Lipopolysaccharide Pretreatment Research Articles

Dietary copper deficiency increases inducible nitric oxide synthase‐mediated vascular dilation in rat aorta

AbstractThe attenuation of endothelium‐dependent nitric oxide (NO)‐mediated vasodilation is a consistent finding in both conduit and resistance vessels during copper deficiency. However, there is often no effect on systemic blood pressure in experimental animals, suggesting that peripheral vascular resistance is not altered. We hypothesized that baseline vascular smooth muscle relaxation may be maintained by a chronic increase in inducible NO synthase (iNOS) expression, as has been documented in hearts of copper‐deficient rats. We used endothelium‐denuded rat aortic rings to examine the role of iNOS in the regulation of vascular reactivity during dietary copper deficiency. Male weanling rats were fed a copper‐adequate (CuA, 5.6 mg Cu/kg diet) or copper‐deficient diet (CuD, 0.33 mg Cu/kg diet) for 4 weeks. The induction of “functional” iNOS was indicated by a relaxation response to the NO precursor L‐arginine or to Cu,Zn‐superoxide dismutase (SOD), which preserves basal NO. Time to 50% relaxation in response to either compound was significantly shorter in the CuD than in the CuA aortas. The maximal relaxation response to L‐arginine was blocked by the iNOS inhibitor L‐NIL, and the relaxation response to Cu,Zn‐SOD was blocked by the NO‐sensitive guanylate cyclase inhibitor ODQ. Maximal activation of iNOS expression with lipopolysaccharide pretreatment did not cause a difference in vascular relaxation between dietary groups in response to L‐arginine. Expression of the iNOS protein in the aortas was also not different between groups. These results suggest that although there is no apparent increase in protein expression, copper deficiency increases baseline iNOS activity in the vascular wall. J. Trace Elem. Exp. Med. 15:85–95, 2002. © 2002 Wiley‐Liss, Inc.

LPS activation of leukocytes attenuates adhesion to endothelial cells under shear stress

Leukocyte adhesion is triggered by upregulation of cell surface adhesion molecules and counteracted by the shear forces of the flowing blood. Since both factors, inflammatory response and flow dynamics are severely altered during endotoxemia, we studied the effects of endotoxin on leukocyte–endothelial interactions under different levels of shear stress in vitro. In order to perform the experiments at precisely adjustable levels of shear stress, we used a parallel plate flow chamber as has been employed in a number of adhesion studies previously. Within this chamber, endothelial cells (HUVEC) could be perfused with neutrophils (PMN) at 0.25–3 dynes/cm2. FACS analysis of adhesion molecules, trypan blue exclusion and PMN migration showed that the cells were not altered during cell separation. To determine the effects of lipopolysaccharide (LPS) on shear-dependent adhesion, we studied HUVEC and PMN in a native state and following activation with LPS at 100 ng/ml and 10 ng/ml. All flow experiments were videotaped and the results were analysed by the paired t-test (P < 0.05). The effects of LPS on leukocyte–endothelial interactions strongly depended on the site of activation. Whereas LPS pretreatment of HUVEC increased PMN adhesion by 5–10 fold, pre-activation of the PMN resulted in a 50% reduction of adherent cells. In addition, after pre-activation of PMN, adhesion became increasingly dependent on shear stress and, thus, inhibition by LPS was most pronounced at a normal, postcapillary shear stress of 2–3 dynes/cm2. As demonstrated by addition of antibodies against selectins, the effect of LPS was due to a functional loss of L-Selectin and P-Selectin mediated interactions. In addition, integrin-mediated adhesion was impaired despite upregulation of CD11b on activated PMN. In summary, our results show that leukocyte–endothelial interactions become increasingly dependent on shear stress as soon as PMN are activated. Once PMN have undergone LPS activation, leukocyte tethering to the endothelium becomes entirely dependent on endothelial E-Selectin. Furthermore, adhesion efficiency decreases despite upregulated expression of CD11b. Since LPS increased the rigidity of PMN, it seems that the increased stiffness attenuated PMN adhesion by hampering PMN flattening and, thereby, reducing the number of available bonds to the endothelium.

Effect of endotoxin pretreatment on hepatic stellate cell response to ethanol and acetaldehyde.

