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PubMed2002

Tumor necrosis factor-alpha and interleukin-1beta mediate endothelial permeability induced by lipopolysaccharide-stimulated whole blood.

OBJECTIVE: To investigate the role of endotoxin-induced inflammatory mediators in blood on the permeability of endothelial monolayers. DESIGN: Whole blood of healthy volunteers was treated with bacterial lipopolysaccharide (Escherichia coli, B55:05), and the resultant plasma was added to human umbilical venular endothelial cells (HUVEC) cultured on semipermeable membrane inserts (Transwells). SETTING: University hospital laboratory. SUBJECTS: Whole blood of healthy volunteers. INTERVENTIONS: Donor plasma was treated with excess antibodies against either tumor necrosis factor-alpha, interleukin-1beta, or both, before the incubation on HUVEC. MEASUREMENTS AND MAIN RESULTS: The permeability of HUVEC monolayers to fluorescent-labeled albumin and dextran was measured over a 6-hr period, after removal of the stimulus. The production of tumor necrosis factor-alpha and interleukin-1beta in lipopolysaccharide-treated whole blood was determined by radioimmunoassay. Individually, lipopolysaccharide (10 microg/mL), tumor necrosis factor-alpha (10 ng/mL), and interleukin-1beta (50 ng/mL) all increased endothelial permeability by about 2.5-fold. A much larger increase could be achieved by preincubation of lipopolysaccharide (10 microg/mL) in whole blood: the resultant plasma induced a ten-fold increase of the permeability. The permeability response after preincubation of lipopolysaccharide in whole blood was time- and dose-dependent. Moreover, this treatment increased the sensitivity of endothelial monolayers to lipopolysaccharide by a factor of several thousand-fold: Whereas high doses of lipopolysaccharide were required for direct stimulation of the permeability, picomolar amounts of lipopolysaccharide in whole blood induced a similar increase. Significant amounts of tumor necrosis factor-alpha and interleukin-1beta were produced in blood at similar doses of lipopolysaccharide. The addition of antibodies against tumor necrosis factor-alpha or interleukin-1beta to plasma partially but significantly abrogated the permeability increase. However, a complete inhibition could be achieved by the simultaneous addition of anti-tumor necrosis factor-alpha and anti-interleukin-1beta to plasma. CONCLUSIONS: Although lipopolysaccharide is capable of directly inducing endothelial permeability, blood-borne tumor necrosis factor-alpha and interleukin-1beta mediate lipopolysaccharide-induced endothelial permeability at low endotoxin concentrations. These findings support the idea that multifactorial inhibition of inflammatory mediators may improve survival in septic patients.

Capillary Permeability↗

PubMed1996

Lipopolysaccharide isolated from Porphyromonas gingivalis grown in hemin-limited chemostat conditions has a reduced capacity for human neutrophil priming.

One way prokaryotes respond to environmental stresses is by modifying selected outer membrane components. Iron, in the form of hemin, has been shown to be a significant regulator of Porphyromonas gingivalis growth and virulence and of the expression of outer membrane proteins and lipopoly saccharide. Since lipopoly saccharide has profound effects on host immune cells, this study compared the effect of hemin-restricted and hemin-normal P. gingivalis growth conditions on lipopolysaccharide priming of N-formylmethionyl-leucyl-phenylalanine-induced superoxide generation by human neutrophils. P. gingivalis was grown in a chemostat under normal (5 micrograms hemin/ml) and hemin-restricted (0.08 microgram hemin/ml) conditions. Purified lipopolysaccharide from both P. gingivalis normal and hemin-limited environments increased N-formylmethionyl-leucyl-phenylalanine-induced superoxide release by neutrophils in a dose-dependent manner. Lipopolysaccharide isolated from the hemin-normal conditions was a significantly more potent neutrophil priming agent than the lipopolysaccharide isolated from hemin-restricted conditions. Addition of normal human serum enhanced the priming effect of both lipopolysaccharide preparations; this effect, however, was more evident with the hemin-normal lipopolysaccharide. Further, this enhancing effect of serum was partly reduced in the presence of antibodies raised against the serum lipopolysaccharide-binding protein. The differences in the biological activity of the two lipopolysaccharide preparations could be associated with structural differences detected by sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis. These results indicate that hemin availability affects regulation of an aspect of P. gingivalis virulence, lipopolysaccharide-human neutrophils priming. The reduced capacity for neutrophil priming by hemin-restricted lipopolysaccharide appears to be related to lipopolysaccharide-neutrophil interactions and not to serum factors Targeting bacterial cell-surface components involved in hemin transport might be effective therapy for P. gingivalis-associated periodontal diseases.

