Suppressive oligodeoxynucleotides protect mice from lethal endotoxic shock

Endotoxic shock is a life-threatening condition caused by exposure to bacterial LPS. LPS triggers the release of acute phase, proinflammatory, and Th1 cytokines that facilitate the development of endotoxic shock. Synthetic oligodeoxynucleotides (ODN) expressing suppressive TTAGGG motifs effectively down-regulate the production of proinflammatory and Th1 cytokines elicited by a variety of immune stimuli. The current results demonstrate that suppressive ODN protect mice from LPS-induced endotoxic shock. Underlying this protective effect is the ability of suppressive ODN to bind to and prevent the phosphorylation of STAT1 and STAT4, thereby blocking the signaling cascade mediated by LPS-induced IFN-beta and IL-12. These findings suggest that suppressive ODN might be of use in the treatment of endotoxic shock.     

The Novel Role of Platelet-Activating Factor in Protecting Mice against Lipopolysaccharide-Induced Endotoxic Shock

Background

Platelet-activating factor (PAF) has been long believed to be associated with many pathophysiological processes during septic shock. Here we present novel activities for PAF in protecting mice against LPS-mediated endotoxic shock.

Principal Findings

In vivo PAF treatment immediately after LPS challenge markedly improved the survival rate against mortality from endotoxic shock. Administration of PAF prominently attenuated LPS-induced organ injury, including profound hypotension, excessive polymorphonuclear neutrophil infiltration, and severe multiple organ failure. In addition, PAF treatment protects against LPS-induced lymphocytes apoptosis. These protective effects of PAF was correlated with significantly decreases in the production of the inflammatory mediators such as TNF-α, IL-1β, IL-12, and IFN-γ, while increasing production of the anti-inflammatory cytokine IL-10 in vivo and in vitro.

Conclusions

Taken together, these results suggest that PAF may protect mice against endotoxic shock via a complex mechanism involving modulation of inflammatory and anti-inflammatory mediators.     

Divergent results induced by different types of septic shock in transglutaminase 2 knockout mice

Acute sepsis can be induced by cytokines such as TNF-α and biological products such as LPS. All of these agents cause systemic inflammation, which is characterized by hemodynamic shock and liver toxicity. However, the outcomes of different septic shock models were totally opposite in transglutaminase 2 knockout (TGase 2^?/?) mice. The aim of our study was to clarify the role of TGase 2 in liver injury. Therefore, we explored the role of TGase 2 in liver damage using two different stress models: LPS-induced endotoxic shock and TNF-α/actinomycin D (ActD)-induced sepsis. TNF-α-dependent septic shock resulted in increased liver damage in TGase 2^?/? mice compared with wild-type (WT) mice, and was accompanied by increased levels of caspase 3 and cathepsin D (CTSD) in the damaged liver. Conversely, LPS-induced septic shock resulted in ablation of inflammatory endotoxic shock in TGase 2^?/? mice and decreased liver injury. We found that TGase 2 protected liver tissue from TNF-α-dependent septic shock by reducing the expression of caspase 3 and CTSD. However, TGase 2 differently participated in increased the hemodynamic shock in LPS-induced septic shock through macrophage activation rather than protecting direct liver damage. Therefore, these findings demonstrate that septic shock caused by different agents may induce different results in TGase 2^?/? mice depending on the primary target organs affected.     

Apoptotic cells protect mice against lipopolysaccharide-induced shock

LPS is a main causative agent of septic shock. There is a lack of effective therapies. In vitro studies have shown that uptake of apoptotic cells actively inhibits the secretion by activated macrophages (Mphi) of proinflammatory mediators such as TNF-alpha and that such uptake increases the antiinflammatory and immunosuppressive cytokine TGF-beta. We therefore investigated the protective effect of apoptotic cells against LPS-induced endotoxic shock in mice. The current report is the first study to demonstrate that administration of apoptotic cells can protect mice from LPS-induced death, even when apoptotic cells were administered 24 h after LPS challenge. The beneficial effects of administration of apoptotic cells included 1) reduced circulating proinflammatory cytokines, 2) suppression of polymorphonuclear neutrophil infiltration in target organs, and 3) decreased serum LPS levels. LPS can quickly bind to apoptotic cells and these LPS-coated apoptotic cells can be recognized and cleared by Mphi in a CD14/thrombospondin/vitronectin receptor-dependent manner, accompanied with suppression of TNF-alpha and enhancement of IL-10 expression by LPS-activated Mphi. Apoptotic cells may therefore have therapeutic potential for the treatment of septic shock.     

