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PubMed1980

Endotoxin-like activities of mycoplasmal lipopolysaccharides (lipoglycans).

Lipoglycans (previously designated lipopolysaccharides) from several species of Acholeplasma and from Thermoplasma acidophilum were examined for endotoxin-like activities as measured by the standard rabbit fever test and the Limulus amoebocyte lysate assay. The lipoglycans from Acholeplasma granularum, Achloplasma laidlawii, Acholeplasma modicum, and Acholeplasma oculi caused a febrile response at concentrations of 1 ng/ml per kg or greater, whereas with control Escherichia coli EC-2 lipopolysaccharides, 6.25 ng/ml per kg was required. Similar results were obtained in the Limulus amoebocyte lysate test. The minimum concentrations in nanograms per milliliter required to stimulate formation of a solid clot were: Acholeplasma axanthum, 0.22; A. granularum, 0.85; A. modicum, 0.51; A. laidlawii, 1.05; A. oculi, 0.74. Standard E. coli 1B lipopolysaccharide required a concentration of 0.125 ng/ml. Thermoplasma lipoglycan was least active, requiring 4.25 ng/ml. Clotting of the Limulus lysate proceeds by the activation by lipopolysaccharide plus Ca(2+) of a proenzyme which cleaves an arginine-lysine peptide bond of the coagulogen. The clotting and amidase activities are inactivated by deoxycholate and can be reactivated by addition of lipopolysaccharide and Ca(2+). As with E. coli 1B lipopolysaccharide, acholeplasmal lipoglycans were shown to restore both clotting and amidase activities of the deoxycholate-inactivated Limulus clotting enzyme. The degree of restoration of amidase activity by mycoplasmal lipoglycans relative to E. coli lipopolysaccharide (1.00) were: A. axanthum, 1.71; A. modicum, 1.22; A. granularum, 0.61; and Thermoplasma, 0.37. The coagulating enzyme, restored with either E. coli lipopolysaccharide or mycoplasmal lipoglycans, was able to react with the synthetic peptide benzoyl-Ile-Glu-(gamma-OCH(3))-Gly-p-nitroaniline (an analog of the coagulogen) or with the purified coagulogen itself to form the clot. The mycoplasmal lipoglycans alone were incapable of promoting these reactions when incubated with the synthetic peptide or with the purified coagulogen, thereby ruling out the contamination of these lipoglycans with proteases capable of cleaving the same Arg-Lys peptide bond of the coagulogen. These results show that acholeplasmal lipoglycans possess endotoxin-like activities. Their passive or active role in disease remains to be established.

Acholeplasma↗

PubMed1985

Lipopolysaccharide characteristics of pathogenic campylobacters.

Most Campylobacter jejuni strains are sensitive and most Campylobacter fetus strains are resistant to the bactericidal activity in normal human serum. We purified lipopolysaccharides from Campylobacter strains to determine whether their composition and structure relate to serum susceptibility. The lipopolysaccharide of two serum-sensitive strains was best isolated by the Galanos procedure, but for two serum-resistant strains a cold-ethanol extraction was optimal. For each lipopolysaccharide preparation, the ratio of 2-keto-3-deoxyoctonate to protein was increased by 100 to 1,000-fold over that of whole cells. For serum-resistant strains, total carbohydrates was a high proportion of lipopolysaccharide weight; for serum-sensitive strains, 2-keto-3-deoxyoctanate was a high proportion of total carbohydrates. By polyacrylamide gel electrophoresis, the lipopolysaccharide of serum-sensitive strains appeared rough, but for serum-resistant strains a smooth-type ladder was seen, with a minimal core region and several high-molecular-weight complexes. Proteinase K-treated whole-cell lysates showed polyacrylamide gel electrophoresis profiles similar to that of pure lipopolysaccharide. Proteinase K-treated whole-cell lysates from seven serum-sensitive C. jejuni strains all had rough profiles, and five serum-resistant C. fetus strains all had smooth profiles. These studies indicate that lipopolysaccharide composition may be an important determinant of serum susceptibility among Campylobacter species and that serum resistance is usually associated with a smooth-type lipopolysaccharide.