Culture Media, Serum-Free↗

PubMed1987

Reversible binding of Salmonella typhimurium lipopolysaccharides by immobilized protamine.

The ability of agarose-linked protamine to bind Salmonella typhimurium lipopolysaccharides was investigated. Radioactively labelled lipopolysaccharides were isolated both from a smooth strain (SH6749, labelled with [14C]galactose) and from a rough strain (SH5014, lipopolysaccharide chemotype Rb2, labelled with [3H]acetate). From 50-micrograms samples of the lipopolysaccharides, protamine-agarose columns bound 99.5-99.9% of the input radioactivity. The binding efficacy was not affected by pH in the range from 3.7 to 10.5. Maximal binding capacity of protamine-agarose for highly soluble (triethylamine form) lipopolysaccharide of SH5014 was estimated to be 13.5 mg/ml packed adsorbent. The bound lipopolysaccharides could be totally released from the columns and recovered by elution with the anionic detergent sodium deoxycholate, or with 0.5 M NaCl in the presence of the uncharged detergent Triton X-100. By analysis in sodium dodecyl sulfate/polyacrylamide gels, the macromolecular quality of the recovered lipopolysaccharide was shown to be identical to that of the original. Protamine-agarose chromatography can thus be applied to purify lipopolysaccharide preparations, and to separate as well as concentrate lipopolysaccharides from dilute solutions without altering their composition. This application was challenged with water as well as insulin solution experimentally contaminated with radiolabelled lipopolysaccharide. While the insulin protein did not bind to the protamine-agarose, the contaminating lipopolysaccharide was effectively trapped.

Binding Sites↗

PubMed1995

Hepatocyte-derived interleukin-6 and tumor-necrosis factor alpha mediate the lipopolysaccharide-induced acute-phase response and nitric oxide release by cultured rat hepatocytes.

The regulation of acute-phase protein production and nitric oxide (NO) release in lipopolysaccharide-induced liver injury is thought to occur in response to monocytes/macrophages and Kupffer-cell-derived cytokines. In this study, we used primary cultured rat hepatocytes maintained as a differentiated phenotype to investigate the direct effects of endotoxin (lipopolysaccharide) on the production of the acute-phase proteins and on NO release. Lipopolysaccharide (10 micrograms/ml) increased the production of alpha 2-macroglobulin 2.5-fold compared to untreated cultures and decreased the production of albumin by 50%. The effect of lipopolysaccharide was mimicked by adding interleukin-6 (IL-6) and tumor-necrosis factor alpha (TNF-alpha), cytokines being induced by treatment of hepatocytes with lipopolysaccharide. Maximal TNF-alpha (600 pg/ml) and IL-6 (1800 pg/ml) concentrations were observed 4 h and 6 h after lipopolysaccharide stimulation, respectively. The lipopolysaccharide-induced acute-phase protein response was blocked by anti-(IL-6) but not by anti-(TNF-alpha) IgG. The latter reduced the lipopolysaccharide-induced IL-6 production by 60%. Besides its effects on the acute-phase proteins, endotoxin caused a significant increase in NO production in cultured rat hepatocytes. Unlike anti-(IL-6) IgG, anti-(TNF-alpha) IgG reduced the lipopolysaccharide-induced NO production by 50% indicating that endotoxin-induced NO production is partially mediated by TNF-alpha but not by IL-6. Preculture with gadolinium chloride (GdCl3), an inhibitor of Kupffer cells, did not change the response of hepatocytes to lipopolysaccharide indicating that the observed findings are direct endotoxin effects on hepatocytes. The data demonstrate that by their production of TNF-alpha and IL-6 rat hepatocytes respond to lipopolysaccharide treatment with an IL-6 mediated acute-phase protein and a TNF-alpha-mediated NO production. These features have previously been attributed to monocytes/macrophages and Kupffer cells.