CCK-8对内毒素休克大鼠肺脏细胞因子的抑制效应

观察八肽胆囊收缩素(cholecystokinin-octapeptide,CCK-8）改善脂多糖(lipopolysaccharide,LPS）引起的大鼠内毒素性休克（endotoxic shock,ES）过程中血清及肺脏细胞因子的变化,探讨p38比裂素活化蛋白激酶（p38 mito-gen-activated protein kinase,p38 MAPK）的信号转导作用。用生理多道记录仪观察尾静脉注入LPS(p38 mito-gen-activated protein kinase,p38 MAPK）的信号转导作用。用生理多道记录仪观察尾静脉注入 LPS（8mg/kg i.v.）复制的SD大鼠ES模型、LPS注入前10min尾静脉注入CCK-8(40ug/kg i.v.）、单独注入CCK-8(40Uug/kg i.v.）或生理盐水（对照）的四组大鼠平均动脉血压（MAP）的改变,应用ELISA试剂盒检测血清和肺脏中炎性细胞因子（TNF-a、IL-1β和IL-6）的变化。用Western blot检测肺脏p38 MAPK的表达。结果显示：CCK-8可改善LPS引起的大鼠MAP的下降。与对照组相比,LPS可显著增加血清和肺脏TNF-a、IL-1β和IL-6含量;CCK-8可显著抑制LPS诱导的血清和肺脏TNF-a、IL-1β和IL-6的增加。CCK-8可增加ES大鼠肺脏磷酸化p38 MAPK的表达。结果提示CCK-8可改善ES大鼠MAP的降低,并对肺脏促炎性细胞因子过量产生有抑制作用,p38MAPK可能参与了其信号转导机制。     

Characterization of mice deficient in interleukin-1β converting enzyme

Interleukin-1β converting enzyme (ICE) processes the inactive proIL-1β to the proinflammatory mature IL-1β. ICE belongs to a family of cysteine proteases that have been implicated in apoptosis. To address the biological functions of ICE, we generated ICE-deficient mice through gene targeting technology. ICE-deficient mice developed normally, appeared healthy, and were fertile. Peritoneal macrophages from ICE-deficient mice underwent apoptosis normally upon ATP treatment. Thymocytes from young ICE-deficient mice also underwent apoptosis when triggered by dexamethasone, gamma irradiation, or aging. ICE-deficient mice had a major defect in the production of mature IL-1β and had impaired IL-1α production on LPS stimulation in vitro and in vivo. ICE-deficient mice were resistant to LPS-induced endotoxic shock. J. Cell. Biochem. 64:27–32. © 1997 Wiley-Liss, Inc.     

Several functions of immune cells in mice changed by oxidative stress caused by endotoxin

We have studied natural killer (NK) activity, lymphoproliferative response, the release of several cytokines (IL-2, TNF alpha and IL-1 beta) and the ROS production in peritoneal leukocytes obtained 0, 2, 4, 12 and 24 h after lipopolysaccharide (LPS) injection. Lethal septic shock (100 % mortality occurred at 30 h after LPS administration) was caused in female BALB/c mice by intraperitoneal injection of 100 mg/kg of E. coli LPS. Cytotoxicity and lymphoproliferation assay were preformed together with the measurement of IL-1 beta, IL-2 and TNF alpha production, and quantification of ROS. Natural killer activity, spontaneous lymphoproliferative response, IL-2, TNF alpha, IL-beta release and ROS production were increased after LPS injection. In conclusions, ROS and proinflammatory mediators produced by immune cells in response to LPS are involved in the oxidative stress of endotoxic shock. This oxidative state alters some functional characteristics of leukocytes (proliferation and NK activity).     

Suppression of murine endotoxic shock by sPLA2 inhibitor, indoxam, through group IIA sPLA2-independent mechanisms.

Endotoxic shock is a systemic inflammatory process, involving a variety of proinflammatory mediators. Two types of secretory phospholipase A2 (sPLA2) have been implicated in this process. Group IB sPLA2 (PLA2-IB) binds to the PLA2 receptor (PLA2R), and PLA2R-deficient mice exhibit resistance to endotoxin-induced lethality with reduced plasma levels of proinflammatory cytokines, such as TNF-alpha. Group IIA sPLA2 (PLA2-IIA) is found in many tissues and cell types, and local and systemic levels are elevated under numerous inflammatory conditions including sepsis. In this study, we investigated the effect of a specific sPLA2 inhibitor, indoxam, on murine endotoxic shock. Indoxam suppressed the elevation of plasma TNF-alpha with a similar potency in PLA2-IIA-expressing and PLA2-IIA-deficient mice after LPS challenge. In PLA2-IIA-deficient mice, indoxam also suppressed the elevation of plasma IL-1beta, IL-6 and NO, and prolonged survival after LPS challenge. Indoxam was found to block the PLA2-IB binding to murine PLA2R with a high potency (Ki=30 nM). The inhibitory effects of indoxam on the LPS-induced elevation of plasma TNF-alpha levels could not be observed in mice deficient in PLA2R. These findings suggest that indoxam blocks the production of proinflammatory cytokines during endotoxemia through PLA2-IIA-independent mechanisms, possibly via blockade of the PLA2R function.     