The role of endotoxin in alcohol-induced liver damage is well recognized. How pre-exposure to endotoxin might affect alcohol injury is not known. We herein studied the effect of endotoxin pretreatment on hepatic stellate cell (HSC) response to ethanol and acetaldehyde. Rat HSC (CFSC-2G) were exposed to media supplemented with 1 microg/mL lipopolysaccharide (LPS). This was followed by a 24 h exposure to media containing LPS plus 50 mmol/L ethanol or 175 micromol/L acetaldehyde. Lipid peroxidation, collagen, and tumor necrosis factor (TNF)-alpha, interleukin (IL)-1beta, IL-6 and transforming growth factor (TGF)-beta1 secretion were determined at the end of both periods of exposure. Lipopolysaccharide pretreatment did not modify lipid peroxidation induced by ethanol or acetaldehyde alone. Glutathione (GSH) content decreased to 4.2 +/- 0.5 and 16.3 +/- 0.8 nmol protein after exposure to ethanol or acetaldehyde alone, and decreased further with LPS pretreatment (2.4 +/- 0.2 and 2.7 +/- 0.3 nmol/mg protein, respectively). Oxidized GSH (GSSG) content increased in ethanol and acetaldehyde LPS-pretreated cells only. Collagen secretion increased to 988 +/- 82 and 1169 +/- 91 microg/10(6) cells after exposure to acetaldehyde or LPS alone. Lipopolysaccharide pretreatment enhanced collagen secretion significantly in both ethanol- and acetaldehyde-treated cells (969 +/- 56 and 1360 +/- 72 microg/10(6) cells, respectively). Interleukin-6 production increased to 288 +/- 48, 1195 +/- 86 and 247 +/- 35 pg/mL per 10(6) cells after ethanol, acetaldehyde and LPS exposure, and increased further with LPS pretreatment in ethanol-exposed cells (680 +/- 23 pg/mL 10(6) cells). Lipopolysaccharide pretreatment of HSC adds to the damage produced by ethanol and acetaldehyde by diminishing GSH content and increasing GSSG content, collagen and IL-6 secretion.

Effect of cross-tolerance between endotoxin and TNF-α or IL-1β on cellular signaling and mediator production

Endotoxin [lipopolysaccharide (LPS)] tolerance suppresses macrophage/monocyte proinflammatory-mediator production. This phenomenon also confers cross-tolerance to other stimuli including tumor necrosis factor (TNF) alpha and interleukin (IL)-1beta. Post-receptor convergence of signal transduction pathways might occur after LPS, IL-1beta, and TNF-alpha stimulation. Therefore, it was hypothesized that down-regulation of common signaling molecules induces cross-tolerance among these stimuli. LPS tolerance and cross-tolerance were examined in THP-1 cells. Phosphorylation of MAP kinases and degradation of inhibitor kappaBalpha (IkappaBalpha) DNA binding of nuclear factor-kappaB (NF-kappaB), and mediator production were examined. In naive cells, LPS, TNF-alpha, and IL-1beta induced IkappaBalpha degradation, kinase phosphorylation, and NF-kappaB DNA binding. LPS stimulation induced production of TNF-alpha or TxB2 and degradation of IRAK. However, neither TNF-alpha nor IL-1beta induced IRAK degradation or stimulated TNF-alpha or TxB2 production in naive cells. Pretreatment with each stimulus induced homologous tolerance to restimulation with the same agonist. LPS tolerance also suppressed LPS-induced TxB2 and TNF-alpha production. LPS pretreatment induced cross-tolerance to TNF-alpha or IL-1beta stimulation. Pretreatment with TNF-alpha induced cross-tolerance to LPS-induced signaling events and TxB2 production. Although pretreatment with IL-1beta did not induce cross-tolerance to LPS-induced signaling events, it strongly inhibited LPS TNF-alpha and TxB2 production. These data demonstrate that IL-1beta induces cross-tolerance to LPS-induced mediator production without suppressing LPS-induced signaling to MAP kinases or NF-kappaB activation.

Peritonitis induces rat cardiac myocytes to promote polymorphonuclear leukocyte emigration and activate endothelial cells: effect of lipopolysaccharide pretreatment.