Acute-Phase Reaction↗

PubMed1994

Detoxification of lipopolysaccharide (LPS) by egg white lysozyme.

Recent studies carried out by our group suggest that lysozyme binds to bacterial lipopolysaccharide with a high affinity to produce a complex, and inhibits various biological activities of lipopolysaccharide. Although the basic structure of lipopolysaccharide is independent of the species and strains of Gram-negative bacteria, many structural factors such as O-antigenic polysaccharide, lipid A, substituted groups, and associated molecules, affect the biological activities of lipopolysaccharide. In this study, we prepared lysozyme/lipopolysaccharide complexes using various structures of lipopolysaccharide and compared the activity and physicochemical properties. Native and dansylated lysozyme were found to bind to all tested lipopolysaccharides. The mitogenic activity and TNF production by all tested lipopolysaccharides were significantly reduced by complex formation in vitro. Administration of the complex prepared by various lipopolysaccharides produced significantly less quantities of TNF in the septic shock model. These results suggested that binding of lysozyme to lipopolysaccharide is important for the host both in pathophysiological responses to lipopolysaccharides and in the modification of lipopolysaccharide biological activity.

Animals↗

PubMed1967

Lipopolysaccharides of Salmonella T mutants.

The composition of lipopolysaccharides derived from various Salmonella T forms was studied. All T1-form lipopolysaccharides examined contained 14 to 22% each of both d-galactose and pentose in addition to 4 to 9% each of ketodeoxyoctonic acid, heptose, d-glucosamine, and d-glucose. The pentose was identified as d-ribose. The T2-form lipopolysaccharide examined did not contain a significant amount of pentose, nor more than the usual amounts of d-galactose. Periodate oxidation of T1 (lipo) polysaccharides followed by NaBH(4) reduction revealed that ribose was almost quantitatively protected, galactose was destroyed, and threitol and mannose were newly formed. The latter two products probably originated from 4-linked galactose and heptose, respectively. Ribose and galactose were found in specific precipitates of T1 lipopolysaccharide with anti-T1 antiserum but were not found in specific precipitates of alkali-treated T1 lipopolysaccharide and of Freeman degraded polysaccharide with anti-T1 serum Ribose and galactose are present in these degraded preparations in the form of nondialyzable polymers. The T1-form mutant lipopolysaccharides lacked the O-specific sugars constituting the side-chains in the wild-type antigens. They did not produce the soluble O-specific haptenic polysaccharide known to be accumulated in RI strains. With these properties, T1 lipopolysaccharides resemble RII lipopolysaccharides. Like RII degraded polysaccharides, T1-degraded polysaccharides also contained glucosamine. Furthermore, strong cross-reactions were found to exist between T1 and RII lipopolysaccharides in both hemagglutination inhibition assays and in precipitation tests. It is proposed that T1 lipopolysaccharides represent RII lipopolysaccharides to which polymers consisting of ribose and galactose are attached.

Carbohydrates↗

PubMed1975

Reactivity of lipopolysaccharides from various salmonella SR and R chemotypes Ra-Re mutants with concanavalin A.

