In vivo heat shock protein assembles with septic liver NF-kappaB/I-kappaB complex regulating NF-kappaB activity

This study elucidates the mechanism through which heat shock treatment influences the outcome of sepsis. Post-heat shock sepsis was induced in rats by CLP 24 h after whole-body hyperthermia. Liver cytosolic and nuclear fractions were collected and analyzed in early and late sepsis rats (sacrificed 9 and 18 h after CLP, respectively). During sepsis, levels of I-kappaB and nuclear factor-kappaB (NF-kappaB) declined in the cytosol of liver, whereas NF-kappaB increased in nucleus. NF-kappaB activity was significantly enhanced during sepsis, and the products of NF-kappaB target genes, such as TNF-alpha and inducible nitric oxide synthase (iNOS), were overexpressed. Heat shock treatment, inducing heat shock protein synthesis, prevented down-regulation of cytosolic I-kappaB and decreased translocation of NF-kappaB into the nucleus. Therefore, the sepsis-induced acceleration of NF-kappaB activation was inhibited. Expression of TNF-alpha and iNOS mRNA was also down-regulated. Coimmunoprecipitation with anti-NF-kappaB (p65) and anti-IkappaB antibodies verified an assembling phenomenon of heat shock protein (HSP) 72 with NF-kappaB and I-kappaB. We suggest that the mechanism preventing septic activation of NF-kappaB is that oversynthesized HSP72 forms a complex with NF-kappaB/I-kappaB, thus inhibiting nuclear translocation of NF-kappaB. HSP72 appears to play a crucial protective role in modulating the gene expression controlled by NF-kappaB in sepsis.     

Receptor-interacting protein kinase 3 deficiency inhibits immune cell infiltration and attenuates organ injury in sepsis

Introduction

Sepsis is defined as a systemic hyper-inflammatory immune response, with a subsequent immune-suppressive phase, which leads to multiple organ dysfunction and late lethality. Receptor-interacting protein kinase 3 (RIPK3)-dependent necrosis is implicated in driving tumor necrosis factor alpha (TNF-α)- and sepsis-induced mortality in mice. However, it is unknown if RIPK3 deficiency has any impact on immune cell trafficking, which contributes to organ damage in sepsis.

Methods

To study this, male wild-type (WT) and RIPK3-deficient (Ripk3^-/-) mice on C57BL/6 background were subjected to sham operation or cecal ligation and puncture (CLP)-induced sepsis. Blood and tissue samples were collected 20 hours post-CLP for various measurements.

Results

In our severe sepsis model, the mean survival time of Ripk3^-/- mice was significantly extended to 68 hours compared to 41 hours for WT mice. Ripk3^-/- mice had significantly decreased plasma levels of TNF-α and IL-6 and organ injury markers compared to WT mice post-CLP. In the lungs, Ripk3^-/- mice preserved better integrity of microscopic structure with reduced apoptosis, and decreased levels of IL-6, macrophage inflammatory protein (MIP)-2 and keratinocyte-derived chemokine (KC), compared to WT. In the liver, the levels of MIP-1, MIP-2 and KC were also decreased in septic Ripk3^-/- mice. Particularly, the total number of neutrophils in the lungs and liver of Ripk3^-/- mice decreased by 59.9% and 66.7%, respectively, compared to WT mice post-CLP. In addition, the number of natural killer (NK) and CD8T cells in the liver decreased by 64.8% and 53.4%, respectively, in Ripk3^-/- mice compared to WT mice post-sepsis.

Conclusions

Our data suggest that RIPK3 deficiency modestly protected from CLP-induced severe sepsis and altered the immune cell trafficking in an organ-specific manner attenuating organ injury. Thus, RIPK3 acts as a detrimental factor in contributing to the organ deterioration in sepsis.     