Peritonitis induced by cecal ligation and perforation results in inflammation and dysfunction of the rat myocardium, an organ remote from the locus of infection. This peritonitis-induced pathology can be prevented by pretreating these animals with lipopolysaccharide before cecal ligation and perforation. In the present study, we assessed a) whether cardiomyocytes obtained from rats subjected to cecal ligation and perforation could induce polymorphonuclear leukocyte transendothelial migration, b) whether these cardiomyocytes could activate endothelial cells (increased proadhesive phenotype), and c) whether these responses could be attenuated by lipopolysaccharide pretreatment. Prospective animal study. Experimental animal laboratory. Male Sprague Dawley rats. Lipopolysaccharide pretreated and nonpretreated rats were subjected to cecal ligation and perforation or to laparotomy. Myocytes were isolated 6 hrs after surgery and used for in vitro experiments. Myocytes isolated from cecal ligation and perforation rats promoted migration of polymorphonuclear leukocytes across a rat endothelial cell monolayer, an effect prevented by platelet activating factor receptor antagonists. Myocytes isolated from these animals also increased surface level expression of intercellular adhesion molecule-1 on rat endothelial cells, an effect also prevented by platelet activating factor receptor antagonists. Myocytes isolated from rats pretreated with lipopolysaccharide and then subjected to cecal ligation and perforation did not a) promote polymorphonuclear leukocyte transendothelial migration or b) increase intercellular adhesion molecule-1 surface expression on endothelial cells. Our findings indicate that induction of peritonitis results in a systemic response that induces cardiac myocytes to become proinflammatory (i.e., these myocytes produce chemotactic factors and activate endothelial cells). This effect of cecal ligation and perforation is abrogated by pretreating animals with lipopolysaccharide before induction of peritonitis.

Oxidized low density lipoproteins downregulate LPS-induced platelet-activating factor receptor expression in human monocyte-derived macrophages: implications for LPS-induced nuclear factor-kappaB binding activity.

Monocytes/macrophages play a key role in atherogenesis due to their inflammatory properties including formation of lipid mediators such as platelet-activating-factor (PAF). We investigated the effect of oxidized low-density lipoprotein (oxLDL) on lipopolysaccharide (LPS)-induced PAF receptor (PAF-R) expression in human macrophages and the implication of the nuclear factor (NF)-kappaB in this regulation. LPS-treatment (1 microg.mL(-1)) of macrophages increased PAF binding and PAF-R mRNA expression by 56% (P < 0.05) and twofold (P < 0.01), respectively. In contrast, highly oxidized low-density lipoprotein [ox24hLDL; 100 microg.mL(-1); thiobarbituric acid reacting substances: 31 +/- 3 nmol equiv. malondialdehyde (MDA).mg protein LDL-1] diminished PAF-R expression (-69%; P < 0.05) and mRNA level (- 45%; P < 0.01). LPS pretreatment induced the activated form of p65 in the nuclear compartment of macrophages (detected by Western blotting) and NF-kappaB binding activity (by electrophoretic mobility shift assay). Treatment of macrophages with ox24hLDL suppressed the LPS-induced binding of NF-kappaB to DNA. In addition, treatment of macrophages with lysophosphatidylcholine (2 and 10 microM), a major component of oxLDL, inhibited the LPS-induced NF-kappaB binding to DNA and reduced PAF binding by 30 and 70%, respectively. In conclusion, oxLDL may downregulate PAF-R expression in human macrophages by inhibiting LPS-induced NF-kappaB binding to DNA.

Major roles of prostanoid receptors IP and EP 3 in endotoxin-induced enhancement of pain perception

To know the roles of prostaglandin I (IP) and prostaglandin E (EP) receptors in pain perception, we compared the acetic acid-induced writhing response in mice deficient in prostaglandin receptors, i.e. IP, EP 1, EP 2, EP 3, or EP 4, with or without lipopolysaccharide (LPS) pretreatment. Without LPS pretreatment, IP-receptor deficient mice showed a significantly smaller number of responses, as previously reported, whereas mice deficient in any of the EP-receptor subtypes showed a number of writhings similar to those of wild-type mice. When mice were pretreated with LPS for 24 hr to induce cyclooxygenase-2 expression, the wild-type as well as EP 1-, EP 2-, or EP 4-receptor-deficient mice showed a similar enhanced writhing response, whereas IP- and EP 3-receptor-deficient mice had a significantly less enhanced number of writhings. These results indicate that IP and EP 3 are the major prostaglandin receptors mediating the enhanced acetic acid-induced writhing response in mice pre-exposed to LPS, i.e. in endotoxin-enhanced inflammatory nociception.