Lipopolysaccharides from different R mutants of Salmonella minnesota and Salmonella typhimurium belonging to chemotypes Ra to Re, as well as from three SR mutants of Salmonella typhimurium were selected for a study of their precipitability with Concanavalin A. Predictions as to the outcome of the reaction could be made since both the chemical structure of the Salmonella R lipopolysaccharides and structural requirements for a positive reaction with Concanavalin A are well established. Precipitation studies in the immuno-electrophoretic assay and in the microcapillary test were carried out with alkali-treated lipopolysaccharides as untreated lipopolysaccharide is too highly aggregated to allow a sufficient migration in agarose layers. Lipopolysaccharides of all mutants--except the SR mutants--were obtained by the phenol/chloroform/petroleum ether method in order to avoid contaminations by glucans or glycogen which are known to occur in phenol/water extracted lipopolysaccharides and which would lead to erroneous results. Additional precipitation studies were carried out with two other lectins of different polysaccharide specificity: Wheat Germ Agglutinin and Soybean Agglutinin. As expected, lipopolysaccharides of chemotypes Ra, Rb1, and RcP- mutants reacted strongly with Concanavalin A, whereas no reaction was demonstrable with lipopolysaccharides of chemotypes Rb2, Rb3, Rd and Re mutants. The lipopolysaccharide of an RcP+ mutant unexpectedly failed to precipitate unless it was dephosphorylated with HF. This artificially prepared RcP-lipopolysaccharide showed a strong reaction, thus demonstrating that negative charges in the direct neighborhood of reactive sugar units as in RcP+ LPS may prevent precipitation with Concanavalin A. No reactivity demonstrable by precipitation could be obtained using either Wheat Germ Agglutinin or Soybean Agglutinin with alkali-treated lipopolysaccharide even of those chemotypes which had the supposedly reactive sugar in a terminal position, such as N-acetyl-D-glucosamine in Ra mutants (Wheat Germ Agglutinin) or D-galactose in Rb2 or Rb3 mutants (Soybean Agglutinin).

Acetylgalactosamine↗

PubMed2026

Acyloxyacyl Hydrolase-Mediated Lipopolysaccharide Inactivation Limits Macrophage Endotoxin Tolerance and Promotes Inflammation and Fibrosis in Metabolic Dysfunction-Associated Steatohepatitis.

BACKGROUND & AIMS: Metabolic dysfunction-associated steatohepatitis, a chronic liver disease, is characterized by persistent low-grade inflammation, partially driven by gut-derived lipopolysaccharide. Although repeated lipopolysaccharide exposure can induce endotoxin tolerance in innate immune cells, its role in chronic liver diseases remains unclear. Acyloxyacyl hydrolase is an endogenous enzyme that inactivates lipopolysaccharide, potentially modulating this process. We aimed to investigate how acyloxyacyl hydrolase regulates endotoxin tolerance in Kupffer cells and how this affects hepatic inflammation and fibrosis during metabolic dysfunction-associated steatohepatitis progression. METHODS: Acyloxyacyl hydrolase-deficient mice and wild-type controls were subjected to multiple dietary metabolic dysfunction-associated steatohepatitis models. Inflammatory responses, fibrosis, and transcriptomic changes in liver tissues and isolated Kupffer cells were analyzed. Endotoxin tolerance was modulated through β-glucan administration or lipopolysaccharide preconditioning. Lipopolysaccharide bioactivity was assessed using Toll-like receptor 4-reporter cell assays. RESULTS: Lipopolysaccharide-preconditioned Kupffer cells exhibited reduced proinflammatory cytokine production and transcriptional suppression of inflammatory pathways, indicating tolerance. Despite slight elevation of plasma lipopolysaccharide levels in metabolic dysfunction-associated steatohepatitis, upregulation of hepatic acyloxyacyl hydrolase positively correlated with disease severity, suggesting enhanced lipopolysaccharide inactivation but impaired establishment of tolerance. In contrast, acyloxyacyl hydrolase-deficient Kupffer cells displayed reinforced endotoxin tolerance, leading to diminished hepatic inflammation and fibrosis. Reversal of tolerance using β-glucan reactivated inflammatory and fibrogenic responses in acyloxyacyl hydrolase-deficient mice, whereas tolerance induction by low-dose lipopolysaccharide preconditioning mitigated metabolic dysfunction-associated steatohepatitis pathology, supporting the protective role of macrophage tolerance in chronic liver injury. CONCLUSIONS: Endotoxin tolerance in Kupffer cells represents a protective mechanism against chronic liver inflammation and fibrosis. Acyloxyacyl hydrolase regulates this state by limiting bioactive lipopolysaccharide, thereby modulating the establishment of endotoxin tolerance and downstream inflammatory and fibrotic responses. Enhancing macrophage tolerance by utilizing lipopolysaccharide may offer a novel therapeutic avenue to control the progression of metabolic dysfunction-associated steatohepatitis.

AOAH↗

PubMed1995

Lipopolysaccharide induces upregulation of neutral endopeptidase 24.11 on human neutrophils: involvement of the CD14 receptor.

