Endotoxin Pretreatment Research Articles

Modulation of intestinal afferent nerve sensitivity to inflammatory mediators following systemic endotoxin in mice.

Endotoxin exposure may be followed by visceral hypersensitvity but potential mechanisms are not fully explored. We aimed to test the hypothesis that mast cells and the cyclooxygenase pathway (COX) mediate modulation of afferent nerve sensitivity following systemic endotoxin. C57Bl6 mice received endotoxin injection i.p. to induce systemic inflammation. Control animals received normal saline. Extracellular multi-unit afferent nerve discharge was recorded from jejunal mesenteric nerves in vitro. Afferent nerve response to 5-hydroxytryptamine (5-HT, 250 μmol/L), bradykinin (BK, 0.5 μmol/L), and to mechanical ramp distension of the intestinal lumen from 0 to 60 cmH2O were recorded 2 h following endotoxin administration. Following endotoxin administration peak afferent discharge to 5-HT and BK was increased compared to controls (p < 0.05). Pre-perfusion with the mast cell stabilizer Doxantrazole (10(-4) M), or the cyclooxygenase inhibitor Naproxen inhibited the increased response to 5-HT and BK (p < 0.05 vs endotoxin pretreatment). Mechanosensitivity during luminal ramp distension from 10 to 60 cmH2O was increased following endotoxin pretreatment compared to controls (p < 0.05). This increase in sensitivity following endotoxin was no longer observed after Doxantrazole or Naproxen administration for pressures from 10 to 30 cmH2O (p < 0.05). Selective COX-2 inhibition by NS398 (10 μM) but not COX-1 inhibition by SC560 (300 μM) reduced increased afferent discharge in endotoxin pretreated animals to 5-HT, BK and mechanical ramp distension from 10 to 40 cmH2O (all p < 0.05). Systemic endotoxin sensitizes mesenteric afferent nerve fibers to 5-HT, BK and mechanical stimuli. The underlying mechanism responsible for this sensitization seems to involve mast cells and the COX-2 pathway.

IRAK4 kinase activity is not required for induction of endotoxin tolerance but contributes to TLR2-mediated tolerance

Prior exposure to LPS induces "endotoxin tolerance" that reprograms TLR4 responses to subsequent LPS challenge by altering expression of inflammatory mediators. Endotoxin tolerance is thought to limit the excessive cytokine storm and prevent tissue damage during sepsis but renders the host immunocompromised and susceptible to secondary infections. Tolerance initiated via one TLR can affect cellular responses to challenge via the same TLR ("homotolerance") or through different TLRs ("heterotolerance"). IRAK4, an essential component of the MyD88-dependent pathway, functions as a kinase and an adapter, activating subsets of divergent signaling pathways. In this study, we addressed mechanistically the role of IRAK4 kinase activity in TLR4- and TLR2-induced tolerance using macrophages from WT versus IRAK4(KDKI) mice. Whereas IRAK4 kinase deficiency decreased LPS signaling, it did not prevent endotoxin tolerance, as endotoxin pretreatment of WT and IRAK4(KDKI) macrophages inhibited LPS-induced MAPK phosphorylation, degradation of IκB-α and recruitment of p65 to the TNF-α promoter, expression of proinflammatory cytokines, and increased levels of A20 and IRAK-M. Pretreatment of WT macrophages with Pam3Cys, a TLR2-TLR1 agonist, ablated p-p38 and p-JNK in response to challenge with Pam3Cys and LPS, whereas IRAK4(KDKI) macrophages exhibited attenuated TLR2-elicited homo- and heterotolerance at the level of MAPK activation. Thus, IRAK4 kinase activity is not required for the induction of endotoxin tolerance but contributes significantly to TLR2-elicited homo- and heterotolerance.

Effects of a “two-hit” model of organ damage on the systemic inflammatory response and development of laminitis in horses