TLR ligand decreases mesenteric ischemia and reperfusion injury-induced gut damage through TNF-alpha signaling

Ischemic gut contributes to the development of sepsis and organ failure in critically ill patients. Toll-like receptors (TLRs) have been reported to mediate the pathophysiology of organ damage following ischemia/reperfusion (I/R) injury. We hypothesize that LPS, a ligand for TLR4, decreases mesenteric I/R injury-induced gut damage through tumor necrosis factor alpha (TNF-alpha) signaling. First, wild-type (WT) mice were fed with oral antibiotics for 4 weeks to deplete the intestinal commensal microflora. At week 3, drinking water was supplemented with LPS (10 microg/microL) to trigger TLRs. The intestinal mucosa was harvested for TLR4 protein, caspase 3 activity, and terminal deoxynucleotide transferase labeling assay. Second, WT and Tnfrsf1a mice received 30-min ischemia and 30-min reperfusion (30I-30R) or 30I-180R of the intestine; intestinal permeability and lipid peroxidation of the intestine were examined. Third, WT and Tnfrsf1a mice were fed with oral antibiotics with or without LPS and received 30I-180R of the intestine. The intestinal mucosa was harvested for lipid peroxidation; glutathione (GSH) level; nuclear factor kappaB (NF-kappaB) and AP-1 DNA-binding activity; Bcl-w, TNF-alpha, and CXCR2 mRNA expression; and HSP70 protein assay. Commensal depletion increased caspase 3 activity as well as villi apoptosis and decreased TLR4 expression of the intestinal mucosa. LPS increased TLR4 expression and decreased villi apoptosis. Commensal depletion augmented 30I-180R-induced intestine permeability as well as lipid peroxidation and decreased GSH level in WT mice but not in Tnfrsf1a mice. LPS decreased 30I-180R-induced intestinal permeability as well as lipid peroxidation and increased GSH level of the intestinal mucosa in WT mice but not in Tnfrsf1a mice. Commensal depletion with 30I-180R increased NF-kappaB and AP-1 DNA-binding activity, HSP70 protein expression, and decreased Bcl-w and TNF-alpha mRNA expression of the intestinal mucosa in WT mice but not in Tnfrsf1a mice. Collectively, commensal microflora induces TLR4 expression and decreases apoptosis of the intestinal mucosa. Commensal depletion enhances I/R-induced gut damage. LPS prevents I/R-induced intestinal permeability, lipid peroxidation, and decrease in GSH level. Given that the preventive effect of LPS on I/R-induced gut damage and NF-kappaB activity of the intestine is abolished in Tnfrsf1a mice, we conclude that TLR ligand decreases mesenteric I/R injury-induced gut damage through TNF-alpha signaling.     

核转录因子-κB在热休克预处理抑制过氧化氢所致心肌细胞损伤中的作用

目的探讨热休克预处理抑制氧化应激损伤心肌细胞的分子机制。方法采用1mmol/L的过氧化氢(H2O2)作用于原代培养的新生大鼠心肌细胞;用总抗氧化能力检测试剂盒及福林酚法检测心肌细胞中总抗氧化能力的变化;用蛋白质免疫印迹法(Westernblot)检测热休克蛋白70(HSP70)、αB晶状体蛋白、核转录因子κB(NFκB)抑制物(IκB)含量;免疫组化检测NFκB及HSP70在细胞内的分布情况。结果1与H2O2(1mmol/L,3h)损伤组比,热休克预处理组(43℃1h,恢复6h)的总抗氧化能力明显增加(P均<0.01);2心肌细胞经热休克预处理(43℃1h)后3h,HSP70、αB晶状体蛋白、IκBα的表达均增加,6～12h达高峰,并可维持至24h;3与正常心肌细胞比较,H2O2(1mmol/L)处理的心肌细胞中IκBα的含量明显下降,而热休克预处理则可抑制H2O2所致的这种变化;4H2O2(1mmol/L,30min)可使心肌细胞胞质中的NF-κB及HSP70向胞核移位,若先经热休克预处理(43℃1h,恢复6h)则可使这种移位显著减少。结论热休克预处理可抑制H2O2所致的心肌细胞损伤,其保护作用可能与其诱导HSP70、αB晶状体蛋白及IκBα的表达,从而抑制NF-κB的活化有关。     

Calcium flux and endothelial dysfunction during acute lung injury: a STIMulating target for therapy