Blood Bactericidal Activity↗

PubMed1996

Augmented immunological activities of recombinant lipopolysaccharide possessing the mannose homopolymer as the O-specific polysaccharide.

Recombinant lipopolysaccharide possessing the mannose homopolymer as the O-specific polysaccharide was manufactured genetically by transforming Escherichia coli K-12 with various rfb genes capable of synthesizing the mannose homopolymer. Recombinant lipopolysaccharide exhibited levels of anticomplement activity, adjuvant activity, and regional lymph node-enlarging activity much higher than those exhibited by the original rough-type lipopolysaccharide from E. coli K-12 or lipopolysaccharide possessing the heteropolysaccharide from E. coli O111. Immunological activities of recombinant lipopolysaccharide were as strong as those of wild-type lipopolysaccharide possessing the mannose homopolymer. Characteristic activities of wild-type lipolysaccharide possessing the mannose homopolymer were exhibited by recombinant lipopolysaccharide. The abilities of lipopolysaccharide to activate B cells polyclonally and to produce cytokines did not seem to be related to the presence of the mannose homopolymer. Therefore, it was suggested that the mannose homopolymer in the O-specific polysaccharide might exclusively enhance anticomplement activity, adjuvant activity, and regional lymph node-enlarging activity among various lipid A activities.

Adjuvants, Immunologic↗

PubMed1986

Physical properties of short- and long-O-antigen-containing fractions of lipopolysaccharide from Escherichia coli 0111:B4.

Aggregates of short- and long-chain O-antigen-containing fractions of lipopolysaccharide were analyzed by electron spin resonance probing to reveal differences in their physical properties. The fluidities of the lipid regions of the two fractions were quite similar, although the long-chain lipopolysaccharide aggregates appeared to be more hydrated as reflected by the polarity determined with a lipid probe. In contrast, the head-group region of the long-chain fraction was dramatically more mobile than that of the short-chain sample. The binding of polycations (e.g., polymyxin B, spermine) to lipopolysaccharide aggregates was measured by the partitioning of a cationic spin probe. Less probe was displaced from the long-chain fraction and unseparated lipopolysaccharide than from the short-chain fraction by the addition of cations, suggesting that the long O-antigen masks anionic sites on lipopolysaccharide. These results indicate that the aggregate shape and reactivity of lipopolysaccharide are affected by O-antigen length. Thus, the biological activity of lipopolysaccharide may be modulated directly by the presence of O-antigen and indirectly by the effects of O-antigen on the lipopolysaccharide aggregate structure.

Antigens, Bacterial↗

PubMed1989

Characterization of lipopolysaccharide from Myxococcus xanthus by use of monoclonal antibodies.

Lipopolysaccharide is a major constituent of the cell surface of the gram-negative procaryote Myxococcus xanthus. We have purified lipopolysaccharide from M. xanthus and have shown by silver staining that the lipopolysaccharide contains a heterogeneous population of molecules which migrate as a broad low-molecular-mass band (approximately 5 kilodaltons) and as a stepladder of about 30 higher-molecular-mass bands (15- to 70-kilodalton range). The broad band consists of lipopolysaccharide molecules with just lipid A and core regions. The stepladder bands contain lipopolysaccharide molecules with lipid A, core regions, and various numbers of O-antigen units. Monoclonal antibodies generated against the cell surface of developing M. xanthus cells (J. S. Gill and M. Dworkin, Proc. Natl. Acad. Sci. USA 84:4505-4508, 1987) were used to help characterize the lipopolysaccharide molecules. Five monoclonal antibodies bound to carbohydrate epitopes on the stepladder but not to the broad band, indicating that these monoclonal antibodies recognize carbohydrates on the O antigen of the lipopolysaccharide molecules. Four of these five monoclonal antibodies bound to doublet bands in the stepladder, while the other monoclonal antibody bound to singlet bands in the stepladder. One monoclonal antibody bound to a carbohydrate epitope on both the broad band and the stepladder, indicating that it bound to the core of the lipopolysaccharide.