The role of endotoxemia in the development of laminitis remains unclear. Although systemic inflammation is a risk factor for laminitis in hospitalized horses, experimental endotoxin administration fails to induce the disease. While not sufficient to cause laminitis by itself, endotoxemia might predispose laminar tissue to damage from other mediators during systemic inflammation. In “two-hit” models of organ damage, sequential exposure to inflammatory stimuli primes the immune system and causes exaggerated inflammatory responses during sepsis. Acute laminitis shares many characteristics with sepsis-associated organ failure, therefore an equine “two-hit” sepsis model was employed to test the hypothesis that laminitis develops with increased frequency and severity when repeated inflammatory events exacerbate systemic inflammation and organ damage. Twenty-four light breed mares (10) and geldings (14) with chronic disease conditions or behavioral abnormalities unrelated to laminitis that warranted euthanasia were obtained for the study. Horses were randomly assigned to receive an 8-h intravenous infusion of either lipopolysaccharide (5ng/kg/h) or saline beginning at −24h, followed by oligofructose (OF; 5g/kg) via nasogastric tube at 0h. Euthanasia and tissue collection occurred at Obel grade 2 laminitis, or at 48h if laminitis had not developed. Liver biopsies were performed at 24h in laminitis non-responders. Blood cytokine gene expression was measured throughout the study period. Lipopolysaccharide and OF administration independently increased mean rectal temperature (P<0.001), heart rate (P=0.003), respiratory rate (P<0.001), and blood interleukin (IL)-1β gene expression (P<0.0016), but responses to OF were not exaggerated in endotoxin-pretreated horses. The laminitis induction rate did not differ between treatment groups and was 63% overall. When horses were classified as laminitis responders and non-responders, area under the blood IL-1β expression curve (P=0.010) and liver and lung gene expression of IL-1β, IL-6, IL-8, IL-10, and tumor necrosis factor-α (P<0.05) were higher in responders following OF administration. The results indicate that endotoxin pretreatment did not enhance responses to OF. However, systemic inflammation was more pronounced in laminitis responders compared to non-responders, and tissue-generated inflammatory mediators could pose a greater risk than those produced by circulating leukocytes.

Pulmonary mechanical responses to intestinal ischaemia-reperfusion and endotoxin preconditioning

During intestinal ischaemia-reperfusion, endotoxin can be translocated. Pretreatment with sublethal doses of endotoxin develops tolerance to ischaemia-reperfusion in different organs; however, the tolerance to intestinal ischaemia-reperfusion in the lung has rarely been investigated. Our aim was to study the role of endotoxin pretreatment in the mechanical responses and inflammatory activation induced by intestinal ischaemia-reperfusion in the lung. Wistar rats were preconditioned with a sublethal dose of endotoxin on day -3 or -1. On day 0, anesthetized, paralyzed and mechanically ventilated rats were subjected to a 60-min occlusion of the superior mesenteric artery and a subsequent 240-min reperfusion. The low-frequency forced oscillation technique was employed to characterize the separate mechanical responses of the airways and respiratory tissues. Intestinal ischaemia-reperfusion caused a significant decrease in airway resistance and increases in tissue resistance and elastance, nitric oxide synthase and myeloperoxidase activities. Pretreatment with endotoxin modified both the pulmonary mechanical responses and the inflammatory markers in the lung during intestinal ischaemia-reperfusion. We conclude that endotoxin or the endotoxin-induced processes (and humoral mediators) have significant roles in the pathomechanism of the remote pulmonary effect of intestinal ischaemia-reperfusion.

Turning down the heat: the potential role of RIP140 in inflammation

Turning down the heat: the potential role of RIP140 in inflammation

Effects of endotoxaemia and carbohydrate overload on glucose and insulin dynamics and the development of laminitis in horses

Insulin resistance (IR) is a risk factor for pasture-associated laminitis in equids and alimentary carbohydrate overload may trigger laminitis. Whether glucose metabolism responses to carbohydrate overload are more pronounced in insulin-resistant horses requires further study. Horses pretreated with endotoxin to alter insulin sensitivity differ significantly in their glucose and insulin responses to carbohydrate overload. Horses (n=24) were divided into 3 groups. A lipopolysaccharide (LPS; n=8) group that received endotoxin as an 8 h 7.5 ng/kg bwt/h i.v. continuous rate infusion, an oligofructose (OF; n=8) group that received an infusion of saline followed by 5 g/kg bwt OF via nasogastric intubation, and a LPS/OF (n=8) group that received LPS followed 16 h later by OF. Glucose and insulin dynamics were evaluated at -24 h and 48 h using the frequently sampled i.v. glucose tolerance test and minimal model analysis. Physical examinations and haematology were performed and the severity of laminitis assessed. Horses receiving LPS developed leucopenia and both LPS and OF induced clinical signs consistent with systemic inflammation. Insulin sensitivity significantly decreased (P < 0.001) over time, but responses did not differ significantly among groups. Time (P < 0.001) and treatment x time (P = 0.038) effects were detected for the acute insulin response to glucose, with mean values significantly increasing in LPS and LPS/OF groups, but not the OF group. Five horses in the LPS/OF group developed clinical laminitis compared with 0 and 2 horses in the LPS and OF groups, respectively. Endotoxaemia and carbohydrate overload reduce insulin sensitivity in horses. Endotoxin pretreatment does not affect the alterations in glucose metabolism induced by carbohydrate overload. Insulin sensitivity decreases after carbohydrate overload in horses, which may be relevant to the development of pasture-associated laminitis.

