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PubMed2000

Kupffer cell activation by lipopolysaccharide in rats: role for lipopolysaccharide binding protein and toll-like receptor 4.

Lipopolysaccharide (LPS) binding protein (LBP) is a key serum factor that mediates LPS activation of mononuclear cells. In the presence of LBP, 1/1,000 the concentration of LPS is sufficient to activate peripheral blood monocytes. Previous studies with Kupffer cells have shown a variable effect of serum on LPS activation of these cells and led to the conclusion that, unlike extrahepatic mononuclear cells, Kupffer cells do not respond to LPS in an LBP-dependent fashion. Because there are multiple components in serum other than LBP that might affect LPS activation, these reports with serum are difficult to interpret. To investigate the specific role of LBP in LPS activation of Kupffer cells, we produced a functional recombinant rat LBP using a baculovirus expression system, which we used to selectively examine the role of LBP's on Kupffer-cell function. Isolated Kupffer cells exposed to increasing concentrations of LPS (0, 1, 10 ng/mL) showed a dose-dependent increase in TNF-alpha production, which was augmented and accelerated by the presence of LBP. The effects of LBP on Kupffer cell activation by LPS are dependent on a functional Toll-like receptor 4 (Tlr 4) because Kupffer cells from C3H/HeJ mice failed to respond to LPS in the presence of LBP. LBP plays an important role in mediating Kupffer cell activation by LPS, and these effects are dependent on the presence of functioning Tlr 4.

Acute-Phase Proteins↗
PubMed1993

Competition between bactericidal/permeability-increasing protein and lipopolysaccharide-binding protein for lipopolysaccharide binding to monocytes.

The bactericidal/permeability-increasing protein (BPI) inhibits the lipopolysaccharide (LPS)-mediated activation of monocytes. Due to its inhibitory activity for various LPS, BPI has therapeutic potential in endotoxic shock. To be efficient in vivo, BPI should overcome the action of LPS-binding protein (LBP), a serum molecule that increases the expression of LPS-inducible genes via CD14 of monocytes, rBPI23, a recombinant fragment of BPI, prevented in a dose-dependent manner the binding and the internalization of LPS mediated by LBP. Consequently, rBPI23 also inhibited LPS-induced tumor necrosis factor (TNF alpha) synthesis from monocytes. LPS- and LBP-mediated activation of monocytes was totally inhibited when LPS was preincubated with rBPI23. Adding rBPI23 at the same time as LBP resulted in an important but partial inhibition of TNF alpha release, but this inhibition vanished with delaying the time of addition of rBPI23. These studies suggest that the inhibitory activity of BPI is related to its ability to compete with LBP for LPS.

Acute-Phase Proteins↗
PubMed2003

Expression patterns of the lipopolysaccharide receptor CD14, and the FCgamma receptors CD16 and CD64 on polymorphonuclear neutrophils: data from patients with severe bacterial infections and lipopolysaccharide-exposed cells.

In polymorphonuclear neutrophils (PMN) CD14, one of the receptors for lipopolysaccharides (LPS) is stored intracellularly as a preformed protein, with only few receptors expressed on the surface. We now report that in patients with severe bacterial infections, CD14 expression is profoundly upregulated, as is CD64 (FcgammaRI), the high-affinity receptor for IgG, whereas CD16 (FcgammaRIII) was partly lost from the surface. To further analyze regulation of these receptors, PMN of healthy donors were exposed to low doses of LPS. By brief exposure (10-120 min) to LPS, CD14 was transferred to the surface in a cytochalasin B-sensitive manner, as were CD16 and CD64. Prolonged culture (up to 48 h) resulted in a further upregulation of CD14, sustained expression of CD64, and profound decline of CD16, yielding a similar pattern of receptor expression as seen in the patients. Subsequent studies revealed that LPS induced de novo synthesis of CD14: the increase of surface expression could be inhibited by cycloheximide and by interfering with a known LPS-induced signaling event, the translocation of NFkappaB. Moreover, an up to 10-fold increase of specific mRNA was seen, as was incorporation into CD14 of 35S-methionine. The de novo synthesis prolonged expression of CD14, whereas the CD16 expression declined, generating a PMN phenotype characteristic for severe infection and indicative of escape from apoptosis of a PMN subpopulation.