Antibodies, Monoclonal↗

PubMed1992

Relation between the bronchial obstructive response to inhaled lipopolysaccharide and bronchial responsiveness to histamine.

BACKGROUND: Bronchoconstriction has developed after inhalation of lipopolysaccharide in a dose of 20 micrograms in asthmatic patients and of 200 micrograms in normal subjects. This study set out to determine whether the bronchial response to lipopolysaccharide was related to non-specific bronchial responsiveness and atopy. METHODS: Sixteen subjects with a fall in specific airway conductance of 40% (PD40sGaw) after inhaling up to 900 micrograms histamine inhaled 20 micrograms lipopolysaccharide (from Escherichia coli type 026:B6) a week after bronchial challenge with a control solution of saline. The bronchial response over five hours was measured as change in FEV1 and area under the FEV1-time curve. RESULTS: FEV1 fell significantly more after lipopolysaccharide than after diluent inhalation, the difference in mean (SE) FEV1 being 4.6% (5.4%); response was maximal 60 minutes after lipopolysaccharide inhalation and lasted more than five hours. Histamine PD20FEV1 and PD40sGaw correlated with the fall in FEV1 after lipopolysaccharide inhalation. There was no difference in the proportions of responders and non-responders to lipopolysaccharide who were atopic. CONCLUSION: Lipopolysaccharide induced bronchial obstruction is associated with non-specific responsiveness but not with atopy.

Administration, Inhalation↗

PubMed1994

Lipopolysaccharides of Legionella erythra and Legionella oakridgensis.

The chemical composition of lipopolysaccharides from Legionella erythra and Legionella oakridgensis was analysed. Sodium dodecyl sulfate polyacrylamide gel electrophoresis showed both lipopolysaccharides to have a smooth-type character. The polysaccharide part of both lipopolysaccharides contained D-mannose, D-glucose, D-glycero-D-mannoheptose, L-glycero-D-manno-heptose, 2-keto-3-deoxyoctonic acid, L-fucosamine, D-glucosamine, and glucosamine phosphate. In addition, L-rhamnose, glycerol phosphate, and glucose phosphate were identified in the polysaccharide part of L. erythra lipopolysaccharide. The main sugar identified in the lipid A part of both lipopolysaccharides, 2,3-diamino-2,3-dideoxy-D-glucose, was found to be substituted with a complex fatty acid composition including at least 16 different amide-linked 3-hydroxy fatty acids. Both lipopolysaccharides contained nonhydroxy fatty acids and the uncommon 27-oxo-octacosanoic acid, 29-oxotriacontanoic acid, and 27-hydroxyoctacosanoic acid. The lipopolysaccharide of L. oakridgensis also contained 29-hydroxytriacontanoic acid. The dioic long-chain acids heptacosane-1,27-dioic acid and nonacosane-1,29-dioic acid were only present in the lipopolysaccharide of L. erythra.

Carbohydrates↗

PubMed1990

Studies on the lipopolysaccharide of a virulent and an avirulent strain of Vibrio vulnificus.

Vibrio vulnificus is a marine bacterium associated with both primary septicemias and wound infections in humans. The lipopolysaccharides of a virulent and an avirulent strain of Vibrio vulnificus were compared with respect to their chemical constituents and electrophoretic characteristics. 2-Keto-3-deoxyoctonic acid, a normal constituent of the lipopolysaccharide of typical Enterobacteriaceae, was not found in the lipopolysaccharide of either strain. Hexadecenoate (C16:1) was the predominant fatty acid of the lipid A moiety of the lipopolysaccharides and of the membrane phospholipids of both strains. Hydroxy fatty acids composed 44% of the total fatty acids of the lipid A of the avirulent and 40% of those in the virulent strain. In addition, odd-numbered fatty acids were detected in both lipopolysaccharides. The electrophoretic profile was similar for both strains, but demonstrated no "ladder-like" pattern characteristic of "smooth" lipopolysaccharides. The result of this study showed no significant differences between the lipopolysaccharides of the virulent and avirulent strains of Vibrio vulnificus. The possible role for lipopolysaccharide in pathogenesis of Vibrio vulnificus infections is discussed.