Improved innate immunity of endotoxin-tolerant mice increases resistance to Salmonella enterica serovar typhimurium infection despite attenuated cytokine response.

During infection with gram-negative bacteria, exposure of immune cells to lipopolysaccharide (LPS) from the bacterial cell membrane induces a rapid cytokine response which is essential for the activation of host defenses against the invading pathogens. Administration of LPS to mice induces a state of hyporesponsiveness, or tolerance, characterized by reduced cytokine production upon subsequent LPS challenge. In the model of experimental Salmonella enterica serovar Typhimurium infection of mice, we assessed the question of whether complete LPS tolerance induced by repetitive doses of LPS interfered with cytokine production and host defense against gram-negative bacteria. Although production of various cytokines in response to serovar Typhimurium was attenuated by LPS pretreatment, LPS-tolerant mice showed improved antibacterial activity, evidenced by a prolongation of survival and a continuously lower bacterial load. We attribute this protective effect to three independent mechanisms. (i) Peritoneal accumulation of leukocytes in the course of LPS pretreatment accounted for enhanced defense against serovar Typhimurium during the first 6 h of infection but not for decreased bacterial load in late-stage infection. (ii) LPS-tolerant mice had an increased capacity to recruit neutrophilic granulocytes during infection. (iii) LPS-tolerant mice showed threefold-increased Kupffer cell numbers, enhanced phagocytic activity of the liver, and strongly improved clearance of blood-borne serovar Typhimurium. These results demonstrate that despite attenuated cytokine response, acquired LPS tolerance is associated with enhanced resistance to infections by gram-negative bacteria and that this effect is mainly mediated by improved effector functions of the innate immune system.

Plasma extravasation mediated by lipopolysaccharide-induction of kinin B1 receptors in rat tissues.

The present study was performed to: (a) evaluate the effects of kinin B1 (Sar[D-Phe8]-des-Arg9-BK; 10 nmol/kg) and B2 (bradykinin (BK); 10 nmol/kg) receptor agonists on plasma extravasation in selected rat tissues; (b) determine the contribution of a lipopolysaccharide (LPS) (100 microg/kg) to the effects triggered by B1 and B2 agonists; and (c) characterize the selectivity of B1 ([Leu8]desArg9-BK; 10 nmol/kg) and B2 (HOE 140; 10 nmol/kg) antagonists as inhibitors of this kinin-induced phenomenon. B1 and B2 agonists were shown to increase plasma extravasation in the duodenum, ileum and also in the urinary bladder of the rat. LPS pretreatment enhanced the plasma extravasation mediated only by the B1 agonist in the duodenum, ileum, trachea, main and segmentar bronchi. These effects were prevented by the B1. but not the B2 antagonist. In normal rats, the B2 antagonist inhibited the effect of B2 agonist in all the tissues analyzed. However, in LPS-treated rats, the B2 antagonist was ineffective in the urinary bladder. These results indicate that kinins induce plasma extravasation in selected rat tissues through activation of B1 and B2 receptors, and that LPS selectively enhances the kinin effect on the B1 receptor in the duodenum, ileum, trachea and main and segmentar bronchi, and may increase B1 receptor expression in these tissues.

Endotoxin tolerance from lipopolysaccharide pretreatment induces nuclear factor-kappaB alterations not present in C3H/HeJ mice.

Lipopolysaccharide (LPS) activation of macrophage (MO) cytokine secretion requires activation and translocation of nuclear factor-kappaB (NF-kappaB). Endotoxin tolerance induced in LPS-responsive C3H/HeN MOs by LPS pretreatment results in decreased tumor necrosis factor (TNF) secretion and altered NF-kappaB activation. C3H/HeJ MOs have a genetic defect that renders them tolerant to LPS activation. We hypothesized that the alterations of NF-kappaB activation seen with LPS tolerance in HeN MOs would be present in HeJ mice. MOs from C3H/HeJ and C3H/HeN mice were cultured with +/- 10 ng/mL LPS pretreatment for 24 hours and then stimulated with 1 to 1,000 ng/mL LPS. Activation of NF-kappaB was assayed by gel shift using a 32P-labeled specific oligonucleotide 30 minutes after LPS activation. TNF secretion 6 hours after LPS stimulation was measured by bioassay. LPS stimulation activated NF-kappaB in both HeN and HeJ MOs. We observed decreased NF-kappaB activation and a characteristic mobility shift in endotoxin-tolerant MOs from HeN mice that were not present in HeJ MOs. In contrast with the results in HeN mice, LPS pretreatment did not induce any alterations in NF-kappaB activation in HeJ MOs. LPS-stimulated TNF secretion was decreased in HeN MOs after LPS pretreatment. There was no change in TNF secretion in HeJ MOs, but, overall, TNF secretion by these cells was much less than that seen in HeN cells. MOs from C3H/HeN mice rendered LPS-tolerant by low-dose LPS pretreatment have alterations in activation of NF-kappaB not present in LPS-hyporesponsive C3H/HeJ mice.