DIURNAL VARIATION IN ENDOTOXIN-INDUCED MORTALITY IN MICE: CORRELATION WITH PROINFLAMMATORY FACTORS

Many immune parameters exhibit daily and circadian oscillations, including the number of circulating cells and levels of cytokines in the blood. Mice also have a differential susceptibility to lipopolysaccharide (LPS or endotoxin)-induced endotoxic shock, depending on the administration time in the 24?h light-dark (LD) cycle. We replicated these results in LD, but we did not find temporal differences in LPS-induced mortality in constant darkness (DD). Animals challenged with LPS showed only transient effects on their wheel locomotor activity rhythm without modification of circadian period and phase. Levels of several key factors involved in the pathology of sepsis and septic shock were tested in LD. We found that LPS-induced levels of interleukin (IL)-1β, IL-6, JE (MCP-1), and MIP1α were significantly higher at zeitgeber time (ZT) 11 (time of increased mortality) than at ZT19 (ZT12?=?time of lights-off in the animal quarters for the 12L:12D condition). Our results indicate that the differences found in mortality that are dependent on the time of LPS-challenge are not directly related to an endogenous circadian clock, and that some relevant immune factors in the development of sepsis are highly induced at ZT11, the time of higher LPS-induced mortality, compared to ZT19. (Author correspondence: dgolombek@unq.edu.ar)     

Lipopolysaccharide-induced apoptosis of endothelial cells and its inhibition by vascular endothelial growth factor

Endothelial injury is a major manifestation of septic shock induced by LPS. Recently, LPS was shown to induce apoptosis in different types of endothelial cells. In this study, we observed that pretreatment with vascular endothelial growth factor (VEGF), a known cell survival factor, blocked LPS-induced apoptosis in endothelial cells. We then further defined this LPS-induced apoptotic pathway and its inhibition by VEGF. We found that LPS treatment increased caspase-3 and caspase-1 activities and induced the cleavage of focal adhesion kinase. LPS also augmented expression of the pro-apoptotic protein Bax and the tumor suppressor gene p53. The pro-apoptotic Bax was found to translocate to the mitochondria from the cytosol following stimulation with LPS. Pretreatment of endothelial cells with VEGF inhibited the induction of both Bax and p53 as well as the activation of caspase-3. These data suggest that VEGF inhibits LPS-induced endothelial apoptosis by blocking pathways that lead to caspase activation.     

Glucocorticoids as cytokine inhibitors: role in neuroendocrine control and therapy of inflammatory diseases

Glucocorticoids are potent inhibitors of inflammation and endotoxic shock. This probably occurs through an inhibition of the synthesis of pro-inflammatory cytokines as well as of many of their toxic activities. Therefore, endogenous glucocorticoids (GC) might represent a major mechanism in the control of cytokine mediated pathologies. GC inhibit the synthesis of cytokines in various experimental models. Adrenalectomy or GC antagonists potentiate TNF, IL-1 and IL-6 production in LPS treated mice. GC inhibit the formation of arachidonic acid metabolites and the induction of NO synthase. They also inhibit various activities of cytokines including toxicity, haemodynamic shock and fever. Adrenalectomy sensitizes to the toxic effects of LPS, TNF and IL-1. On the other hand, GC potentiate the synthesis of several cytokine induced APP by the liver. Since many of these proteins have anti-toxic activities (antioxidant, antiprotease etc.) or bind cytokines, this might well represent a GC mediated protective feedback mechanism involving the liver. Not only do GC inhibit cytokines, but in vivo LPS and various cytokines (TNF, IL-1, IL-6) increase blood GC levels through a central mechanism involving the activation of the HPA. Thus, this neuroendocrine response to cytokines constitutes an important immunoregulatory feedback involving the brain.     

Taurolidine, an analogue of the amino acid taurine, suppresses interleukin 1 and tumor necrosis factor synthesis in human peripheral blood mononuclear cells.