1. As lipopolysaccharide is a major stimulator of neutrophil responses during Gram-negative bacterial infections, we studied its effect on the membrane expression of neutral endopeptidase 24.11/CD10 on neutrophils in a model of endotoxaemia in vitro. Lipopolysaccharide added to human whole-blood induced a marked and sustained CD10/neutral endopeptidase upregulation that was already detectable at 0.1 ng/ml and was maximal at a lipopolysaccharide concentration of 10 ng/ml. 2. We observed that neither tumour necrosis factor-alpha nor any newly synthesized protein was involved in the upregulation observed after 1 h incubation with 10 ng/ml lipopolysaccharide. 3. We further studied whether the lipopolysaccharide-induced CD10/neutral endopeptidase upregulation was mediated by lipopolysaccharide binding to the neutrophil CD14 receptor. Incubation of whole blood with an anti-CD14 monoclonal antibody before the addition of 0.1 ng/ml or 0.5 ng/ml lipopolysaccharide resulted in complete inhibition of CD10/neutral endopeptidase upregulation. In contrast, at a lipopolysaccharide concentration of 10 ng/ml, the anti-CD14 monoclonal antibody had an incomplete blocking effect. 4. The differential requirement for the CD14 receptor, depending on the lipopolysaccharide dose, was confirmed by the study of a patient suffering from paroxysmal nocturnal haemoglobinuria (in whom a complete defect in neutrophil CD14 expression was previously documented). 5. We finally confirmed these results using purified neutrophils, demonstrating that lipopolysaccharide-induced CD10/neutral endopeptidase upregulation depends on direct interaction with neutrophil CD14.

Antigens, CD↗

PubMed1989

The role of bile acids in the reduction in lipopolysaccharide uptake by cultured rat Kupffer cells.

The influence of bile salts on the binding and uptake of Salmonella abortus equi lipopolysaccharide by cultured Kupffer cells was studied. In control preparations, the percentage of cell-associated lipopolysaccharide increased with time and reached a plateau after about 2 h incubation at 37 degrees C. About 1.2 micrograms lipopolysaccharide was associated with 10(6) Kupffer cells at this time interval. In the presence of 0.3, 0.6 and 1 mumol bile salts/ml the cell-associated lipopolysaccharide was respectively, about 5%, 13% and 29% lower than in control cultures. In the presence of 1 mumol bile salts/ml, the association of lipopolysaccharide to cells at 0 degrees C was about 25% lower than in controls. Preincubation of Kupffer cells with 1 mumol bile salts/ml, with or without lipopolysaccharide, did not affect cell-associated lipopolysaccharide after removal of the bile salts. The rate of secretion of radioactivity by Kupffer cells was not influenced by the presence of bile salts during the uptake or the secretion periods. Bile acids proved to inactivate lipopolysaccharide. From these observations it was concluded that low concentrations of bile salts influence the binding and uptake of lipopolysaccharide by Kupffer cells. It was, therefore, considered likely that, in patients with obstructive jaundice, the high serum bile acid level accounts for spill-over of portal lipopolysaccharide into the systemic blood.

Animals↗

PubMed2003

Propofol reduces nitric oxide biosynthesis in lipopolysaccharide-activated macrophages by downregulating the expression of inducible nitric oxide synthase.

Nitric oxide is an active oxidant that contributes to the physiology and pathophysiology of macrophages. Propofol has been widely used in intravenous anesthesia. It possess antioxidant and immunomodulating effects. This study aimed to evaluate the effects of propofol on nitric oxide production in lipopolysaccharide-activated macrophages. Exposure of macrophages to propofol (25, 50 and 75 micro M), to lipopolysaccharide (0.5, 1, 1.5 and 2 ng/ml) or to a combination of propofol and lipopolysaccharide did not affect cell viability. However, propofol at 100 micro M led to significant cell death ( P<0.05). The levels of nitrite, an oxidative product of nitric oxide, were increased in lipopolysaccharide-treated macrophages in a concentration-dependent manner ( P<0.01), while propofol could concentration-dependently decrease the lipopolysaccharide-enhanced nitrite levels ( P<0.01). Immunoblotting analysis revealed that lipopolysaccharide increased the protein level of inducible nitric oxide synthase (iNOS). The co-treatment of propofol and lipopolysaccharide significantly reduced this lipopolysaccharide-induced iNOS protein (357+/-49 x 10(3) versus 92+/-6 x 10(3) arbitrary units, P<0.01). Analysis by reverse transcriptase-polymerase chain reaction showed that lipopolysaccharide induced mRNA of iNOS, but that the inductive effect was inhibited by propofol (95+/-7 x 10(2) versus 30+/-4 x 10(2) arbitrary units, P<0.01). This study has demonstrated that propofol, at therapeutic concentrations, could suppress nitric oxide biosynthesis by inhibiting iNOS expression in lipopolysaccharide-activated macrophages. The mechanism of suppression was at a pretranslational level.