Defective lipopolysaccharide-dependent ERK 1/2 activation in endotoxin tolerant murine macrophages is reversed by direct protein kinase C stimulation.

Lipopolysaccharide (LPSp) pretreatment inhibits TNF secretion in endotoxin-tolerant macrophages via alterations in signal transduction pathways of LPS activation (LPSa). Protein kinase C inhibitors prevent TNF release in response to LPSa and direct protein kinase C activation with phorbol myristate acetate (PMA) restores TNF secretion after LPSp. In the current experiments the effect of protein kinase C modulation on LPSa-stimulated ERK 1/2 activation was investigated. Murine macrophage TNF production was determined after stimulation with 100 ng/mL of LPSa, +/- 24 h pretreatment with 10 ng/mL of LPSp. Direct protein kinase C activators (PMA or indolactam) or inhibitors (H7 or bisindolylmaleimide) were added 1 h before LPSa. Diphosphorylated ERK 1/2 was assayed after LPSa stimulation by Western blot. LPS tolerance after LPSp was characterized by inhibition of LPSa-stimulated TNF and accompanied by impaired ERK 1/2 activation by LPSa. Protein kinase C activation with PMA or indolactam restored ERK 1/2 activation and TNF secretion. Inhibition of protein kinase C with H7 or bisindolylmaleimide prevented TNF secretion and ERK 1/2 activation by LPSa. These findings suggest that both ERK 1/2 and protein kinase C are required for TNF production in nontolerant macrophages and that LPS tolerance may be associated with an inability to phosphorylate ERK 1/2.

Evaluation of iNOS-dependent and independent mechanisms of the microvascular permeability change induced by lipopolysaccharide.

1. Subcutaneous injection of lipopolysaccharide (LPS) increases plasma leakage in mouse skin. Pretreatment with LPS conditions mice tolerant to the LPS-induced plasma leakage. Nitric oxide (NO) has been suggested to be involved in these LPS effects. A specific role of inducible NO synthase (iNOS) was investigated in the LPS-induced plasma leakage using iNOS deficient mice. 2. Plasma leakage in mouse skin was measured by the local accumulation of Pontamine sky blue at the site of subcutaneous injection of LPS (Sal. typhimurium). LPS (100 - 400 microg site(-1)) produced a dose-related increase in dye leakage in both iNOS deficient and wild-type mice with about 40% less dye leakage in iNOS deficient mice. 3. Indomethacin (5 mg kg(-1)), N-[-2-cyclohexyloxy]-4-nitrophenyl methanesulphonamide (NS-398) (1 mg kg(-1)), diphenhydramine (10 mg kg(-1)) and anti-TNF-alpha antibody (dilution 1 : 400, 10 ml kg(-1)) inhibited the LPS-induced dye leakage in both iNOS deficient and wild-type mice, whereas N(G)-nitro-L-arginine methyl ester (L-NAME) (10 mg kg(-1)) or aminoguanidine (10 mg kg(-1)) inhibited that in wild-type but not in iNOS deficient mice. 4. Pretreatment with LPS (0.15 mg kg(-1) i.p.) 4 h before decreased the LPS-induced dye leakage in wild-type but not in iNOS deficient mice. LPS pretreatment increased serum corticosterone levels in both mice, while it increased the serum nitrate/nitrite levels in wild-type but not in iNOS deficient mice. 5. These studies indicate that an increase in vascular permeability induced by LPS is mediated by NO produced by iNOS, eicosanoids, histamine and TNF-alpha. The tolerance against LPS-induced vascular permeability change may be mediated by iNOS induction but not by an increased release of endogenous corticosteroids.