Apoptosis↗
PubMed1998

Low endotoxic potential of Legionella pneumophila lipopolysaccharide due to failure of interaction with the monocyte lipopolysaccharide receptor CD14.

Legionella pneumophila, a gram-negative bacterium causing Legionnaires' disease and Pontiac fever, was shown to be highly reactive in in vitro gelation of Limulus lysate but not able to induce fever and the local Shwartzman reaction in rabbits and mice. We analyzed the capacity of purified L. pneumophila lipopolysaccharide (LPS-Lp) to induce activation of the human monocytic cell line Mono Mac 6, as revealed by secretion of proinflammatory cytokines and desensitization to subsequent LPS stimulation. We showed that despite normal reactivity of LPS-Lp in the Limulus amoebocyte lysate assay, induction of cytokine secretion in Mono Mac 6 cells and desensitization to an endotoxin challenge required LPS-Lp concentrations 1,000 times higher than for LPS of Salmonella enterica serovar Minnesota. Therefore, we examined the interaction of LPS-Lp with the LPS receptor CD14. We demonstrated that LPS-Lp did not bind to membrane-bound CD14 expressed on transfected CHO cells, nor did it react with soluble CD14. Our results suggest that the low endotoxic potential of LPS-Lp is due to a failure of interaction with the LPS receptor CD14.

Animals↗
PubMed2001

Down-modulation of L-selectin by lipopolysaccharide is not required for lipopolysaccharide-induced expression of CD14 in mouse bone marrow granulocytes.

We established in previous studies that a constitutive lipopolysaccharide (LPS) receptor of low affinity is present on mouse bone marrow granulocytes (BMG). This yet-unidentified receptor is involved in the LPS-induced expression of a second LPS receptor, CD14. Because it has been claimed that L-selectin (CD62L) is a low-affinity LPS receptor in mature granulocytes (polymorphonuclear leukocytes), it may be asked whether this molecule could be the constitutive LPS receptor in BMG. We show in this study that L-selectin is constitutively present on BMG and is down-regulated after exposure of the cells to LPS. A phorbol ester induced a down-regulation of CD62L and blocked the LPS-induced expression of CD14. However, a metalloproteinase inhibitor (BB-3103) blocked the former but not the latter effect of PMA. We also observed an absence of cross-reactivity between LPS and a CD62L ligand (fucoidan) in binding studies with radiolabeled derivatives of the two agents. Furthermore, BMG from L-selectin-deficient mice expressed normal levels of CD14 in response to LPS. Taken together, these results demonstrate that in BMG, L-selectin is not the constitutive LPS receptor required for the LPS-induced expression of CD14.

Animals↗
PubMed2005

Lipopolysaccharide is transferred from high-density to low-density lipoproteins by lipopolysaccharide-binding protein and phospholipid transfer protein.

Lipopolysaccharide (LPS), the major outer membrane component of gram-negative bacteria, is a potent endotoxin that triggers cytokine-mediated systemic inflammatory responses in the host. Plasma lipoproteins are capable of LPS sequestration, thereby attenuating the host response to infection, but ensuing dyslipidemia severely compromises this host defense mechanism. We have recently reported that Escherichia coli J5 and Re595 LPS chemotypes that contain relatively short O-antigen polysaccharide side chains are efficiently redistributed from high-density lipoproteins (HDL) to other lipoprotein subclasses in normal human whole blood (ex vivo). In this study, we examined the role of the acute-phase proteins LPS-binding protein (LBP) and phospholipid transfer protein (PLTP) in this process. By the use of isolated HDL containing fluorescent J5 LPS, the redistribution of endotoxin among the major lipoprotein subclasses in a model system was determined by gel permeation chromatography. The kinetics of LPS and lipid particle interactions were determined by using Biacore analysis. LBP and PLTP were found to transfer LPS from HDL predominantly to low-density lipoproteins (LDL), in a time- and dose-dependent manner, to induce remodeling of HDL into two subpopulations as a consequence of the LPS transfer and to enhance the steady-state association of LDL with HDL in a dose-dependent fashion. The presence of LPS on HDL further enhanced LBP-dependent interactions of LDL with HDL and increased the stability of the HDL-LDL complexes. We postulate that HDL remodeling induced by LBP- and PLTP-mediated LPS transfer may contribute to the plasma lipoprotein dyslipidemia characteristic of the acute-phase response to infection.