Bacterial pathogen-associated molecular pattern molecules (PAMPs) such as LPS activate the endothelium and can lead to lung injury, but the signaling pathways mediating endothelial injury remain incompletely understood. In a recent issue of the JCI, Gandhirajan et al. identify STIM1, an ER calcium sensor, as a key link between LPS-induced ROS, calcium oscillations, and endothelial cell (EC) dysfunction. In addition, they report that BTP2, an inhibitor of calcium channels, attenuates lung injury. This study identifies a novel endothelial signaling pathway that could be a future target for the treatment of lung injury. During infection, circulating bacterial products and endogenous cytokines stimulate the endothelium, setting off a cascade of vascular activation, including the increased expression of vascular adhesion molecules and regional increases in endothelial permeability. This response, the result of millennia of warfare between mammals and microbes, is beneficial during compartmentalized infections such as those of soft tissue and pneumonia. The alterations in vascular adhesion and permeability enable neutrophils and monocytes to penetrate infected tissues, where they sequester and kill pathogens. However, similar physiological responses in vulnerable vascular beds, such as the capillary networks of the lungs and kidneys, can lead to multiple organ failure in critically ill patients. Achieving a deeper understanding of the signals that regulate vascular integrity during host defense and organ injury would be an important step in reducing vascular injury during human sepsis.     

Maturational differences in lung NF-kappaB activation and their role in tolerance to hyperoxia

Neonatal rodents are more tolerant to hyperoxia than adults. We determined whether maturational differences in lung NF-kappaB activation could account for the differences. After hyperoxic exposure (O2 > 95%), neonatal (<12 hours old) lung NF-kappaB binding was increased and reached a maximum between 8 and 16 hours, whereas in adults no changes were observed. Additionally, neonatal NF-kappaB/luciferase transgenic mice (incorporating 2 NF-kappaB consensus sequences driving luciferase gene expression) demonstrated enhanced in vivo NF-kappaB activation after hyperoxia in real time. In the lungs of neonates, there was a propensity toward NF-kappaB activation as evidenced by increased lung I-kappaB kinase protein levels, I-kappaBalpha phosphorylation, beta-transducin repeat-containing protein levels, and total I-kappaBalpha degradation. Increased lung p-JNK immunoreactive protein was observed only in the adult lung. Inhibition of pI-kappaBalpha by BAY 11-7085 resulted in decreased Bcl-2 protein levels in neonatal lung homogenates and decreased cell viability in lung primary cultures after hyperoxic exposure. Furthermore, neonatal p50-null mutant (p50(-/-)) mice showed increased lung DNA degradation and decreased survival in hyperoxia compared with WT mice. These data demonstrate that there are maturational differences in lung NF-kappaB activation and that enhanced NF-kappaB may serve to protect the neonatal lung from acute hyperoxic injury via inhibition of apoptosis.     

Endothelial dysfunction is a potential contributor to multiple organ failure and mortality in aged mice subjected to septic shock: preclinical studies in a murine model of cecal ligation and puncture

Introduction

The goal of the current study was to investigate the effect of aging on the development of endothelial dysfunction in a murine model of sepsis, and to compare it with the effect of genetic deficiency of the endothelial isoform of nitric oxide synthase (eNOS).

Methods

Cecal ligation and puncture (CLP) was used to induce sepsis in mice. Survival rates were monitored and plasma indices of organ function were measured. Ex vivo studies included the measurement of vascular function in thoracic aortic rings, assessment of oxidative stress/cellular injury in various organs and the measurement of mitochondrial function in isolated liver mitochondria.

Results

eNOS deficiency and aging both exacerbated the mortality of sepsis. Both eNOS-deficient and aged mice exhibited a higher degree of sepsis-associated multiple organ dysfunction syndrome (MODS), infiltration of tissues with mononuclear cells and oxidative stress. A high degree of sepsis-induced vascular oxidative damage and endothelial dysfunction (evidenced by functional assays and multiple plasma markers of endothelial dysfunction) was detected in aortae isolated from both eNOS^−/− and aged mice. There was a significant worsening of sepsis-induced mitochondrial dysfunction, both in eNOS-deficient mice and in aged mice. Comparison of the surviving and non-surviving groups of animals indicated that the severity of endothelial dysfunction may be a predictor of mortality of mice subjected to CLP-induced sepsis.

Conclusions

Based on the studies in eNOS mice, we conclude that the lack of endothelial nitric oxide production, on its own, may be sufficient to markedly exacerbate the severity of septic shock. Aging markedly worsens the degree of endothelial dysfunction in sepsis, yielding a significant worsening of the overall outcome. Thus, endothelial dysfunction may constitute an early predictor and independent contributor to sepsis-associated MODS and mortality in aged mice.     