Inducffile nitric oxide synthase (INOS) involvement in sphincteric smooth muscle function changes following lipopolysaccharide (LPS) pretreatment

Inducffile nitric oxide synthase (INOS) involvement in sphincteric smooth muscle function changes following lipopolysaccharide (LPS) pretreatment

Kainic acid-induced seizures cause neuronal death in infant rats pretreated with lipopolysaccharide.

A major controversy in human epilepsy is whether severe seizures in infants or young children cause brain damage and subsequent epilepsy. Kainic acid (KA) produces severe seizures in infant rats, but hippocampal neuronal death and mossy fibre sprouting have not been previously demonstrated. There are similarities between lipopolysaccharide (LPS) pretreatment and KA-induced seizures in rats and the febrile convulsion of young children, in that both processes are associated with an immune stimulus and seizures. Infant rats, co-treated with LPS and KA, showed hippocampal neuronal death and mossy fibre sprouting. Taken together, our results suggest that severe febrile convulsion of young children may cause hippocampal damage and synaptic reorganization.

Lipopolysaccharide Pretreatment Protects from Renal Ischemia/Reperfusion Injury: Possible Connection to an Interleukin-6-Dependent Pathway

In vivo administration of low doses of lipopolysaccharide (LPS) to rodents can protect these animals from subsequently administrated, usually lethal doses of endotoxin or LPS. In this study we tested the effects of LPS pretreatment on ischemia/reperfusion injury in the kidney. Male C57/B1 mice were pretreated with different doses of LPS or phosphate-buffered saline on days -4 and -3. The right kidney was removed, and the vessels of the left kidney were clamped for 30 or 45 minutes on day 0. Creatinine levels and survival of animals were monitored. To test the involvement of cytokines, additional animals were harvested before ("time 0") and 15 minutes, 1, 2, 8, and 16 hours after reperfusion for histology, immunohistochemistry, terminal deoxynucleotidyltransferase-mediated UTP end-labeling assay, and reverse transcriptase-polymerase chain reaction analysis (including tumor necrosis factor (TNF)-alpha, interleukin (IL)-1, IL-6, inducible nitric oxide synthase (iNOS), and interferon (IFN)-gamma messenger RNA (mRNA)). In controls, renal ischemia of 30 minutes was nonlethal, whereas 73% of the animals died within 48 +/- 18 hours, after 45 minutes of ischemia. All different doses of LPS protected the animals from lethal renal ischemia/reperfusion injury. Starting at similar levels, serum creatinine increased significantly in controls but not in LPS-pretreated animals over time. As early as 2 hours after reperfusion, tubular cell damage was significantly more pronounced in controls than in LPS-treated mice. In controls, tubules deteriorated progressively until 8 hours of reperfusion. At this time, more than 50% of tubular cells were destroyed. This destruction was accompanied by a pronounced leukocytic infiltration, predominantly by macrophages. In contrast, LPS pretreatment prevented the destruction of kidney tissue and infiltration by leukocytes. The terminal deoxynucleotidyltransferase-mediated UTP end-labeling assay revealed significantly more apoptotic cells in controls compared with LPS-pretreated animals. IL-1, IFN-gamma, and iNOS mRNA expression did not differ between the groups throughout the time points examined. However, the expression of TNF-alpha mRNA was significantly increased at 2 hours and IL-6 mRNA was significantly down-regulated before ischemia and shortly after reperfusion in the LPS-pretreated kidneys. Therefore, we found that sublethal doses of LPS induced cross-tolerance to renal ischemia/reperfusion injury. Our data suggest that increased TNF-alpha and reduced IL-6 mRNA expression might be responsible. However, more studies are needed to decipher the exact mechanism.

Protective Action of Lipopolysaccharidesin Rat Caerulein-Induced Pancreatitis: Role of Nitric Oxide