Antigens, Bacterial↗

PubMed1977

Structural features of Salmonella typhimurium lipopolysaccharide required for activation of tissue factor in human mononuclear cells.

Activation of mononuclear cell tissue factor was examined utilizing lipopolysaccharides obtained from wild-type and both Rc and Re mutants of Salmonella typhimurium. Wild-type (smooth) lipopolysaccharide, galactose-deficient (Rc) lipopolysaccharide, heptose-deficient (Re) lipopolysaccharide, and lipid A preparations were all active in their ability to generate tissue factor activity in human mononuclear cells grown in tissue culture. Polymyxin B has been reported to prevent some of the lethal effects of endotoxin in vivo, and the drug reportedly binds to the 2-keto-3-deoxyoctulosonate-lipid A region of the lipopolysaccharide molecule. Polymyxin B was effective in inhibiting the tissue factor generating activity of wild-type lipopolysaccharide, Re lipopolysaccharide, and lipid A in a dose-dependent fashion. Treatment of lipid A preparations with mild alkali abolished the ability of these preparations to activate tissue factor in cells. Analogous to many of the other biologic properties of lipopolysaccharide, tissue factor activation in human mononuclear cells appears to depend upon the integrity of the lipid A portion of the molecule.

Cells, Cultured↗

PubMed2005

Maturational effects of lipopolysaccharide on white-matter injury in fetal sheep.

White-matter damage has been associated with the development of cerebral palsy in children born both prematurely and at term, and it has been suggested that intrauterine infection can contribute to the brain injury. However, the relative importance of age on white-matter injury following infectious exposure in utero remains unclear. In this study, fetal sheep were exposed to systemic endotoxemia by administration of Escherichia coli lipopolysaccharide (88.7 +/- 7.7 ng/kg) at 65% or 85% of gestation. These gestational ages approximately correspond to human brain development in preterm and near-term infants respectively. White-matter injury was evaluated 3 days after lipopolysaccharide exposure with regard to microglia activation and loss of neurofilament and myelin basic protein. The expression of oligodendrocytes at different maturational stages was demonstrated in preterm and near-term fetuses with the oligodendroglial markers O4 and 2 ,3 -cyclic nucleotide 3 -phospodiesterase. Forty percent of the fetuses in the preterm group and 22% in the near-term group died within 8 hours of the endotoxin exposure. Three of six preterm and two of seven near-term surviving fetuses demonstrated pathologic changes in the brain with regard to increased microglia activation and loss of neurofilament staining. The number of activated microglia was enhanced in the subcortical white matter in both the preterm lipopolysaccharide-exposed fetuses (lipopolysaccharide: 235 +/- 64 cells/mm2; control: 72 +/- 28 cells/mm2; P = .0374) and the near-term fetuses (lipopolysaccharide: 180 +/- 40 cells/mm2; control 23 +/- 16 cells/mm2; P = .0152). There was a loss of neurofilament staining in both preterm fetuses (lipopolysaccharide: 2.20 +/- 0.77 pixel units; control: 0.20 +/- 0.10 pixel units; P = .0306) and near-term fetuses (lipopolysaccharide: 1.15 +/- 0.48 pixel units; control: 0.06 +/- 0.06 pixel units; P = .0285). O4-positive cells were detected at both gestational ages, whereas 2,3-cyclic nucleotide 3-phospodiesterase-positive cells and myelin basic protein staining were mainly detected in the near-term fetuses. In summary, we found white-matter injury in a proportion of both preterm and near-term fetuses after administration of lipopolysaccharide. These results are in agreement with clinical evidence suggesting that both preterm and term infants are at risk of periventricular leukomalacia in association with intrauterine infection.

Animals↗

PubMed2000

Up-regulation of endothelin-1 in gastric mucosal inflammatory responses to Helicobacter pylori Lipopolysaccharide: effect of omeprazole and sucralfate.