Animals↗

PubMed1988

Assay of bacterial endotoxin (lipopolysaccharide) in human amniotic fluid: potential usefulness in diagnosis and management of preterm labor.

The long-range goal of our research is to determine whether the presence of bioactive agents of infection in amniotic fluid may serve as sensitive indexes of the existence of infection as the cause of preterm labor in a given pregnancy. The aim of this study was to explore the possibility that bacterial endotoxin (lipopolysaccharide) could be detected and quantified in amniotic fluid. In particular, we sought to ascertain (1) if amniotic fluid could be collected in a manner to prevent endotoxin contamination, (2) whether there were inhibitors of lipopolysaccharide in amniotic fluid, and (3) if Limulus amebocyte lysate-based assays could be used to identify and quantify lipopolysaccharide in this fluid. We found that the Limulus amebocyte lysate assays (gelation assay and chromophore generation assay) were useful in the qualitative and quantitative analysis of lipopolysaccharide in amniotic fluids. Lipopolysaccharide-mediated generation of chromophore in the presence of amniotic fluid was accelerated strikingly compared with that of lipopolysaccharide in endotoxin-free water. In three amniotic fluid samples obtained during preterm labor, lipopolysaccharide was detectable in aliquots of 1 to 10 microliters by use of the gelation assay and lipopolysaccharide was quantifiable by use of chromophore generation assays. Two of these amniotic fluid samples were sterile as determined by bacteriologic examination; in the third sample, Fusobacterium species was identified. We suggest that these assays may be extremely useful in the identification of lipopolysaccharide in amniotic fluid. Indeed, lipopolysaccharide may serve as one marker of infection useful in establishing the cause of preterm labor.

Amniotic Fluid↗

PubMed1996

Lipopolysaccharides from Porphyromonas gingivalis, Prevotella intermedia and Actinobacillus actinomycetemcomitans promote osteoclastic differentiation in vitro.

Bacterial lipopolysaccharides possess bone-resorbing activity. Here, lipopolysaccharides from three putative periodontopathic bacteria were examined for effects on osteoclast-like cell formation of bone marrow cells from lipopolysaccharide-responsive C3H-HeN and non-responsive C3H/HeJ mice. The bone marrow cells were cultured with or without various doses of lipopolysaccharide in the presence of 1,25-dihydroxyvitamin D3 and dexamethasone. These lipopolysaccharide preparations significantly increased the number of osteoclast-like cells formed in the culture of C3H/HeN marrow cells; the same as lipopolysaccharides from Escherichia coli and a synthetic lipid A with E. coli-type structure (LA-15-PP), at doses from 0.1 to 1 microgram/ml. This stimulating effect of each lipopolysaccharides was uniformly abrogated by the addition of polymyxin B at 5 micrograms/ml. All the lipopolysaccharide and the synthetic lipid A had no effect on osteoclast formation of the C3H/HeJ marrow cells, whereas lipopolysaccharide from Porphyromonas gingivalis and Prevotella intermedia showed significant mitogenic activity on C3H/HeJ spleen cells. It seems likely that the activity of lipopolysaccharides to augment osteoclast-like cell formation in the bone marrow cell cultures is derived from a common structure of the lipid A portion.

Acid Phosphatase↗

PubMed1978

Interaction of lipopolysaccharide with detergents and its possible role in the detergent resistance of the outer membrane of Gram-negative bacteria.