Lipopolysaccharides (LPS), the component of the cell wall of gram-negative bacteria, have been implicated in the pathogenesis of acute pancreatitis, but the mechanism of their action on the pancreas has not been fully explored. The aim of this study was to investigate the effects of various doses of LPS on the integrity of intact pancreas and that involved in acute caerulein-induced pancreatitis (CIP) in the rat and to compare these effects with those of nitric oxide (NO) donor, S-nitrose-acetylpenicillamine (SNAP). The expression of constitutive NO synthase (cNOS) and inducible NO synthase (iNOS) mRNA was also examined in the isolated pancreatic acini obtained from the inflamed pancreas of rats treated with LPS. CIP was produced by subcutaneous (s.c.) infusion of caerulein (5 μg/kg·h for 5 h) to conscious rats. Bolus injections of various doses of LPS (0.1, 1, 10, 20 or 40 mg/kg) or SNAP (1.5, 3 or 6 mg/kg) were made intraperitoneally (i.p.) either alone or 30 min prior to s.c. infusion of caerulein to induce CIP. Infusion of caerulein produced acute pancreatitis confirmed by histological examination and manifested by an increase of pancreatic mass (by about 200%). Blood levels of amylase and lipase were augmented by 400 and 800% respectively, whereas the pancreatic blood flow (PBF) was decreased by 50% in rats with CIP. Injection of low doses of LPS (0.1–1 mg/kg i.p.) or SNAP (1.5–3 mg/kg i.p.) 30 min prior to caerulein infusion reversed the harmful effects of pancreatic overstimulation with caerulein and reduced significantly the histological manifestations of CIP such as edema, neutrophil infiltration and vacuolization of the acinar cells. These protective effects of low doses of LPS pretreatment on the pancreas were completely antagonized by the suppression of the activity of NO synthase (NOS) with N^G-nitro-L-arginine (L-NNA) applied (20 mg/kg i.p.) 15 min prior to the LPS injection. Combination of L-arginine (100 mg/kg i.p.), a substrate for NOS, with L-NNA given prior to low doses of LPS, restored the LPS-induced protection of the pancreas in rats with CIP. In contrast, higher doses of LPS (20–40 mg/kg i.p.) or SNAP (6 mg/kg i.p.), which produced a significant fall of the PBF, did not protect the pancreas against CIP. Administration of various doses of LPS to rats with CIP resulted in significant and dose-dependent stimulation of NO biosynthesis in the isolated acini obtained from the pancreas of these animals. LPS enhanced the expression of both cNOS and iNOS in the pancreatic acini obtained from rats subjected to CIP. The signal for cNOS mRNA was detected in all samples, reaching peak at the protective dose of LPS (1 mg/kg i.p.), while iNOS was overexpressed only at the highest doses of LPS that failed to exhibit the protective activity. We conclude that the pretreatment with low doses of LPS protects the pancreas against the damage provoked by CIP and this effect could be attributed, at least in part, to the activation of L-arginine-NO system in the pancreas.

Lipopolysaccharides and plant responses to phytopathogenic bacteria

Abstract Treatment of the leaves of pepper (Capsicum annuum) cv. ECW10R with lipopolysaccharides (LPS) from both plant pathogenic and enteric bacteria alters several aspects of the plant response to subsequent inoculation with phytopathogenic xanthomonads. LPS pre-treatment prevents the hypersensitive reaction caused by strains of Xanthomonas campestris pv. vesicatoria carrying the avirulence gene avrBs1 (a gene-for-gene interaction) and by X. campestris pv. campestris (a non-host interaction). Associated with this effect are the earlier synthesis of feruloyl- and coumaroyl-tyramine, phenolic conjugates that are potentially antimicrobial, and alterations in the expression patterns of genes for some pathogenesis-related (PR) proteins. Similar effects on the timing of phenolic conjugate synthesis are also seen in the compatible interaction with X. campestris pv. vesicatoria, although the level of the response is lower. Recognition of LPS by plants may allow expression of resistance in the absence of catastrophic tissue damage. However phytopathogenic bacteria may have evolved mechanisms to suppress the effects of LPS (and of other non-specific bacterial elicitors) on plant cells.

Enhanced macrophage resistance to Pseudomonas exotoxin A is correlated with decreased expression of the low-density lipoprotein receptor-related protein.