BACKGROUND: Helicobacter pylori is recognized as a primary etiologic factor in the development of gastric disease and the product of particular significance to the virulent action of the bacterium is its cell wall lipopolysaccharide. We applied the animal model of H. pylori lipopolysaccharide-induced acute gastritis to study the effect of antiulcer agents, omeprazole and sucralfate, on the course of mucosal inflammatory responses by analyzing the interplay between the extent of epithelial cell apoptosis and the mucosal expression of endothelin-1 (ET-1), tumor necrosis factor-alpha (TNF-alpha), and the activity of constitutive (cNOS) and inducible (NOS-2) nitric oxide synthase. METHODS: Rats pretreated twice daily for 3 consecutive days with omeprazole at 40 mg/kg, sucralfate at 100 mg/kg or the vehicle, were subjected to intragastric application of H. pylori lipopolysaccharide at 50 microg/animal, and after 2, 4, and 10 additional days on the antiulcer drug or vehicle regimen their mucosal tissue used for histologic and biochemical assessment. RESULTS: In the absence of antiulcer agents, H. pylori lipopolysaccharide elicited within 2 days a pattern of acute mucosal inflammatory responses accompanied by a massive epithelial cell apoptosis, a 2.9-fold increase in the mucosal expression of ET-1, an 11.7-fold enhancement in TNF-alpha, and a 9.3-fold increase in NOS-2, while cNOS activity showed a 5.5-fold decrease. The extent of mucosal inflammatory involvement reached a maximum by the 4th day and showed a decline by the 10th day. This was reflected in a marked reduction in epithelial cell apoptosis, decrease in the mucosal expression of ET-1, TNF-alpha and NOS-2, and the recovery in cNOS activity. Comparing to the vehicle controls, treatment with proton pump inhibitor, omeprazole, led at the end of a 10 day period to a 48.3% reduction in the extent of mucosal inflammatory involvement elicited by H. pylori lipopolysaccharide, while a 74.2% reduction in the mucosal inflammatory involvement was achieved with gastroprotective agent, sucralfate. Moreover, this advantage of sucralfate over omeprazole in countering the lipopolysaccharide-induced changes was reflected at the end of 10 day treatment period in a 20.4% greater decrease in apoptosis, a 47.5% greater reduction in TNF-alpha and a 50.7% greater reduction in ET-1. However, both agents exerted similar influence on the restoration of gastric mucosal cNOS activity and showed a comparable effect at the end of a 10 day treatment in countering the lipopolysaccharide-induced increase in the expression of NOS-2. CONCLUSIONS: The findings suggest that an increase in the mucosal ET-1 level elicited by H. pylori lipopolysaccharide, combined with a decline in cNOS may be responsible for the induction of TNF-alpha and triggering the inflammatory process. We also show that sucralfate exhibits greater efficacy than omeprazole in suppressing the H. pylori-induced mucosal inflammatory responses. This property of sucralfate may well be due to its ability to suppress the mucosal rise in ET-1.

Acute Disease↗

PubMed1984

Complement activation via the alternative pathway by purified Salmonella lipopolysaccharide is affected by its structure but not its O-antigen length.

Salmonellae, the lipopolysaccharide of which differ in the chemical structure of their O-antigenic side chains, were previously shown to activate C3 at differential rates via the alternative pathway. We wanted to test whether lipopolysaccharide isolated from these strains yields identical results, and also the effect of the polysaccharide chain length, which varies from 0 to 40 or more repeating units in a single strain. Lipopolysaccharide was purified from the above strains, hydrolyzed (0.1 N NaOH, 56 degrees C, 30 min), and used to coat sheep erythrocytes to different densities, and C3 activation in C4-deficient guinea pig serum was measured. C3 activation was proportional to lipopolysaccharide density and time, and the relative rates and extents of activation by this bacteria-free system were the same as for the original bacteria. Activation was reduced 10 to 15% when the serum was preabsorbed with strains either containing or lacking O-antigen side chain, suggesting augmentation by antibody; however, even after multiple absorptions, activation varied with O-antigen structure as expected. This differential activation was not due to differences in the average length of the O-antigenic polysaccharide chains, because the size was similar for all three lipopolysaccharides. Moreover, the extent of activation by lipopolysaccharide that had been fractionated on a column of Sephadex G-200 was independent of the polysaccharide chain length for lengths greater than 3 repeating units. The results prove that C3 activation by lipopolysaccharide via the alternative pathway is sensitive to slight variations in the chemical structure, but not to large variations in length of the O-antigen polysaccharide side chain of lipopolysaccharide.