In the presence of MgCl2, amounts of detergents which disrupted phospholipid vesicles caused lipopolysaccharide I from Proteus mirabilis to aggregate and form vesicular, membrane-like structures. Vesicle formation with P. mirabilis lipopolysaccharide II containing longer O-polysaccharide chains was extremely poor. Lipopolysaccharides of Salmonella minnesota R mutants (chemotypes Ra, Rc and Re) displayed a growing tendency for vesicle formation with increasing deficiency of the R core polysaccharide. Lipopolysaccharides of chemotypes Rc and Re produced vesicles even in the absence of MgCl2 and detergent. Spherical aggregates consisting of P. mirabilis lipopolysaccharide I MgCl2 and detergent were unable to either entrap or retain [14C]-sucrose, [3H=inulin or [3H]dextran. On the other hand, S. minnesota R mutant lipopolysaccharides of chemotypes Rc and Re could entrap all three saccharides and retain them for at least short periods of time. Leakage of [3H]-inulin out of re-lipopolysaccharide vesicles was greatly retarded by addition of MgCl2 to the vesicle system. Incorporation of P. mirabilis lipopolysaccharide I or S. minnesota Rc lipopolysaccharide into phospholipid vesicles protected these model membranes from disruption by detergent. This suggested a similar protective function of lipopolysaccharide in the outer membrane of enteric bacteria against the action of surfactants occurring in their normal intestinal habitat.

Biological Transport↗

PubMed1993

Nitric oxide, but not interleukin-1, mediates the local blood flow response to lipopolysaccharide in rabbit skin.

Lipopolysaccharide can mimic many aspects of the inflammatory response to gram-negative bacteria. In rabbit and rat skin, lipopolysaccharide induces neutrophil accumulation and an increase in blood flow. Interleukin-1, tumor necrosis factor-alpha and the vasodilator, nitric oxide (NO) have been implicated in the biological responses to lipopolysaccharide and gram-negative bacteria. In the present study, we characterized the local vascular response to E. coli lipopolysaccharide in rabbit skin and investigated its dependence on de novo protein synthesis, NO, interleukin-1 and neutrophils. The local vascular response to the intradermal injection of lipopolysaccharide was determined by measuring changes in local blood flow with a laser-Doppler flowmeter. Injections of lipopolysaccharide (3, 10 and 30 micrograms/site) induced a dose-related increase in blood flow, with a maximum at 2 h. The response to 10 micrograms/site lipopolysaccharide was abolished by co-injection of actinomycin D (4 x 10(-9) mol/site), reduced by 60% by NG-nitro-L-arginine methyl ester (10(-7) mol/site) and suppressed by 82% in rabbits pretreated with dexamethasone (2 mg/kg, i.v.). However, the lipopolysaccharide-induced increase in blood flow was not affected by co-injection of recombinant human interleukin-1 receptor antagonist (7.0 x 10(-11) mol/site) or by systemic neutrophil depletion. These results demonstrate that lipopolysaccharide causes delayed vasodilatation of the rabbit cutaneous microvasculature which depends on protein synthesis and involves in part the release of NO. However, this response does not depend on interleukin-1 or neutrophils. The vasodilator effect of lipopolysaccharide may involve the expression of the inducible form of NO synthase in the vessel wall, through a mechanism independent of interleukin-1.

Animals↗

PubMed2005

Influence of feeding status on neuronal activity in the hypothalamus during lipopolysaccharide-induced anorexia in rats.

Fasting attenuates disease-associated anorexia, but the mechanisms underlying this effect are not well understood. In the present study, we investigated the extent to which a 48 h fast alters hypothalamic neuronal activity in response to the anorectic effects of lipopolysaccharide in rats. Male rats were fed ad libitum or fasted, and were injected with i.p. saline or lipopolysaccharide (250 microg/kg). Immunohistochemistry for Fos protein was used to visualize neuronal activity in response to lipopolysaccharide within selected hypothalamic feeding regulatory nuclei. Additionally, food intake, body weight, plasma interleukin-1 and leptin levels, and the expression of mRNA for appetite-related neuropeptides (neuropeptide Y, proopiomelanocortin and cocaine-amphetamine-regulated transcript) were measured in a time-related manner. Our data show that the pattern of lipopolysaccharide-induced Fos expression was similar in most hypothalamic nuclei whatever the feeding status. However, we observed that fasting significantly reduced lipopolysaccharide-induced Fos expression in the paraventricular nucleus, in association with an attenuated lipopolysaccharide-induced anorexia and body weight loss. Moreover, lipopolysaccharide reduced fasting-induced Fos expression in the perifornical area of the lateral hypothalamus. Lipopolysaccharide-induced circulating levels of interleukin-1 were similar across feeding status. Finally, fasting, but not lipopolysaccharide, affected circulating level of leptin and appetite-related neuropeptides expression in the arcuate nucleus. Together, our data show that fasting modulates lipopolysaccharide-induced anorexia and body weight loss in association with neural changes in specific hypothalamic nuclei.