Cellular intoxification by exotoxin A of Pseudomonas aeruginosa (PEA) begins when PEA binds to its cellular receptor, the low-density lipoprotein receptor-related protein (LRP). This receptor is particularly abundant on macrophages. We hypothesize here that inducible changes in cellular expression levels of the LRP represent an important mechanism by which macrophage susceptibility to PEA is regulated by the host. We have examined the effect of lipopolysaccharide (LPS) on LRP expression and PEA sensitivity in the macrophage-like cell line HS-P. Using a [(3)H]leucine incorporation assay to measure inhibition of protein synthesis, we have demonstrated that HS-P macrophages are highly sensitive to PEA and that PEA toxicity is decreased by the LRP antagonist receptor-associated protein. LPS pretreatment decreases HS-P PEA sensitivity in a time- and dose-dependent manner. The dose of toxin required to inhibit protein synthesis by 50% increased from 11.3 +/- 1.2 ng/ml in untreated cells to 25.7 +/- 2.0 ng/ml in cells treated with LPS. In pulse experiments, involving brief exposure to saturating concentrations of PEA, [(3)H]leucine incorporation was more than threefold higher in cells pretreated with LPS than in untreated macrophages. These changes in HS-P PEA sensitivity following LPS treatment were consistently associated with a fivefold decrease in HS-P LRP mRNA expression as measured by Northern blot analysis and a three-and-a-half-fold decrease in HS-P LRP-specific ligand internalization as determined by activated alpha(2)-macroglobulin internalization studies. These data demonstrate for the first time that modulation of LRP levels by extracellular signaling molecules can alter cellular PEA sensitivity.

Enhancement by galactosamine of lipopolysaccharide(LPS)-induced tumour necrosis factor production and lethality: its suppression by LPS pretreatment.

1. D-Galactosamine (GalN) depletes UTP primarily in the liver, resulting in decreased RNA synthesis in hepatocytes. Co-injection of GalN and lipopolysaccharide (LPS) into mice produces fulminant hepatitis with severe hepatic congestion, resulting in rapid death. Although the underlying mechanism is uncertain, GalN enhances the sensitivity to tumour necrosis factor (TNF). Administration of uridine (a precursor of UTP) prior injection of either LPS itself or interleukin-1 (IL-1) reduces the lethality of GalN+LPS. The present study focused on the effects of these agents on TNF production. 2. Intraperitoneal injection of GalN+LPS into mice greatly elevated serum TNF. Although large doses of LPS alone also greatly elevated serum TNF, LPS itself induced neither hepatic congestion nor rapid death. Administration of a macrophage depletor, liposomes encapsulated with dichloromethylene bisphosphonate, reduced both the TNF production and mortality induced by GalN+LPS. 3. Uridine, when injected 0.5 h after the injection of GalN+LPS, reduced the production of TNF. Prior injection of LPS, but not of IL-1, also reduced this TNF production. 4. Serum from LPS-injected mice reduced the TNF production induced by GalN+LPS, but it was less effective at reducing the lethality. Its ability to reduce TNF production was abolished by heat-treatment. 5. We hypothesize that a factor inhibiting TNF production by macrophages is produced by hepatocytes in response to LPS. Possibly, production of this hepatocyte-derived TNF-down-regulator (TNF-DRh) may be: (i) inhibited by GalN, causing over-production of TNF by macrophages and (ii) stimulated by LPS-pretreatment (and restored by uridine), causing reduced TNF production.

Cerebrovascular hemodynamics and ischemic tolerance: lipopolysaccharide-induced resistance to focal cerebral ischemia is not due to changes in severity of the initial ischemic insult, but is associated with preservation of microvascular perfusion.

Lipopolysaccharide (LPS), administered 72 hours before middle cerebral artery (MCA) occlusion, confers significant protection against ischemic injury. For example, in the present study, LPS (0.9 mg/kg intravenously) induced a 31% reduction in infarct volume (compared with saline control) assessed 24 hours after permanent MCA occlusion. To determine whether LPS induces true tolerance to ischemia, or merely attenuates initial ischemic severity by augmenting collateral blood flow, local CBF was measured autoradiographically 15 minutes after MCA occlusion. Local CBF did not differ significantly between LPS- and saline-pretreated rats (e.g., 34 +/- 10 and 29 +/- 15 mL x 100 g(-1) x min(-1) for saline and LPS pretreatment in a representative region of ischemic cortex), indicating that the neuroprotective action of LPS is not attributable to an immediate reduction in the degree of ischemia induced by MCA occlusion, and that LPS does indeed induce a state of ischemic tolerance. In contrast to the similarity of the initial ischemic insult between tolerant (LPS-pretreated) and nontolerant (saline-pretreated) rats, microvascular perfusion assessed either 4 hours or 24 hours after MCA occlusion was preserved at significantly higher levels in the LPS-pretreated rats than in controls. Furthermore, the regions of preserved perfusion in tolerant animals were associated with regions of tissue sparing. These results suggest that LPS-induced tolerance to focal ischemia is at least partly dependent on the active maintenance of microvascular patency and hence the prevention of secondary ischemic injury.