Animals↗

PubMed1993

Examination of Lipopolysaccharide (O-Antigen) Populations of Thiobacillus ferrooxidans from Two Mine Tailings.

Net acid-generating capacities of 39.74 kg of H(2)SO(4) per ton (ca. 0.05 kg/kg) (pH 2.68) for the Lemoine copper mine tailings (closed ca. 8 years ago; located 40 km west of Chibougamau, Quebec, Canada) and 16.07 kg of H(2)SO(4) per ton (ca. 0.02 kg/kg) (pH 3.01) for the Copper Rand tailings (in current use and 50 km distant [east] from those of Lemoine) demonstrate that these sulfide tailings can support populations of acidophilic thiobacilli. Oxidized regions in both tailings environments were readily visible, were extremely acidic (Lemoine, pH 2.36; Copper Rand, pH 3.07), and provided natural isolates for our study. A 10% (wt/vol) oxalic acid treatment, which solubilizes both ferric sulfate and ferric hydroxide precipitates (B. Ramsay, J. Ramsay, M. deTremblay, and C. Chavarie, Geomicrobiol. J. 6:171-177, 1988), enabled the recovery of intact bacterial cells from the tailings material and from liquid synthetic medium for lipopolysaccharide analysis. No viable cells could be cultured after this oxalic acid treatment. Sodium dodecyl sulfate-polyacrylamide gel electro-phoretic profiles of lipopolysaccharides extracted from the Lemoine tailings were complex, indicating a heterogeneous population of Thiobacillus ferrooxidans. Six T. ferrooxidans subspecies as identified by lipopolysaccharide analysis (i.e., lipopolysaccharide chemotypes) were eventually isolated from a total of 112 cultures from the Lemoine tailings. Using the same isolate and lipopolysaccharide typing techniques, we identified only a single lipopolysaccharide chemotype from 20 cultures of T. ferrooxidans isolated from the Copper Rand tailings. This homogeneity of lipopolysaccharide chemotype was much different from what was found for the older Lemoine tailings and may reflect a progressive lipopolysaccharide heterogeneity of Thiobacillus isolates as tailings leach and age.

Journal Article↗

PubMed1995

Increased in vitro production and serum levels of the soluble lipopolysaccharide receptor sCD14 in liver disease.

Elevated concentrations of lipopolysaccharide have been found in serum of patients with severe parenchymal liver disease and its toxic effect is thought to involved in hemodynamic disturbances seen in cirrhosis. A soluble form of the receptor (sCD14) for lipopolysaccharide is present in serum and is released by stimulated macrophages, indicating macrophage activation. We investigated sCD14 serum levels and in vitro production by lipopolysaccharide stimulated peripheral blood monocytes in patients with various liver diseases. In acute viral hepatitis serum sCD14 levels were significantly higher during the first 2 weeks after onset of jaundice (n=11; 3.6 +/- 0.9 microg /ml (mean +/- SD)) than in healthy control individuals (n=52; 2.5 +/- 0.7 microg/ml; p<0.001). These elevated serum levels corresponded to enhanced in vitro production of sCD14 by lipopolysaccharide-stimulated peripheral blood monocytes (n=11; 365 +/- 262 ng/ml) as compared to control monocytes (n=52; 228 +/- 74 ng/ml; p=0.02). Similarly, patients with alcoholic cirrhosis had significantly increased sCD14 serum levels (n=31; 4.5 +/- 3.2 microg/ml; p<0.001) and in vitro sCD14 production by lipopolysaccharide-stimulated monocytes (291 +/- 153 ng/ml; p=0.014). Serum sCD14 levles correlated with the severity of disease (Child A: 2.6 +/- 1.0 microg/ml; Child B: 4.4 +/- 2.1 microg/ml; Child C: 7.8 +/- 5.1 micro/ml; Anova: p=0.001). Patients with chronic viral hepatitis had only slightly elevated serum sCD14 levels (n=17; 2.9 +/- 0.7 microg/ml; p=0.01) and increased in vitro production of sCD14 by peripheral blood monocytes (320 +/- 128 ng/ml; p<0.001). The elevated serum concentration of sCD14 in alcoholic cirrhosis and acute and chronic viral hepatitis points to an incrased macrophage activation in these diseases. sCD14 from serum is able to associate with cells not expressing membrane CD14, such as endothelial cells, allowing those cells to bind and respond to lipopolysaccharide. Elevated levels of sCD14 could in this way contribute to the toxic effects of lipopolysaccharide in severe liver disease.