Animals↗

PubMed1999

Brain dysfunction associated with an induction of nitric oxide synthase following an intracerebral injection of lipopolysaccharide in rats.

We investigated the pathophysiological role of nitric oxide synthesized by inducible nitric oxide synthase in the brain, by injecting lipopolysaccharide directly into the rat cerebral cortex/hippocampus. The levels of nitric oxide metabolites, nitrite and nitrate, began to increase in a dose-dependent manner with a 3-h lag, and reached approximately seven-fold the basal levels 8 h after the direct injection of lipopolysaccharide (5 microg). The lipopolysaccharide-induced increase in nitrite and nitrate levels was inhibited by treatment with the specific inducible nitric oxide synthase inhibitor aminoguanidine. The protein synthesis inhibitor cycloheximide delayed the onset of the increase in nitric oxide metabolite levels, and reduced the peak levels. Lipopolysaccharide increased Ca2+-independent, but not Ca2+-dependent, nitric oxide synthase activity in the brain. Intense nicotinamide adenine dinucleotide phosphate-diaphorase activity was observed in round cells in the vicinity of the site of injection of lipopolysaccharide 8 h after the injection. Neuronal death was observed seven days after the injection of lipopolysaccharide. Spatial memory, as assessed by performance in a water maze task and spontaneous alternation behavior in a Y-maze, was significantly impaired in rats which had had previous bilateral injections of lipopolysaccharide into the hippocampus. The lipopolysaccharide-induced neuronal death and spatial memory impairments were prevented by aminoguanidine. These results suggest that direct injection of lipopolysaccharide into the brain causes an induction of inducible nitric oxide synthase in vivo. Furthermore, it is suggested that nitric oxide produced by inducible nitric oxide synthase is responsible for the lipopolysaccharide-induced brain dysfunction.

Analysis of Variance↗

PubMed2000

Lipopolysaccharide induces distinct alterations in the microtubule cytoskeleton of monocytes.

Microtubules are obligate functional elements of almost all eukaryotic cells. They are involved in a broad range of essential cellular functions and structural changes of this system may trigger cell death. Recently, we have reported that lipopolysaccharides inhibit in vitro microtubule formation due to exclusion of microtubule-associated proteins. The distinct epitopes of lipopolysaccharides responsible for these effects and the in vivo relevance of these data are unknown. Therefore, this study was conducted to elucidate the effects of lipid A, the biologically active motif of lipopolysaccharides, on microtubule formation in vitro and to prove whether lipopolysaccharides affect the microtubule architecture of cultured human monocytes in vivo. Despite a dose- and pH-dependent inhibition of microtubule formation by lipopolysaccharides, inhibition of microtubule assembly could be mimicked by lipid A. Near-infrared two-photon microscopy revealed that human peripheral blood monocytes accumulate lipopolysaccharides. A vesicular distribution pattern of lipopolysaccharides within the monocytes was observed. Confocal laser scanning microscopy demonstrated alterations in the microtubule architecture of monocytes after incubation with lipopolysaccharides. Lipid A seems to be responsible for the observed crosstalk between lipopolysaccharides and microtubule proteins. Furthermore, our data indicate that lipopoly-saccharides may affect the microtubule architecture in human monocytes after intracellular accumulation directly. Therefore, we conclude, that the microtubule cytoskeleton is an essential intracellular target for sepsis-relevant bacterial components such as lipopolysaccharides.

Animals↗