoxin lethality. We previously reported that thrombomodulin (TM), an endothelial thrombin-binding protein, bound to HMGB1, thereby protecting mice from lethal endotoxemia. However, the fate of HMGB1 bound to TM remains to be elucidated. TM enhanced thrombin-mediated cleavage of HMGB1. N-terminal amino acid sequence analysis of the HMGB1 degradation product demonstrated that thrombin cleaved HMGB1 at the Arg10-Gly11 bond. Concomitant with the cleavage of the N-terminal domain of HMGB1, proinflammatory activity of HMGB1 was significantly decreased (P<0.01). HMGB1 degradation products were detected in the serum of endotoxemic mice and in the plasma of septic patients with disseminated intravascular coagulation (DIC), indicating that HMGB1 could be degraded under conditions in which proteases were activated in the systemic circulation. TM not only binds to HMGB1 but also aids the proteolytic cleavage of HMGB1 by thrombin. These findings highlight the novel antiinflammatory role of TM, in which thrombin-TM complexes degrade HMGB1 to a less proinflammatory form.

Blockage of receptor-interacting protein 2 expression by small interfering RNA in murine macrophages

This study aims to demonstrate that blocking the receptor-interacting protein2 (Rip2) expression can decrease inflammatory cytokine production by macrophage and protect mice from endotoxin lethality. Murine Rip2 small interfering RNA (siRNA) plasmids were constructed and transfected into macrophage and Rip2 expression was detected with reverse transcription-polymerase chain reaction (RT-PCR) and western blot. Cell proliferation was assayed with MTT. TNF-α concentration was assayed with ELISA and high-mobility group box 1 protein (HMGB1) level with semi-quantitative western blot after lipopolysaccharide (LPS) stimulation. LPS challenge was given after the plasmids were injected into mice and the survival rate was calculated. Rip2 siRNA plasmid could block the mRNA and protein expression of Rip2 and promote cell proliferation. Blocking Rip2 could attenuate LPS-induced TNF-α and HMGB1 production. The HMGB1 expression in the liver decreased to (40.21 ± 11.03) pg/g, and serum TNF-α level decreased to (300.43 ± 59.26) ng/L (P < 0.05). The survival rate of mice from endotoxemia was also improved (P < 0.05). The results demonstrate that Rip2 siRNA plasmid can block the expression of Rip2, decrease the production of TNF-α and HMGB1 and protect mice from fatal endotoxemia.

Neuropeptide Modulators of High Mobility Group Box 1 Secretion as Potential Therapeutic Agents for Severe Sepsis

This Commentary describes a study suggesting that the endogenous neuropeptides vasoactive intestinal peptide (VIP) and urocortin can improve survival in an animal model of lethal sepsis, possibly through their ability to decrease circulating levels of the cytokine-like protein high mobility group box 1 (HMGB1).

High Mobility Group Box Protein-1 Correlates with Renal Function in Chronic Kidney Disease (CKD)

Chronic kidney disease (CKD) is associated with inflammation and malnutrition and carries a markedly increased risk of cardiovascular disease (CVD). High Mobility Group Box Protein-1 (HMGB-1) is a 30-kDa nuclear and cytosolic protein known as a transcription and growth factor, recently identified as a proinflammatory mediator of tissue injury. Recent data implicates HMGB-1 in endotoxin lethality, rheumatoid arthritis, and atherosclerosis. The aim of this post-hoc, cross-sectional study was to determine whether HMGB-1 serum levels are elevated in CKD patients. The study groups were categorized as follows: 110 patients starting dialysis defined as CKD 5; 67 patients with moderately to severely reduced renal function or CKD 3-4; and 48 healthy controls. High-sensitivity C-reactive-protein (hs-CRP), interleukin-6 (IL-6), tumor necrosis factor (TNF), serum-albumin (S-albumin), hemoglobin A(1c) (HbA(1c)), hemoglobin, subjective global nutritional assessment (SGA), and glomerular filtration rate (GFR) were analyzed. Kruskal-Wallis test was used to compare groups and Spearman's rank correlation test was used for continuous variables. HMGB-1, measured by Western blot, was significantly (P < 0.001) elevated in CKD 5 (146.7 +/- 58.6 ng/mL) and CKD 3-4 (85.6 +/- 31.8) compared with controls (10.9 +/- 10.5). HMGB-1 levels were correlated positively with TNF (Rho = 0.52; P < 0.001), hs-CRP (Rho = 0.38; P < 0.001), IL-6 (Rho = 0.30; P < 0.001), HbA(1c) (Rho = 0.14; P = 0.02) and SGA (Rho = 0.21; P = 0.002) and negatively correlated with GFR (Rho = -0.69; P = 0.0001), Hb (Rho = -0.60; P < 0.001), S-albumin (Rho = -0.31; P < 0.001). The current study has revealed that HMGB-1 is elevated significantly in CKD patients and correlates with GFR as well as markers of inflammation and malnutrition. Future studies may delineate whether HMGB-1 is also a marker of disease activity and severity as well as a predictor of outcome in CKD.