Deletion of IKK2 in hepatocytes does not sensitize these cells to TNF-induced apoptosis but protects from ischemia/reperfusion injury

The inhibitor of NF-kappaB (I-kappaB) kinase (IKK) complex consists of 3 subunits, IKK1, IKK2, and NF-kappaB essential modulator (NEMO), and is involved in the activation of NF-kappaB by various stimuli. IKK2 or NEMO constitutive knockout mice die during embryogenesis as a result of massive hepatic apoptosis. Therefore, we examined the role of IKK2 in TNF-induced apoptosis and ischemia/reperfusion (I/R) injury in the liver by using conditional knockout mice. Hepatocyte-specific ablation of IKK2 did not lead to impaired activation of NF-kappaB or increased apoptosis after TNF-alpha stimulation whereas conditional NEMO knockout resulted in complete block of NF-kappaB activation and massive hepatocyte apoptosis. In a model of partial hepatic I/R injury, mice lacking IKK2 in hepatocytes displayed significantly reduced liver necrosis and inflammation than wild-type mice. AS602868, a novel chemical inhibitor of IKK2, protected mice from liver injury due to I/R without sensitizing them toward TNF-induced apoptosis and could therefore emerge as a new pharmacological therapy for liver resection, hemorrhagic shock, or transplantation surgery.     

Systemic inflammatory response syndrome (SIRS) and endothelial cell injury

During recent years, evidences have been accumulated demonstrating bidirectional crosstalk between coagulation and inflammation. This review outlines the influences that coagulation and inflammation exert on each other to the endothelium and how these systems induce systemic inflammatory response syndrome (SIRS). Then we discussed the implications of leucocyte-endothelial activation to endothelial cell injury followed by multiple organ dysfunction syndrome (MODS) in patients with sustained SIRS. Last we demonstrated an important role of inflammatory circulation disturbance induced by endothelial cell injury for the pathogenesis of MODS in SIRS and sepsis.     

Thermal injury-induced priming effect of neutrophil is TNF-alpha and P38 dependent

Priming response of neutrophil in clinical-related conditions and its mechanism has not been clarified. This study is to determine if thermal injury-induced priming effect of neutrophil is TNF-alpha and p38 dependent. In Experiment 1, bone marrow neutrophil of wild-type (WT) mice and TNF receptor superfamily, member 1A (Tnfrsf1a-/-) mice were harvested and treated with TNF-alpha, platelet activating factor (PAF) first, then with or without N-formyl-Met-Leu-Phe (fMLP). Reactive oxygen species (ROS) production and p38 phosphorylation were evaluated. In Experiment 2, ROS of neutrophil from WT and Tnfrsf1a-/- mice at 3 or 15 h after thermal injury with or without fMLP treatment were assayed. In Experiment 3, p38 and p44/42 phosphorylation, CXCR2 and macrophage inflammatory protein-2 expression, apoptotic ratio, and activating protein-1 (AP-1) and nuclear factor-kappa B (NF-kappaB) activation of neutrophil from WT and Tnfrsf1a-/- mice at 3 h after thermal injury were tested. FMLP treatment after TNF-alpha or PAF incubation of neutrophil increased ROS of PAF-treated but not TNF-alpha-treated neutrophil. PAF treatment increased ROS of neutrophil in WT and Tnfrsf1a-/- mice. FMLP increased ROS of neutrophil of WT mice at 3 h after thermal but not that of Tnfrsf1a-/- mice. TNF-alpha and PAF increased p38 phosphorylation of neutrophil in WT but not that in Tnfrsf1a-/- mice. Thermal injury increased p38 phosphorylation, NF-kappaB activation, and decreased apoptosis of neutrophil at 3 h after thermal injury in WT but not in Tnfrsf1a-/- mice. Thermal injury also induced AP-1 activation and ROS production on neutrophil at 3 and 15 h after thermal injury, respectively, in WT and Tnfrsf1a-/- mice. Collectively, fMLP stimulates ROS of neutrophil through TNF-alpha signaling; PAF stimulates that of neutrophil through both TNF-alpha-dependent and TNF-alpha-independent pathway. Thermal injury induces a TNF-alpha-dependent priming effect and a TNF-alpha-independent activation effect on neutrophil at 3 and 15 h after thermal injury, respectively. NF-kappaB signaling pathway plays an important role in neutrophil activation. Thermal injury also induces TNF-alpha-dependent delay apoptosis and TNF-alpha-independent AP-1 activation of neutrophil at 3 h after thermal injury. Taken together with the TNF-alpha-dependent p38 and NF-kappaB activation in primed neutrophil, we conclude that thermal injury-induced priming effect of polymorphonuclear neutrophil is TNF-alpha and p38 dependent.     