Protective effects of endotoxin pretreatment on hepatic tissue in rats with endotoxemia

Objective To investigate the protective effects of endotoxin precondition on hepatic tissue in rats with endotoxemia.Method The models of rats with acute endotoxemia were produced by injecting LPS directly.Seventy-two male wistar rats were randomly divided into three groups:saline control group(N,n=24),lipopolysaccharide (LPS)-treated group(L,n=24),LPS pretreated group(P,n=24).Each group was divid-ed into four subgroups:saline control and lipopolysaccharide (LPS)-treated 2 h,4 h,6 h,12 h groups and LPS-pretreated 2 h,4 h,6 h,12 h groups.Rats in group P were first administered with introperitoneal injection of 0.25 mg/kg LPS,and after 24 hours,the rats were injected with 0.5 mg/kg LPS.Rats in group N and group L received with an equivalent amount of saline.After 72 hours,rats in group L and group P were intravenonsly injected with 10 mg/kg LPS,and rats in group N received with an equivalent amount of saline.Six rats were killed at 2,4,6 and 12 hours after injection of LPS in group L and P.The hvers were removed for detecting Toll like receptor-4 (TLR-4),nuclear factor-кB(NF-кB),tumor Necrosis Factor-apha(TNF-α)and malondialdehyde(MDA).The blood was drawn for detecting Alamine aminotmnsferose (ALT) and Aspartate aminotransferose (AST).The patho-logical changes of liver were also examined.Software SPSS13.0 was utilized to do ANOVA for statistical analysis.Results The rats exposed to LPS alone demonstrated an increase in TLR-4.NF-кB and TNF-α activity of the liver tissue.Incontrast.the rats exporsed 10 LPS prelreatment exhibited a significant decrease in TLT-4,NF-кB and TNF-α activity.The contents of TLR-4,NF-кB and TNF-α of LPS-treated 4 h groupwere,(38.76±0.67),170.82 ±31.40),293.16±49.49)and(6.263±0.351),significantly higher than those of the saline control group.The administration of endotoxin pretreatment reduced the indexes to(22.35±1.35),(135.55±26.44)and(234.23±44.96),respectively(P＜0.05).Conclusions TLR-4,NF-кB and TNF-α take part in the progress of hepatic injury in rats with endotoxemia.Endotoxin pretreatment can eliminate hepatic injury and protect the hepatic tissue by downmgulating the levels of TLR-4.NF-кB and TNF-α. Key words: Endotoxin; Endotoxemia; Pretrealment; liver injury; Toll like receptor-4

Endotoxin uptake in mouse liver is blocked by endotoxin pretreatment through a suppressor of cytokine signaling-1–dependent mechanism #

The liver is the main organ that clears lipopolysaccharide (LPS) and hepatocytes are a major cell-type involved in LPS uptake. LPS tolerance, or desensitization, is important in negative regulation of responses to LPS, but little is known about its mechanisms in hepatocytes. Primary isolated C57BL/6 hepatocytes, and liver in vivo, internalized fluorescent LPS, and this was dependent on Toll-like receptor 4 (TLR4) at the cell surface but not on TLR4-TIR signaling through MyD88. LPS clearance from plasma was also TLR4-dependent. Pretreatment of C57BL/6 hepatocytes with LPS prevented uptake of LPS 24 hours later and this LPS-mediated suppression was dependent on TLR4 signaling through MyD88. Many regulators of TLR4 signaling have been identified and implicated in LPS desensitization, including suppressor of cytokine signaling 1 (SOCS1). SOCS1 mRNA and protein expression increased after LPS stimulation in hepatocytes and in whole liver. LPS uptake in hepatocytes and liver was significantly reduced following infection with adenoviral vectors overexpressing SOCS1. Similarly, inhibition of SOCS1 using small interfering (si)RNA-mediated knockdown prevented LPS desensitization in hepatocytes. SOCS1 is known to interact with Toll/IL-1 receptor associated protein (TIRAP) and cause TIRAP ubiquitination and degradation, which regulates TLR signaling. We have also shown previously that TIRAP regulates LPS uptake in hepatocytes. SOCS1 coimmunoprecipitated with TIRAP in wild type hepatocyte cell lysates up to 8 hours after LPS stimulation, but not at later times. In the same samples, ubiquitinated TIRAP was detected after 4 hours and up to 8 hours after LPS stimulation, but not at later times. These data indicate hepatocytes are desensitized by LPS in a TLR4 signaling-dependent manner. LPS-induced SOCS1 upregulation increases degradation of TIRAP and prevents subsequent LPS uptake. The exploitation of these mechanisms of LPS desensitization in the liver may be important in future sepsis therapies.