Taurolidine (Geistlich Pharm, AG, Wolhusen, Switzerland), a derivative of the amino acid taurine, is commonly used in some parts of the world as an adjunctive therapy for various infections. Its mechanism of action is thought to be related to its antimicrobial properties, including its ability to interfere with some of the biological activities of endotoxin (lipopolysaccharide, LPS). For example, taurolidine has been shown to protect animals against endotoxic shock and death. In this study we examined the ability of taurolidine to block LPS-induced tumor necrosis factor (TNF) and interleukin 1 (IL-1) synthesis in human peripheral blood mononuclear cells (PBMC) from 27 donors. We observed a dose-dependent reduction in the synthesis of these two cytokines when taurolidine was preincubated with LPS before being added to PBMC. This reduction was independent of the molar ratio of taurolidine to LPS but was related to the concentration of taurolidine present in the PBMC cultures. There was a 80 to 90% reduction in total IL-1 and TNF synthesis induced by LPS at concentrations of taurolidine of 40 to 100 micrograms/mL; the vehicle was without effect. Following a 30-min preincubation with PBMC, taurolidine could be washed from the cells and still suppress cytokine synthesis induced by LPS. Using release of lactic acid dehydrogenase, 100 micrograms/mL of taurolidine was not toxic for PBMC. Taurolidine also reduced IL-1 and TNF synthesis induced by the Staphylococcus aureus-derived toxic shock syndrome toxin-1 as well as that induced by nontoxic heat-killed Staphylococcus epidermidis organisms.(ABSTRACT TRUNCATED AT 250 WORDS)     

Tim-3 Negatively Mediates Natural Killer Cell Function in LPS-Induced Endotoxic Shock

Sepsis is an exaggerated inflammatory condition response to different microorganisms with high mortality rates and extremely poor prognosis. Natural killer (NK) cells have been reported to be the major producers of IFN-γ and key players in promoting systematic inflammation in lipopolysaccharide (LPS)-induced endotoxic shock. T-cell immunoglobulin and mucin domain (Tim)-3 pathway has been demonstrated to play an important role in the process of sepsis, however, the effect of Tim-3 on NK cell function remains largely unknown. In this study, we observed a dynamic inverse correlation between Tim-3 expression and IFN-γ production in NK cells from LPS-induced septic mice. Blockade of the Tim-3 pathway could increase IFN-γ production and decrease apoptosis of NK cells in vitro, but had no effect on the expression of CD107a. Furthermore, NK cell cytotoxicity against K562 target cells was enhanced after blocking Tim-3 pathway. In conclusion, our results suggest that Tim-3 pathway plays an inhibitory role in NK cell function, which might be a potential target in modulating the excessive inflammatory response of LPS-induced endotoxic shock.     

Electroacupuncture Ameliorates Acute Renal Injury in Lipopolysaccharide-Stimulated Rabbits via Induction of HO-1 through the PI3K/Akt/Nrf2 Pathways

Electroacupuncture at select acupoints have been verified to protect against organ dysfunctions during endotoxic shock. And, heme oxygenase (HO)-1 as a phase II enzyme and antioxidant contributed to the protection of kidney in septic shock rats. The phosphatidylinositol 3-kinase (PI3K)-Akt pathway mediated the activation of NF-E2 related factor-2 (Nrf2), which was involved in HO-1 induction. To understand the efficacy of electroacupuncture stimulation in ameliorating acute kidney injury (AKI) through the PI3K/Akt/Nrf2 pathway and subsequent HO-1 upregulation, a dose of LPS 5mg/kg was administered intravenously to replicate the rabbit model of AKI induced by endotoxic shock. Electroacupuncture pretreatment was handled bilaterally at Zusanli and Neiguan acupoints for five consecutive days while sham electroacupuncture at non-acupoints as control. Results displayed that electroacupuncture stimulation significantly alleviated the morphologic renal damage, attenuated renal tubular apoptosis, suppressed the elevated biochemical indicators of AKI caused by LPS, enhanced the expressions of phospho-Akt, HO-1protein, Nrf2 total and nucleoprotein, and highlighted the proportions of Nrf2 nucleoprotein as a parallel. Furthermore, partial protective effects of elecroacupuncture were counteracted by preconditioning with wortmannin (the selective PI3K inhibitor), indicating a direct involvement of PI3K/Akt pathway. Inconsistently, wortmannin pretreatment made little difference to the expressions of HO-1, Nrf2 nucleoprotein and total protein, which indicated that PI3K/Akt may be not the only pathway responsible for electroacupuncture-afforded protection against LPS-induced AKI. These findings provide new insights into the potential future clinical applications of electroacupuncture for AKI induced by endotoxic shock instead of traditional remedies.     