Lest we forget the endothelial glycocalyx in sepsis

ABSTRACT: Sepsis is the third largest cause of death in industrialised countries, but treatment remains largely supportive and effective therapeutic interventions are urgently needed. Disruption and dysfunction of the microvascular endothelium leading directly or indirectly to multiple organ failure are now recognised to underpin the pathophysiology of sepsis. Biomarkers of endothelial activation may therefore assume an important role in guiding future research efforts. We suggest that integral to this approach is the investigation and evaluation of endothelial glycocalyx biomarkers, not only as indicators of the pathogenic process but also to inform the development of pharmacological and other therapies.     

20-Hydroxyeicosatetraenoic acid stimulates nuclear factor-kappaB activation and the production of inflammatory cytokines in human endothelial cells

Endothelial dysfunction is associated with endothelial cell activation, i.e., up-regulation of surface cell adhesion molecules and the release of proinflammatory cytokines. 20-Hydroxyeicosatetraenoic acid (HETE), a major vasoactive eicosanoid in the microcirculation, has been implicated in the regulation of endothelial cell function through its angiogenic and pro-oxidative properties. We examined the effects of 20-HETE on endothelial cell activation in vitro. Cells transduced with adenovirus containing either CYP4A1 or CYP4A2 produced higher levels of 20-HETE, and they demonstrated increased expression levels of the adhesion molecule intercellular adhesion molecule (ICAM) (4-7-fold) and the oxidative stress marker 3-nitrotyrosine (2-3-fold) compared with cells transduced with control adenovirus. Treatment of cells with 20-HETE markedly increased levels of prostaglandin (PG) E(2) and 8-epi-isoprostane PGF(2alpha), commonly used markers of activation and oxidative stress, and most prominently, interleukin-8, a potent neutrophil chemotactic factor whose overproduction by the endothelium is a key feature of vascular injury. 20-HETE at nanomolar concentrations increased inhibitor of nuclear factor-kappaB phosphorylation by 2 to 5-fold within 5 min, which was followed with increased nuclear translocation of nuclear factor-kappaB (NF-kappaB). Likewise, 20-HETE activated the mitogen-activated protein kinase/extracellular signal-regulated kinase (MAPK/ERK) pathway by stimulating phosphorylation of ERK1/2. Inhibition of NF-kappaB activation and inhibition of ERK1/2 phosphorylation inhibited 20-HETE-induced ICAM expression. It seems that 20-HETE triggers NF-kappaB and MAPK/ERK activation and that both signaling pathways participate in the cellular mechanisms by which 20-HETE activates vascular endothelial cells.     

Interaction between the innate and adaptive immune systems is required to survive sepsis and control inflammation after injury

Substantial clinical and laboratory research has revealed that major injury causes abnormalities in both the innate and adaptive immune systems. However, the relative importance of each of these systems in the immune dysfunction after injury is poorly understood and difficult to establish by clinical studies alone. Rag1 (-/-) C57BL/6 mice (Rag1), which lack an adoptive immune system, and immune-sufficient wild-type (WT) C57BL/6 mice underwent 25% total body surface area burn injury or sham injury under anesthesia and were subjected to cecal ligation and puncture (CLP) at day 10 postinjury, a time of high CLP mortality in this model. To test the effect of adaptive immune deficiency on inflammatory cytokine production after injury, adaptive cell-depleted splenocytes from sham and burn WT and Rag1 mice were stimulated with LPS, and TNF-alpha and IL-6 production were assayed at days 1 and 7 postinjury. Intracellular expression of TNFalpha and IL-6 by F4/80 macrophages was also assessed on day 7 by intracellular cytokine staining. Finally, Rag1 animals were reconstituted with WT splenocytes, and the effect of such reconstitution on CLP survival and cytokine production was determined. Survival of sham WT animals after CLP was significantly higher (P < 0.01) than survival of burn WT and Rag1 sham and burn animals, all of which had equivalently low survival. Reconstitution of Rag1 animals with WT splenocytes restored CLP survival to WT sham levels. Splenocytes from Rag1 burn mice showed significantly augmented cytokine production when compared with WT burn mice on day 7 (P < 0.05). Reconstitution of Rag1 mice with WT splenocytes at the time of injury returned cytokine production to WT levels. Intracellular cytokine expression in F4/80 macrophages was increased to a similar degree after burn, but not sham burn injury in Rag1, reconstituted Rag1 and WT animals. These studies demonstrate that the adaptive immune system is necessary for protection from polymicrobial sepsis and plays a significant role in regulating the inflammatory response to injury.     