Adult↗

PubMed2004

Gamma-interferon enhances expression of CD14/MyD88 and subsequent responsiveness to lipopolysaccharide from Actinobacillus actinomycetemcomitans in human gingival fibroblasts.

OBJECTIVES: CD14, toll-like receptor 4 (TLR4) and MyD88 have been shown to mediate responsiveness in host cells to lipopolysaccharide. We investigated here the regulatory effects of inflammatory cytokines on the expression of membrane CD14 (mCD14), TLR4 and MyD88, and on subsequent responsiveness to lipopolysaccharide from Actinobacillus actinomycetemcomitans in human gingival fibroblasts. MATERIALS AND METHODS: Following treatment with either interleukin-1beta, tumor necrosis factor-alpha (TNF-alpha) or gamma-interferon (IFN-gamma), expression of mCD14/TLR4 and MyD88 was determined by flow cytometry and western blotting, respectively. After pretreatment with IFN-gamma, cells were pre-incubated with either anti-CD14 antibody MY4 or anti-TLR4 antibody HTA125 and subsequently treated with A. actinomycetemcomitans lipopolysaccharide. Then, phosphorylation of mitogen-activated protein (MAP) kinases and IkappaBalpha was examined by western blotting, and production of interleukin-6 and interleukin-8 was measured by their respective enzyme-linked immunosorbent assay (ELISA) kits. RESULTS: IFN-gamma stimulated expression of mCD14, whereas -1beta and TNF-alpha did not. Expression of MyD88 but not TLR4 was also enhanced by IFN-gamma. The lipopolysaccharide activated MAP kinases, such as extracellular signal-regulated kinase (ERK), c-Jun N-terminal kinase (JNK) and p38, and IkappaBalpha and stimulated production of interleukin-6 and interleukin-8. The lipopolysaccharide-stimulated interleukin-6 and interleukin-8 production was markedly inhibited by MY4 or HTA125. Pretreatment with IFN-gamma augmented the following activation of MAP kinases and IkappaBalpha and production of interleukin-6 and interleukin-8 in response to the lipopolysaccharide. CONCLUSIONS: These results suggest that the augmentation by IFN-gamma of the responsiveness to A. actinomycetemcomitans lipopolysaccharide, such as activation of MAP kinases and IkappaBalpha and terminal cytokine production in human gingival fibroblasts, may be partially mediated by up-regulation of CD14 and MyD88 expression.

Adaptor Proteins, Signal Transducing↗

PubMed2002

The toll-like receptor TLR4 is necessary for lipopolysaccharide-induced oligodendrocyte injury in the CNS.