Lipopolysaccharide induces apoptosis in Carassius auratus lymphocytes, a possible role in pathogenesis of bacterial infection in fish

Lipopolysaccharide (LPS), the endotoxin of Gram-negative bacteria, is capable of eliciting a wide variety of pathophysiological effects, including endotoxin shock, tissue injury and lethality in both humans and animals. It is also a potent stimulant to initiate the proliferation, differentiation and activation of B lymphocytes and macrophages, resulting in changes of inflammatory cytokines, such as TNF-alpha, IL1-beta, IL6, IL-8 and IL-12, and enhancement of immune responses. However, little is known about its effect on the induction of apoptosis in lymphocytes. In the present study, the lymphocytes from Carassius auratus were employed for this purpose. The cells were exposed to LPS at various doses for different time periods. By careful apoptotic characteristic analysis, such as condensation of nuclear chromatin, fragmentation of genomic DNA and formation of apoptotic bodies, it provided the first evidence that LPS had apoptotic-inducing effect on fish lymphocytes in a time- and dose-dependent manner. LPS exposure induced significant increase of intracellular reactive oxygen species (ROS), loss of mitochondrial transmembrane potential (DeltaPsi), depletion of ATP production, down-regulation of Bcl-2 expression, up-regulation of Bax and mitochondrial NO-synthase (mNOS) expression, and selective activation of caspase-9 rather than caspase-8. Each of these observations suggests that the LPS-induced apoptosis in C. auratus lymphocytes occurs largely via the mitochondrial apoptotic pathway. This observation was different from the mechanism behind the LPS-induced apoptosis in mammalian macrophages/thymocytes that occurs via the TNF-alpha-mediated death-receptor pathway. Our study suggested the existence of a possible novel role in the pathogenesis of Gram-negative bacterial infection in fish and even in mammals, which may contribute to the therapy of bacterial diseases. Also, it will help to gain more insights into the mechanisms of septic shock and of LPS-induced immunosuppression and autoimmunity.

Caffeic acid phenethyl ester protects mice from lethal endotoxin shock and inhibits lipopolysaccharide-induced cyclooxygenase-2 and inducible nitric oxide synthase expression in RAW 264.7 macrophages via the p38/ERK and NF-κB pathways

Caffeic acid phenethyl ester has been shown to have anti-inflammatory and anti-cancer effects. We examined the effects of caffeic acid phenethyl ester on lipopolysaccharide-induced production of nitric oxide and prostaglandin E 2, and expression of inducible nitric oxide synthase and cyclooxygenase-2 in RAW 264.7 macrophages. We also investigated the effects of caffeic acid phenethyl ester on lipopolysaccharide-induced septic shock in mice. Our results indicate that caffeic acid phenethyl ester inhibits lipopolysaccharide-induced nitric oxide and prostaglandin E 2 production in a concentration-dependent manner and inhibits inducible nitric oxide synthase and cyclooxygenase-2 in RAW 264.7 cells, without significant cytotoxicity. To further examine the mechanism responsible for the inhibition of inducible nitric oxide synthase and cyclooxygenase-2 expression by caffeic acid phenethyl ester, we examined the effect of caffeic acid phenethyl ester on lipopolysaccharide-induced nuclear factor-κB activation and the phosphorylation of mitogen-activated protein kinases. Caffeic acid phenethyl ester treatment significantly reduced nuclear factor-κB translocation and DNA-binding in lipopolysaccharide-stimulated RAW 264.7 cells. This effect was mediated through the inhibition of the degradation of inhibitor κB and by inhibition of both p38 mitogen-activated protein kinase and extracellular signal-regulated kinase phosphorylation, at least in part by inhibiting the generation of reactive oxygen species. Furthermore, caffeic acid phenethyl ester rescued C57BL/6 mice from lethal lipopolysaccharide-induced septic shock, while decreasing serum levels of tumor necrosis factor-α and interleukin-1β. Collectively, these results suggest that caffeic acid phenethyl ester suppresses the induction of cytokines by lipopolysaccharide, as well as inducible nitric oxide synthase and cyclooxygenase-2 expression, by blocking nuclear factor-κB and p38/ERK activation. These findings provide mechanistic insights into the anti-inflammatory and chemopreventive actions of caffeic acid phenethyl ester in macrophages.