Interleukin-6 is a better marker of lethality than tumor necrosis factor in endotoxin treated mice

Abstract We established a mouse model to differentiate between a lethal and non-lethal presentation of endotoxic shock. The model involved injecting different amounts of Escherichia coli LPS into C3H/HeN mice which had been 'primed' with BCG. We found that the mice receiving non-lethal and lethal doses of LPS could not be differentiated in terms of their physical symptoms for the first 8 h post-injection. Tumour necrosis factor (TNF) was detected at concentrations 2–9-fold greater in mice receiving lethal doses of LPA when compared with non-lethally injected mice. However, given that (i) the successful detection of this differential was dependent on the time of sampling and (ii) that TNF was only detected in the first 3–4 h post LPS challenge, we suggest that TNF may not be very useful as a prognostic marker in endotoxic shock. In contrast, circulating IL-6 appeared to mirror the symptoms of the endotoxic mice. The relative disappearance of IL-6 after 10 h in the non-lethally injected mice corresponded with their symptomatic recovery, while IL-6 continued to circulate up to the time of death in the lethally injected mice. Furthermore, there appeared to be a good correlation between the levels of injected LPS and the levels of IL-6 induced into the circulation. Our results suggest that IL-6, rather than TNF, may serve as a prognostic marker for endotoxic shock.     

Activation of virus-specific CD8+ T cells by lipopolysaccharide-induced IL-12 and IL-18

Virus-specific T cells represent a hallmark of Ag-specific, adaptive immunity. However, some T cells also demonstrate innate functions, including non-Ag-specific IFN-gamma production in response to microbial products such as LPS or exposure to IL-12 and/or IL-18. In these studies we examined LPS-induced cytokine responses of CD8(+) T cells directly ex vivo. Following acute viral infection, 70-80% of virus-specific T cells will produce IFN-gamma after exposure to LPS-induced cytokines, and neutralization experiments indicate that this is mediated almost entirely through production of IL-12 and IL-18. Different combinations of these cytokines revealed that IL-12 decreases the threshold of T cell activation by IL-18, presenting a new perspective on IL-12/IL-18 synergy. Moreover, memory T cells demonstrate high IL-18R expression and respond effectively to the combination of IL-12 and IL-18, but cannot respond to IL-18 alone, even at high cytokine concentrations. This demonstrates that the synergy between IL-12 and IL-18 in triggering IFN-gamma production by memory T cells is not simply due to up-regulation of the surface receptor for IL-18, as shown previously with naive T cells. Together, these studies indicate how virus-specific T cells are able to bridge the gap between innate and adaptive immunity during unrelated microbial infections, while attempting to protect the host from cytokine-induced immunopathology and endotoxic shock.     

Protective effects of early treatment with lipoic acid in LPS-induced lung injury in rats.

A lipopolysaccharide (LPS) stimulates the synthesis and releases several metabolites from phagocytes which can lead to an endotoxic shock characterized by multiple organ injury with the earliest to occur in the lungs. Among LPS-induced metabolites, reactive oxygen species are considered to play a crucial pathogenetic role in the lung damage. In this study, the effect of early administration of an antioxidant, alpha-lipoic acid (LA), on pulmonary lipid peroxidation, lung hydrogen peroxide (H(2)O(2)) concentration, and lung sulfhydryl group content was evaluated in rats with endotoxic shock induced by administration of LPS (Escherichia coli 026:B6, 30 mg/kg, i.v.). In addition, lung edema was assessed with wet-to-dry lung weight (W/D) ratio. Animals were treated intravenously with normal saline or LA 60 mg/kg or 100 mg/kg 30 min after LPS injection. After a 5 h observation, animals were killed and the lungs were isolated for measurements. Injection of LPS alone resulted in the development of shock and oxidative stress, the latter indicated by a significant increase in the lung thiobarbituric acid reacting substances (TBARS) and H(2)O(2) concentrations, and a decrease in the lung sulfhydryl group content. The increase in the W/D ratio after the LPS challenge indicated the development of lung edema in response to LPS. Administration of LA after the LPS challenge resulted in an increase in the sulfhydryl group content and a decrease in TBARS and H202 concentration in the lungs as compared with the LPS group. An insignificant decrease in the W/D ratio was observed in rats treated with either dose of LA. These results indicate that the LPS-induced oxidative lung injury in endotoxic rats can be attenuated by early treatment with LA. Administration of LA could be a useful adjunct to conventional approach in the management of septic shock.