Up-Regulation of IRAK-M is Essential for Endotoxin Tolerance Induced by a Low Dose of Lipopolysaccharide in Kupffer Cells

Endotoxin tolerance (ET) is an important mechanism to maintain the homeostasis of Kupffer cells (KCs), because KCs are continually exposed to various pathogen-associated molecular patterns including lipopolysaccharide (LPS). ET involves multiple changes in cell signal transduction pathways; however, not all signaling pathways are down-regulated and some proteins are up-regulated. The latter proteins may be counter regulatory, including interleukin-1 receptor-associated kinase M (IRAK-M) expression. The aim of this study is to clarify weather or not IRAK-M is involved in the mechanisms of ET in KCs through dampening nuclear factor-kappa B (NF-kappaB) mediated pathway. KCs isolated from male C57BL/6J mice were seeded in 24-well plates at 1 x 10(6) cells/well and cultured overnight prior to transfection, were randomly divided into two groups: the pIRAK-M-short hairpin RNA (shRNA) group (transfected with IRAK-M shRNA) and the control group (transfected with control vector); 24 h after transfection, the two groups were further randomly divided into two subgroups: non-endotoxin pretreatment group (incubation in Dulbecco's modified Eagle's medium [Invitrogen, Carlsbad, CA] with 10% fetal bovine serum) and endotoxin pretreatment group (incubation in the same medium containing 10 ng/mL LPS), named pIRAK-M-EP, pIRAK-M-NEP, pCV-EP, and pCV-NEP, respectively. Each subgroup contained 6 wells; 24 h later, fresh media containing LPS (100 ng/mL) was added to each subgroup and incubated for an additional 3 h. The expression of IRAK-M gene and protein level were determined by reverse transcription-polymerase chain reaction and Western blot, the activities of NF-kappaB were estimated by electrophoretic mobility shift assay and enzyme-linked immunosorbent assay, and the supernatant tumor necrosis factor-alpha levels were analyzed by enzyme-linked immunosorbent assay. The recombinant plasmid of pIRAK-M-shRNA specifically inhibited IRAK-M expression after it was transfected into KCs. At 3 h after 100 ng/mL LPS was added to the medium, IRAK-M expression was significantly induced in pCV-EP than that in pCV-NEP; however, there was no difference between pIRAK-M-NEP and pIRAK-M-EP, accompanied with lowest level of NF-kappaB activation and tumor necrosis factor-alpha levels in pCV-EP, and a dramatic enhancement in the other three groups (P < 0.01). Although a primary low dose of LPS stimulation obviously attenuated KCs response to the second LPS stimulation, the inhibitive influences were partly refracted in pIRAK-M-EP than in pCV-EP, indicating that the absence of IRAK-M caused abnormal enhancement of inflammatory effects. IRAK-M negatively regulates toll-like receptors signaling and involves in the mechanisms of ET in KCs through dampening NF-kappaB mediated pathway; therefore it may be a key component of this important control system, and a new target for the clinical treatment of sepsis.