Acute-Phase Proteins↗
PubMed1998

Heterotrimeric G proteins physically associated with the lipopolysaccharide receptor CD14 modulate both in vivo and in vitro responses to lipopolysaccharide.

Septic shock induced by lipopolysaccharide (LPS) triggering of cytokine production from monocytes/macrophages is a major cause of morbidity and mortality. The major monocyte/macrophage LPS receptor is the glycosylphosphatidylinositol (GPI)-anchored glycoprotein CD14. Here we demonstrate that CD14 coimmunoprecipitates with Gi/Go heterotrimeric G proteins. Furthermore, we demonstrate that heterotrimeric G proteins specifically regulate CD14-mediated, LPS-induced mitogen-activated protein kinase (MAPK) activation and cytokine production in normal human monocytes and cultured cells. We report here that a G protein binding peptide protects rats from LPS-induced mortality, suggesting a functional linkage between a GPI-anchored receptor and the intracellular signaling molecules with which it is physically associated.

Animals↗
PubMed2003

Lipopolysaccharide promotes the survival of osteoclasts via Toll-like receptor 4, but cytokine production of osteoclasts in response to lipopolysaccharide is different from that of macrophages.

Lipopolysaccharide is a pathogen that causes inflammatory bone loss. Monocytes and macrophages produce proinflammatory cytokines such as IL-1, TNF-alpha, and IL-6 in response to LPS. We examined the effects of LPS on the function of osteoclasts formed in vitro in comparison with its effect on bone marrow macrophages, osteoclast precursors. Both osteoclasts and bone marrow macrophages expressed mRNA of Toll-like receptor 4 (TLR4) and CD14, components of the LPS receptor system. LPS induced rapid degradation of I-kappaB in osteoclasts, and stimulated the survival of osteoclasts. LPS failed to support the survival of osteoclasts derived from C3H/HeJ mice, which possess a missense mutation in the TLR4 gene. The LPS-promoted survival of osteoclasts was not mediated by any of the cytokines known to prolong the survival of osteoclasts, such as IL-1beta, TNF-alpha, and receptor activator of NF-kappaB ligand. LPS stimulated the production of proinflammatory cytokines such as IL-1beta, TNF-alpha, and IL-6 in bone marrow macrophages and peritoneal macrophages, but not in osteoclasts. These results indicate that osteoclasts respond to LPS through TLR4, but the characteristics of osteoclasts are quite different from those of their precursors, macrophages, in terms of proinflammatory cytokine production in response to LPS.

Animals↗
PubMed2000

[The significance of the expressions of lipopolysaccharide binding protein mRNA and lipopolysaccharide receptor CD14 mRNA in the liver of burned rat].

OBJECTIVE: To explore the significance and the correlation between enteric endotoxin translocation and hepatic endotoxin -- sensitivity -- enhancing system--lipopolysaccharide -- binding protein (LBP)/lipopolysaccharide receptor CD14 (LBP/CD14) after burn. METHODS: Wistar rats subjected to 35% III degree burn were employed as the model. The rats were randomly divided into three groups: i.e. normal control (C, n = 8) group, thermal injury (T, n = 10) group and recombinant bactericidal/permeability -- increasing protein (rBPI(21)) treatment (R, n = 6) group. The rats in T and R groups were sacrificed at 12 postburn hour (PBH) and the liver tissue was collected for the detection of the mRNA expressions of LBP, CD14 and tumor necrosis factor (TNFalpha) and blood samples collected for hepatic functional indices. RESULTS: The hepatic endotoxin content increased significantly postburn (P < 0.01), and the expressions of LBP/CD14 and TNFalpha mRNA in liver tissue increased obviously. However the use of rBPI(21) could evidently lower hepatic endotoxin content and inhibit the expression of tissue LBP/CD14 and TNFalpha. In addition, rBPI(21) could also significantly decrease serum level of glutamic -- pyruvic transaminase (ALT) (P < 0.01). CONCLUSION: Aggregation of endotoxin in liver due to postburn translocation might obviously stimulate the expression of LBP/CD14 mRNA locally. The up -- regulation of the expression of LBP/CD14 might be the principle molecular basis enhandced activity of translocated endotoxin on inflammatory cells.