The immediate or innate immune response is the first line of defense against diverse microbial pathogens and requires the expression of recently discovered toll-like receptors (TLRs). TLR4 serves as a specific receptor for lipopolysaccharide (LPS) and is localized on the surface of a subset of mammalian cells. Although innate immunity is a necessary host defense against microbial pathogens, the consequences of its activation in the CNS can be deleterious, as we show here in a developing neural model. We examined the major non-neuronal cell types in the CNS for expression of TLR4 and found that microglia expressed high levels, whereas astrocytes and oligodendrocytes expressed none. Consistent with TLR4 expression solely in microglia, we show that microglia are the only CNS glial cells that bind fluorescently tagged lipopolysaccharide. Lipopolysaccharide led to extensive oligodendrocyte death in culture only under conditions in which microglia were present. To determine whether TLR4 is necessary for lipopolysaccharide-induced oligodendrocyte death in mixed glial cultures, we studied cultures generated from mice bearing a loss-of-function mutation in the tlr4 gene. Lipopolysaccharide failed to induce oligodendrocyte death in such cultures, in contrast to the death induced in cultures from wild-type mice. Finally, stereotactic intracerebral injection of lipopolysaccharide into the developing pericallosal white matter of immature rodents resulted in loss of oligodendrocytes and hypomyelination and periventricular cysts. Our data provide a general mechanistic link between (1) lipopolysaccharide and similar microbial molecular motifs and (2) injury to oligodendrocytes and myelin as occurs in periventricular leukomalacia and multiple sclerosis.

Animals↗

PubMedDate not supplied

Uptake and modification of 125I-lipopolysaccharide by isolated rat Kupffer cells.

While it is generally believed that hepatic clearance of lipopolysaccharide involves Kupffer cells, the mechanism involved has not been fully elucidated. This study assesses this phenomenon in terms of in vitro uptake and post-uptake modification experiments with an 125I-labeled Salmonella minnesota lipopolysaccharide. 125I-Lipopolysaccharide was added to Kupffer cells in suspension cultures under a variety of conditions. In vitro uptake of 125I-Lipopolysaccharide was not saturable up to concentrations of 33.33 micrograms per ml. Kinetics experiments performed at 16.67 micrograms per ml demonstrated that Kupffer cells were unsaturable after 60 min of incubation. The kinetics of uptake could be inhibited, however, by incubation in the presence of a 10-fold excess of unlabeled lipopolysaccharide, indicating that a component of the uptake process may be limited. Energy dependence in this process was demonstrated by incubation in the presence of 1 mM 2-deoxyglucose which inhibited 125I-lipopolysaccharide uptake by approximately 30%. Pretreatment with 7.5 x 10(-5) M colchicine had no effect on kinetics, implying no role for the cell cytoskeleton in lipopolysaccharide uptake. These results are inconsistent with a receptor-mediated process as previously suggested. Modification of internalized label has been demonstrated by changes in buoyant density in CsCl isopyknic density gradients following overnight incubation with Kupffer cells. These results indicate that Kupffer cells clear bacterial endotoxin in vitro and post-uptake degradation occurs within 20 hr of incubation.

Animals↗

PubMed1992

Lipopolysaccharide treatment of rats alters antigen expression and oxidative metabolism in hepatic macrophages and endothelial cells.

Endothelial cells and macrophages are located within the hepatic sinusoids. These two cell types play an important role in the clearance of bacterially derived lipopolysaccharide from the portal circulation. Our laboratory has previously demonstrated that treatment of rats with lipopolysaccharide results in the accumulation of macrophages in the liver that display properties of activated mononuclear phagocytes. This study was designed to analyze the effects of lipopolysaccharide on hepatic endothelial cells. Female Sprague-Dawley rats were treated with 5 mg/kg of lipopolysaccharide. Macrophages and endothelial cells were isolated from the rats 48 hr later by in situ perfusion of the liver with collagenase and pronase followed by differential centrifugation and centrifugal elutriation. We found that lipopolysaccharide treatment of rats resulted in an increase in the number of both macrophages and endothelial cells recovered from the liver. Using specific monoclonal antibodies and flow cytometry, both macrophages and endothelial cells were found to express cell surface markers for Ia antigen, leukocyte common antigen, CD4 and the macrophage antigen, ED2. Macrophages expressed greater levels of these markers than endothelial cells. Flow cytometric analysis also revealed considerable subpopulation heterogeneity in the endothelial cells in antigen expression, physical characteristics and functional activity. Treatment of rats with lipopolysaccharide decreased expression of cell surface markers on the macrophages but not on the endothelial cells. This may be due to the distinct origin of these cells. To determine whether endothelial cells, like macrophages, were activated by lipopolysaccharide, we examined their ability to produce reactive oxygen intermediates.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