Immune Activation in Patients With Irritable Bowel Syndrome

We set out to test the hypothesis that irritable bowel syndrome (IBS) is characterized by an augmented cellular immune response with enhanced production of proinflammatory cytokines. We further aimed to explore whether symptoms and psychiatric comorbidity in IBS are linked to the release of proinflammatory cytokines. We characterized basal and Escherichia coli lipopolysaccharide (LPS)-induced cytokine production in peripheral blood mononuclear cells (PBMCs) from 55 IBS patients (18 mixed-, 17 constipation-, 20 diarrhea-predominant) and 36 healthy controls (HCs). PBMCs were isolated by density gradient centrifugation and cultured for 24 hours with or without (1 ng/mL) LPS. Cytokine production (tumor necrosis factor [TNF]-alpha, interleukin [IL]-1beta, and IL-6) was measured by enzyme-linked immunosorbent assay. Abdominal symptoms and psychiatric comorbidities were assessed by using the validated Bowel Disease Questionnaire and the Hospital Anxiety and Depression Scale. IBS patients showed significantly (P < .017) higher baseline TNF-alpha, IL-1beta, IL-6, and LPS-induced IL-6 levels compared with HCs. Analyzing IBS subgroups, all cytokine levels were significantly (P < .05) higher in diarrhea-predominant IBS (D-IBS) patients, whereas constipation-predominant IBS patients showed increased LPS-induced IL-1beta levels compared with HCs. Baseline TNF-alpha and LPS-induced TNF-alpha and IL-6 levels were significantly higher in patients reporting more than 3 bowel movements per day, urgency, watery stools, and pain associated with diarrhea compared with patients without these symptoms (all P < .05). LPS-induced TNF-alpha production was associated significantly (r = 0.59, P < .001) with anxiety in patients with IBS. Patients with D-IBS display enhanced proinflammatory cytokine release, and this may be associated with symptoms and anxiety.

Protective Effect of Endotoxin Preconditioning in Pancreas: Analysis With DNA Chip Arrays

Recently we demonstrated a protective effect of endotoxin preconditioning 24 hours before pancreatic ischemia-reperfusion injury, which has also been described for other organs. The mechanisms underlying this phenomenon, such as differential gene expression, are poorly investigated. We chose to approach this question by investigating differential gene expression in the rat pancreas over the time course of endotoxin pretreatment. Male Wistar rats (5 groups, 5 animals per group) were pretreated with endotoxin intraperitoneally (1 mg/kg of body weight). After treatment at 30 minutes, and at 3 and 24 hours the pancreas was removed. Untreated animals and animals with injection of saline solution served as controls. After RNA isolation, RNA was pooled and hybridized to Affymetrix chips to measure the relative mRNA levels of 7000 genes and 1000 expression sequence tags. Three hours after administration of endotoxin there was an activation of proinflammatory transcription factors and other proinflammatory genes. After 24 hours there was a clear decrease of these proinflammatory genes, but a remaining and increasing upregulation of important antiapoptotic genes, antiproteases, and other probably protective genes. There was also a significant upregulation of complement factors. It was surprising that heat-shock proteins and other typical immediate early genes of the AP-1 complex were not upregulated. Our data show that 24 hours after endotoxin stress there is a regulation of a network of genes that represents a multifaceted preconditioning. As most important factors, inhibition of apoptosis and antiproteatic strategies are identified. Heat-shock proteins seem to play no important role in the mechanism of endotoxin preconditioning.

Expression of apoptosis regulatory factors during myocardial dysfunction in endotoxemic rats*

To document the time course of apoptosis pathway activation in sepsis and to determine whether Bcl-2 overexpression would improve endotoxin-induced myocardial dysfunction and mortality rate. Randomized, controlled trial. Experimental laboratory. Male Sprague Dawley rats, wild-type C57BL/6 female mice, C57BL/6 female mice overexpressing Bcl-2. Hearts were isolated from rats treated with endotoxin (10 mg/kg, intravenously) to perform heart function, immunohistochemistry (terminal deoxynucleotidyl transferase-mediated dUTP-biotin nick 3'-end labeling, caspase 3), RNase protection assay, reverse transcriptase polymerase chain reaction, Western blotting (caspase 3), and radiolabeled annexin V studies. Twenty-four hours before endotoxin challenge (10 mg/kg, intravenously), rats were pretreated with saline or endotoxin (0.5 mg/kg, intraperitoneally), with or without parthenolide (1 mg/kg, intraperitoneally). Isolated hearts were used to test myocardial function. Mortality induced by endotoxin (10 mg/kg, intraperitoneally) was tested on wild-type or mice overexpressing Bcl-2. Endotoxin-induced heart dysfunction was maximal at 4 and 8 hrs postinjection, started to improve, and was fully restored at 24 hrs after endotoxin treatment. Endotoxin also induced phosphatidylserine outer leaflet membrane exposure, caspase 3 activation, nuclear apoptosis, and changes in apoptosis gene expression. Bcl-2 overexpression induced by endotoxin pretreatment prevented endotoxin-induced myocardial dysfunction. Mice overexpressing Bcl-2 had dramatic improvement in survival rate compared with wild-type mice. These observations suggest that both death receptor and caspase-mediated apoptosis processes are activated in this sepsis model. Bcl-2 overexpression before endotoxin challenge prevents myocardial dysfunction in rats and improves survival rate in mice.