失血合并内毒素诱发多器官功能障碍综合征的分子病理机制研究

目的 :探讨失血性休克合并内毒素血症致多器官功能障碍综合征 ( MODS)家兔可能的分子病理机制。方法 :采用失血合并内毒素损伤诱发 MODS家兔模型 ,用原位杂交 ( ISH)和凝胶电泳迁移率改变分析法( EMSA)分别检测肺泡巨噬细胞 ( PAM)以及肝枯否氏细胞 ( KC)中 IκΒ激酶 β( IKKβ) m RNA表达和核因子κB( NFкB)的活性 ;用酶联免疫吸附法 ( EL ISA)检测两种细胞培养上清液中肿瘤坏死因子α( TNFα)含量 ;同时进行动脉血气、血生化及肺、肝、小肠病理组织学检查。结果 :诱发 MODS家兔 PAM和 KC中 IKKβm RNA表达 ( 0 .15± 0 .0 3,0 .17± 0 .0 4)、NFκB活性 ( 1.49± 0 .30 ,1.72± 0 .36 )和上清液 TNFα的含量〔( 2 79.74± 2 5 .91) ng/ L 和 ( 30 0 .0 5± 30 .86 ) ng/ L〕均较正常动物〔IKKβ m RNA表达 0 .0 3± 0 .0 1和 0 .0 2±0 .0 1,NFκB活性 0 .2 5± 0 .0 6和 0 .31± 0 .10 ,TNFα含量 ( 137.33± 6 .0 9) ng/ L 和 ( 134.85± 12 .0 9) ng/ L〕明显增高 ( P均 <0 .0 1) ;血尿素氮 ( BU N)和丙氨酸转氨酶 ( AL T)均明显升高 ,氧分压下降 ,内脏组织出现明显炎症病理改变。结论 :IKKβ、NFκB、TNFα在休克合并内毒素诱发 MODS的炎症病理改变中有重要作用。     

A cell wall component from pathogenic and non-pathogenic gram-positive bacteria (peptidoglycan) synergises with endotoxin to cause the release of tumour necrosis factor-alpha, nitric oxide production, shock, and multiple organ injury/dysfunction in the rat

The incidence of sepsis and septic shock due to gram-positive organisms has increased dramatically over the last two decades. Interestingly, many patients with sepsis/septic shock have both gram-positive and gram-negative bacteria present in the bloodstream and these polymicrobial or "mixed" infections often have a higher mortality than infection due to a single organism. The reason for this observation is unclear. The aim of this study was to investigate whether cell wall fragments from gram-positive and gram-negative bacteria could synergise to cause the release of cytokines, shock, and organ injury/ dysfunction in vivo. Male Wistar rats were anaesthetised and received an intravenous bolus of vehicle (saline), lipopolysaccharide (LPS) from Escherichia coli (0.1 mg/kg), peptidoglycan (Pep G) from Staphylococcus aureus (S10 mg/kg), co-administration of LPS (0.1 mg/kg) and PepG from S. aureus (10 mg/kg), LPS (10 mg/kg), PepG from Bacillus subtilis, or co-administration of LPS and PepG from B. subtilis. Blood pressure and heart rate were monitored for 6 h before plasma samples were taken for the measurement of TNF-alpha, total nitrite, and biochemical indices of organ injury. Peptidoglycan from both pathogenic (S. aureus) and non-pathogenic (B. subtilis) gram-positive bacteria synergised with endotoxin to cause formation of TNF-alpha, nitrite, shock, and organ injury. Synergism between PepG and LPS may partly explain the high mortality associated with mixed bacterial infections, as well as the deleterious effects of translocation of bacteria, or their cell wall components from the gut lumen in patients with sepsis.     