Induction and subcellular localization of high‐mobility group box–1 (HMGB1) in the postischemic rat brain

High-mobility group box-1 (HMGB1) was originally identified as a ubiquitously expressed, abundant nonhistone DNA-binding protein. Recently, it was found to act as a cytokine-like mediator of delayed endotoxin lethality and of acute lung injury. Previously, we reported that HMGB1 is massively released extracellularly and plays a cytokine-like function in the postischemic brain. In the present study, we examined the expression profile and cellular distribution of HMGB1 in rat brain after transient focal cerebral ischemia. The expression of HMGB1 in infarction areas in the ipsilateral sides gradually declined over 2 days after 1 hr of middle cerebral artery occlusion (MCAO) to below the basal level. However, after 3 days of reperfusion, HMGB1 level increased to above the basal level, especially in infarction cores, and this delayed induction was then maintained for several days. Immunohistochemistry using a polyclonal antibody against HMGB1 revealed its detailed expression pattern and subcellular localization in the postischemic brain. HMGB1 was found to be widely expressed throughout the normal brain and to be localized to the nuclei of almost all neurons and oligodendrocyte-like cells. After 1 hr of MCAO, HMGB1 immediately translocated from the neuron nuclei to the cytoplasm and subsequently was depleted from neurons during the excitotoxicity-induced acute damaging process. Moreover, beginning 2 days after reperfusion, HMGB1 was notably induced in activated microglia, astrocytes, and in microvascular structures, and these delayed gradual inductions were sustained for several days. These findings suggest that HMGB1 functions as a cytokine-like mediator in a paracrine and autocrine manner in the postischemic brain.

C1 Inhibitor-Mediated Protection from Sepsis

C1 inhibitor (C1INH) protects mice from lethal Gram-negative bacterial LPS-induced endotoxin shock and blocks the binding of LPS to the murine macrophage cell line, RAW 264.7, via an interaction with lipid A. Using the cecal ligation and puncture (CLP) model for sepsis in mice, treatment with C1INH improved survival in comparison with untreated controls. The effect was not solely the result of inhibition of complement and contact system activation because reactive center-cleaved, inactive C1INH (iC1INH) also was effective. In vivo, C1INH and iC1INH both reduced the number of viable bacteria in the blood and peritoneal fluid and accelerated killing of bacteria by blood neutrophils and peritoneal macrophages. In vitro, C1INH bound to bacteria cultured from blood or peritoneal fluid of mice with CLP-induced sepsis, but had no direct effect on bacterial growth. However, both C1INH and iC1INH enhanced the bactericidal activity of blood neutrophils and peritoneal exudate leukocytes. C1INH-deficient mice (C1INH-/- mice) subjected to CLP had a higher mortality than did wild-type littermate mice. Survival of C1INH-/- mice was significantly increased with two doses of C1INH, one given immediately following CLP, and the second at 6 h post-CLP. C1INH may be important in protection from sepsis through enhancement of bacterial uptake by, and/or bactericidal capacity of, phagocytes. Treatment with C1INH may provide a useful additional therapeutic approach in some patients with peritonitis and/or sepsis.

Dendritic Cell Immunotherapy for Acute Inflammatory Diseases

Acute inflammation and the innate immune response to tissue trauma mark a critical pathophysiological challenge within the clinical course of severely injured and critically ill patients. Among the most potent cellular components of the innate host defense system are dendritic cells (DC). This highly effective network of antigen-presenting cells (APC) carries the ability to initiate and amplify diverse immune responses in respect of an appropriate activation signal. Upon activation, DCs direct the immune response towards developing either a Th1 or Th2 response, thus determining the outcome of the inflammatory or infectious stimulus. Advances in the understanding of DC immunobiology have provided new concepts in the treatment of various inflammatory diseases. With gene therapy constantly evolving new methods of celltargeted manipulation of gene expression, DCs have proven to be an exciting new tool in functionally modulating the immune response. In this review we summarize the pivotal functions of DCs and highlight their promising potency of representing an effective immunotherapeutic tool in the treatment of acute inflammation. Keywords: Toll-like receptors, systemic inflammatory response, proinflammatory cytokines, lethal endotoxin, sepsis

TREM‐1 promotes survival during septic shock in mice

Triggering receptor expressed on myeloid (TREM)-1 is integral to the inflammatory response occurring during septic shock, although its precise function has yet to be determined. Here we show that in vivo silencing of TREM-1 using siRNA duplexes in a fecal peritonitis mouse model resulted in a blunted inflammatory response and increased mortality. This was associated with impaired bacterial clearance related to marked inhibition of the neutrophil oxidative burst. By contrast, TREM-1-silenced mice were highly resistant to a lethal endotoxin challenge, while partial silencing of TREM-1 in the bacterial peritonitis model produced a significant survival benefit. These data highlight the crucial role of the TREM-1 pathway in mounting an adequate inflammatory and cytotoxic response to polymicrobial sepsis, and both the therapeutic promise and potential risks of its modulation.