Endotoxin pretreatment modifies peristalsis and attenuates the antipropulsive action of adrenoceptor agonists in the guinea‐pig small intestine

The action of endotoxin to alter gastrointestinal motility in vivo may reflect a direct effect on the gut or result from vascular and other systemic manifestations of this sepsis model. Here we examined whether in vivo pretreatment of guinea-pigs with endotoxin modifies peristalsis in the isolated gut and influences the antipropulsive action of adrenoceptor agonists. Distension-induced peristalsis was recorded in fluid-perfused segments of the small intestine taken from animals pretreated intraperitoneally with endotoxin (1 mg kg(-1)Escherichia coli lipopolysaccharide) or vehicle 4 or 20 h before. Clonidine, adrenaline, noradrenaline, dopamine and dobutamine inhibited peristalsis with differential potency. Endotoxin pretreatment lowered the peristaltic pressure threshold and altered other parameters of baseline peristalsis in a time-related manner. The potency and efficacy of clonidine to inhibit peristalsis were markedly decreased after endotoxin administration, while the potency of the other test drugs was less attenuated. The antipropulsive action of clonidine in control segments was reduced by yohimbine and prazosin, whereas in segments from endotoxin-pretreated animals it was antagonized by yohimbine but not prazosin. We conclude that systemic endotoxin pretreatment of guinea-pigs modifies baseline peristalsis by an action on the gut and inhibits the antipropulsive action of adrenoceptor agonists through changes in adrenoceptor activity.

Rapid up-regulation of IRAK-M expression following a second endotoxin challenge in human monocytes and in monocytes isolated from septic patients

The exposure of human monocytes to the gram-negative endotoxin LPS provokes them to enter a transient state in which they are refractory to further stimulation by LPS. This phenomenon is known as ‘endotoxin tolerance’ (ET) and it is characterized by a decrease in leukocyte proinflammatory cytokine production in response to LPS. In the present study, we have analyzed the expression of IRAK-M mRNA and protein in a human model of ET using human monocytes isolated from peripheral blood. In these monocyte cultures, IRAK-M mRNA was expressed 6 h after stimulation with different doses of LPS. However, endotoxin pretreatment induced a more immediate up-regulation of IRAK-M gene expression, transcripts appearing only one hour after a second LPS-challenge, and the production of high levels of IRAK-M protein in these tolerant monocytes. We also analyzed the response of monocytes isolated from septic patients within a temporal tolerance timeframe when stimulated ex vivo with LPS. In contrast to monocytes from healthy volunteers and patients outside of the tolerance timeframe, monocytes from septic patients rapidly expressed IRAK-M mRNA when stimulated with LPS ex vivo. Moreover, the expression of IRAK-M mRNA was more rapidly induced in the presence of a PI3K inhibitor, suggesting a connection between these two kinases. Thus, our data indicate that IRAK-M could play a pivotal role in the process of ET in human monocytes and provide evidence that PI3K is involved in regulating its expression.

Rapid up-regulation of IRAK-M expression following a second endotoxin challenge in human monocytes and in monocytes isolated from septic patients

The exposure of human monocytes to the gram-negative endotoxin LPS provokes them to enter a transient state in which they are refractory to further stimulation by LPS. This phenomenon is known as ‘endotoxin tolerance’ (ET) and it is characterized by a decrease in leukocyte proinflammatory cytokine production in response to LPS. In the present study, we have analyzed the expression of IRAK-M mRNA and protein in a human model of ET using human monocytes isolated from peripheral blood. In these monocyte cultures , IRAK-M mRNA was expressed 6 h after stimulation with different doses of LPS. However, endotoxin pretreatment induced a more immediate up-regulation of IRAK-M gene expression, transcripts appearing only one hour after a second LPS-challenge, and the production of high levels of IRAK-M protein in these tolerant monocytes. We also analyzed the response of monocytes isolated from septic patients within a temporal tolerance timeframe when stimulated ex vivo with LPS. In contrast to monocytes from healthy volunteers and patients outside of the tolerance timeframe, monocytes from septic patients rapidly expressed IRAK-M mRNA when stimulated with LPS ex vivo. Moreover, the expression of IRAK-M mRNA was more rapidly induced in the presence of a PI3K inhibitor, suggesting a connection between these two kinases. Thus, our data indicate that IRAK-M could play a pivotal role in the process of ET in human monocytes and provide evidence that PI3K is involved in regulating its expression.