Toll-like receptor 4 contributes to microvascular inflammation and barrier dysfunction in thermal injury

Systemic and microvascular inflammation plays a key role in the development of multiple organ failure after infection, sepsis, and traumatic injury. Toll-like receptors (TLRs) regulate host responses to pathogens and sterile, injury-associated inflammatory responses. We investigated whether TLR-4 contributes to microvascular dysfunction during thermal injury in vivo in anesthetized wild-type or TLR-4 (-/-) mice receiving either a 25% total body surface area full-thickness scald burn or sham treatment on the dorsal skin. Using intravital microscopy, we assessed the hemodynamics and leukocyte dynamics in the mesenteric microvasculature as representative of the splanchnic microcirculation at a site remote from the burn wound. The transvascular flux of fluorescein isothiocyanate-albumin across mesenteric venules was measured as an indicator of microvascular permeability. Furthermore, cultured microvascular endothelial cell models were used to evaluate the endothelial-specific mechanisms involved in TLR-4-mediated barrier dysfunction. The results showed significantly elevated microvascular permeability in wild-type mice after burn, whereas this response was markedly attenuated in TLR-4 (-/-) mice. Burn injury also increased leukocyte adhesion in mesenteric venules of wild-type mice, and a blunted leukocyte response was seen in the TLR-4 mice. Treatment of endothelial cell monolayers with burn plasma induced a rapid reduction in the transendothelial electrical resistance measured by electric cell-substrate impedance sensing, indicative of endothelial cell-cell adhesive barrier dysfunction. Reducing expression of TLR-4 with siRNA treatment attenuated this response. Taken together, these data indicate that TLR-4 plays an important role in microvascular leakage and leukocyte adhesion under the inflammatory condition associated with nonseptic thermal injury.     

Pulmonary endothelium in acute lung injury: from basic science to the critically ill

Background Pulmonary endothelium is an active organ possessing numerous physiological, immunological, and metabolic functions. These functions may be altered early in acute lung injury (ALI) and further contribute to the development of acute respiratory distress syndrome (ARDS). Pulmonary endothelium is strategically located to filter the entire blood before it enters the systemic circulation; consequently its integrity is essential for the maintenance of adequate homeostasis in both the pulmonary and systemic circulations. Noxious agents that affect pulmonary endothelium induce alterations in hemodynamics and hemofluidity, promote interactions with circulating blood cells, and lead to increased vascular permeability and pulmonary edema formation.Objective We highlight pathogenic mechanisms of pulmonary endothelial injury and their clinical implications in ALI/ARDS patients.     

The cytokine storm and factors determining the sequence and severity of organ dysfunction in multiple organ dysfunction syndrome

Multiple organ dysfunction syndrome (MODS) is a major cause of morbidity and mortality in intensive care units. It is being encountered frequently in critically ill patients owing to advancements in organ-specific supportive technologies to survive the acute phase of severe sepsis and shock. It is now believed that MODS is the result of an inappropriate generalized inflammatory response of the host to a variety of acute insults. The pathologic mechanisms of MODS were reviewed, and factors determining the sequence and severity of organ dysfunction were discussed in depth. In the early phase of MODS, circulating cytokines cause universal endothelium injury in organs. In the later phase of MODS, overexpression of inflammatory mediators in the interstitial space of various organs is considered a main mechanism of parenchyma injury. The difference in constitutive expression and the upregulation of adhesion molecules in vascular beds and the density and potency of intrinsic inflammatory cells in different organs are the key factors determining the sequence and severity of organ dysfunction. By activating the intrinsic inflammatory cell in a distant organ, organ dysfunctions are linked in a positive feedback loop through circulating inflammatory mediators. Antagonists targeted at adhesion molecules may alleviate the severity of endothelial damage. And nonsteroidial anti-inflammatory drugs or steroids administered judiciously in the early phase of MODS may retard the progress of multiple organ failure.