Endotoxin preconditioning in pancreatic ischemia/reperfusion injury.

Prospective organ donors are exposed to various stress types. The effect of endotoxin pretreatment (ETX) on pancreatic ischemia/reperfusion injury (IRI) is unclear. We investigated, using a rat model of pancreatic IRI of an in situ isolated pancreatic tail segment, the effect of ETX on postischemic microcirculation and organ damage. Twenty-four hours before pancreatic dissection, either intraperitoneal application of ETX (1 mg/kg in 0.9% NaCl) or saline only (control) was performed. Two-hour normothermic ischemia of the pancreatic tail was induced by clamping the splenic vessels and was followed by a reperfusion period of 2 hours. Microcirculatory parameters were measured by intravital epifluorescence microscopy [functional capillary density (FCD), adherent leukocytes (ALs), and histology]. The presented data represent the mean +/- SEM/SD as appropriate. ETX pretreatment caused a significantly greater decrease in FCD (497 +/- 6 cm/cm2 baseline versus 326 +/- 15 cm/cm2 2 hours of reperfusion) compared with controls (498 +/- 8 versus 258 +/- 15 cm/cm2) 2 hours after reperfusion (P < 0.01). Two hours after reperfusion, ALs were significantly decreased in ETX animals compared with controls (ETX: 141 +/- 37 versus 273 +/- 36 cells/mm2, P < 0.05). Histologic damage was less in ETX (6.4 score points +/- 0.32 versus 8.8 +/- 0.33 control, P < 0.05). ETX preconditioning decreases microcirculatory deterioration caused by IRI by means of less loss of nutritive tissue perfusion, decrease in ALs, and less histologic damage. This indicates a protective effect of ETX preconditioning in pancreatic IRI.

Sublethal endotoxin administration evokes super-resistance to systemic hypoxia in rats.

Systemic hypoxia following surgical injury modulates cytokine and catecholamine responses. Endotoxin tolerance develops after pretreatment of animals with sublethal endotoxin doses and is characterized by a reduced inflammatory cytokine response to subsequent endotoxin challenges. The administration of endotoxin also attenuates ischemic injury of rat myocardial tissue following hypoxia, a phenomenon described as cross-tolerance. The objectives of this study were: 1) to determine whether endotoxin evokes a cross-tolerance to systemic hypoxia in rats; and 2) to estimate circulatory and pulmonary performance in rats with systemic hypoxia after endotoxin pretreatment. Seventy-two hours before the experiment, Wistar rats were given an intraperitoneal injection of endotoxin at a dose of 10 microg/kg. Polyethylene catheters were inserted into the femoral vein for infusion, and into the femoral artery for blood sampling and blood pressure monitoring. Systemic hypoxia was achieved by continuous inhalation of a modified gas mixture (9% oxygen+ 91% N2) for 4 hours. Plasma TNF-alpha and IL-6 were measured by ELISA, and norepinephrine (NE) by HPLC. The hypoxic rats that were pretreated with saline showed a significant decrease in mean arterial blood and base excess, as compared with the normoxic rats. Endotoxin pretreatment prevented the drop in mean arterial pressure during hypoxia and reduced the decrease in base excess. Hypoxic conditions markedly stimulated TNF-alpha and IL-6 release and increased NE levels, compared to the normoxic rats. Pretreatment with endotoxin suppressed the hypoxia-induced cytokine production as well as attenuating the increase in NE levels In this rat hypoxia model, endotoxin pretreatment ameliorated the hypoxia-induced inflammatory response as well as suppressing the effects on arterial oxygenation, anaerobic metabolism and NE stimulation.

Endotoxemia prevents the cerebral inflammatory wave induced by intraparenchymal lipopolysaccharide injection: role of glucocorticoids and CD14.

There is a robust and transient innate immune response in the brain during endotoxemia, which is associated with a cascade of NF-kappaB signaling events and transcriptional activation of genes that encode TNF-alpha and the LPS receptor CD14. The present study investigated whether circulating LPS has the ability to modulate the cerebral innate immune response caused by an intrastriatal (IS) injection of the endotoxin. We also tested the possibility that CD14 plays a role in these effects and male rats received an intracerebroventricular injection with an anti-CD14 before the IS LPS administration. The single LPS bolus into the striatum caused a strong and time-dependent transcriptional activation of TNF-alpha, IkappaBalpha, CD14, and monocyte chemoattractant protein-1 mRNA in microglial cells ipsilateral to the site of injection. Surprisingly, this wave of induced transcripts was essentially abolished by the systemic endotoxin pretreatment. Such anti-inflammatory properties of circulating LPS are mediated via plasma corticosterone, because exogenous corticoids mimicked while glucocorticoid receptor antagonist RU486 prevented the effects of systemic endotoxin challenge. Of interest is the partial involvement of CD14 in LPS-induced neuroinflammation; the anti-CD14 significantly abolished the microglial activity at day 3, but not at times earlier. The inflammatory response provoked by an acute intraparenchymal LPS bolus was not associated with convincing neurodegenerative processes. These data provide compelling evidence that systemic inflammation, through the increase in circulating glucocorticoids, has the ability to prevent the cerebral innate immune reaction triggered by an IS endotoxin injection. This study also further consolidates the existence of such system in the brain, which is finely regulated and its transient activation is not harmful for the neuronal elements.

Importance of hypoxic vasoconstriction in maintaining oxygenation during acute lung injury.

To investigate the role of hypoxic pulmonary vasoconstriction in the intrapulmonary blood flow redistribution and gas exchange protection during oleic acid acute lung injury. Prospective, controlled animal study. Research laboratory of an academic institution. Three groups of five mongrel dogs. Induction of acute lung injury by 0.08 mL/kg oleic acid intravenously. Hypoxic pulmonary vasoconstriction inhibition by Escherichia coli endotoxin microdose (15 microg/kg) pretreatment or by metabolic alkalosis (pH 7.60). Pulmonary arterial and venous resistances were determined by flow-pressure curves and by capillary pressure estimation. Regional lung water and pulmonary blood flow were assessed by positron emission tomography. Oleic acid alone increased the arterial and venous resistances, redistributed blood flow away from edematous areas, and decreased the Pao2 from 507 +/- 16 to 373 +/- 60 torr. on Fio2 1.0 and positive end-expiratory pressure 5 cm H2O. Endotoxin pretreatment inhibited the increase in arterial resistance, suppressed the redistribution, and decreased the Pao2 to 105 +/- 22 torr. Alkalosis inhibited the increase in arterial and venous resistances, suppressed the redistribution, and decreased the Pao2 to 63 +/- 12 torr. Reversal of the alkalosis increased the arterial and venous resistances, restored the perfusion redistribution, and improved the Pao2 to 372 +/- 63 torr. Changes in blood gases conformed to predictions of a computer lung model in which hypoxic pulmonary vasoconstriction was suppressed by endotoxin and alkalosis. We conclude that in oleic acid-induced lung injury, a) pulmonary hypertension results from increases in both arterial and venous resistances; b) the increase in arterial resistance is the primary mechanism responsible for the perfusion redistribution and the gas exchange protection; and c) the increase in arterial resistance is most consistent with hypoxic pulmonary vasoconstriction.

Endothelin Receptor Remodeling Induces the Portal Venous Hyper-Response to Endothelin Following Endotoxin Pretreatment

The purpose of this study was to determine the changes in endothelin (ET) receptor subtype expression and their functional significance after endotoxin pretreatment. Rats were pretreated with lipopolysaccharide (LPS) or sterile saline (control). After 24 h, liver samples were homogenized and competitive receptor binding assays were performed. There was no significant difference in ET receptor binding affinity between the control and LPS groups. However, the receptor subtype density showed a significant increase in ET(B) receptors in LPS-treated rats, whereas the amount of ET(A) receptors was almost identical between the two groups. In control, almost all ET receptors (95%) were displaced by using combined ET(A) antagonist (BQ-610) and ET(B) agonist (IRL-1620) as competitors, whereas only 80% (P < 0.05 versus control) was displaced in LPS group, raising the possibility of novel type of ET receptor expression. An infusion of ET(B) agonist (Sarafotoxin 6c, S6c) through portal vein in isolated perfused livers produced the same pressure response in both LPS and control groups; however, the portal pressure increase in response to the ET-1, which binds all ET receptors, was significantly potentiated in LPS-treated rats compared to controls. We conclude that altered regulation of ET receptors, in particular, the appearance of ET binding capacity that is not displaced by ET(A) or ET(B) competitors, may explain the hyper-response of the portal venous system to ET-1 during endotoxemia.