Acute-Phase Proteins↗
PubMed1985

Lipopolysaccharide tolerance inhibits eye inflammation. I. Reduced immune complex or lipopolysaccharide effects.

The effect of endotoxin tolerance on ocular inflammation was studied in rabbits. A single intravenous (IV) injection of endotoxin (bacterial lipopolysaccharide [LPS]) produced a mild acute iridocyclitis. Repeated daily (five to seven days) IV injections of LPS (5 micrograms extracted from Salmonella typhimurium) led to a state of refractoriness or LPS "tolerance," and ocular inflammation was no longer produced. In contrast to controls, in rabbits tolerant to LPS, IV LPS failed to elevate prostaglandin E2, thromboxane B2, or chemotactic factors in the aqueous humor. Rabbits tolerant to LPS also resisted the increase in vascular permeability normally induced by an ocular reversed passive Arthus reaction. These results demonstrated that LPS tolerance can induce anti-inflammatory effects in the eye.

Acute Disease↗
PubMed2003

Separation of Escherichia coli 055:B5 lipopolysaccharide and detoxified lipopolysaccharide by high-performance capillary electrophoresis.

A rapid, highly sensitive and reproducible high-performance capillary electrophoresis (HPCE) method (electrokinetic chromatography with sodium dodecyl sulfate) is described for the determination of the lipopolysaccharide (LPS) and detoxified LPS (D-LPS), produced by both alkaline treatment in anhydrous conditions and mild acid hydrolysis, from Escherichia coli 055:B5 bacteria. LPS and D-LPS are separated and readily determined within 25 min on an uncoated fused-silica capillary using normal polarity at 20 kV and detection at 200 nm. A linear relationship (correlation coefficient greater than about 0.97) was found for the LPS and the two D-LPS species over a wide range of concentrations, from approximately 120 to 360 ng, with a detection sensitivity less than about 100 ng. Furthermore, HPCE was able to separate several molecular species mainly due to the presence of populations with O-specific polysaccharides of distinct and increasing mean chain lengths. This approach could be of great importance for the quantitative determination of LPS and D-LPS during the purification and preparation processes, also considering the importance of D-LPS in the preparation of human vaccines, and for the qualitative evaluation of the heterogeneity of LPS and the O-polysaccharide components.

Buffers↗
PubMed2006

Proteomic analysis of macrophages stimulated by lipopolysaccharide: Lipopolysaccharide inhibits the cleavage of nucleophosmin.

Lipopolysaccharide (LPS) is a complex glycolipid composed of a hydrophilic polysaccharide and a hydrophobic domain that is responsible for the biological activity of LPS. There are many reports about LPS stimulation, and many activated proteins have been detected after LPS stimulation in various cell types. Furthermore, most of the LPS signaling pathways are clear. However, we were interested in examining the changes of LPS-induced total cytosolic proteins expression and the LPS signaling pathway by the proteomics technique during LPS-induced macrophage activation. Our study employed two-dimensional gel electrophoresis and mass spectrometry to analyze the proteins involved in LPS-induced activation in RAW 264.7 cells. We found 11 protein spots whose expression was different between untreated cells and LPS-treated cells. Ten protein spots were identified, seven of which, tubulin beta-4 chain (49.6 kDa, pI 4.78), nucleophosmin (32.6 kDa, pI 4.62, two spots), 40S ribosomal protein SA (P40) (32.7 kDa, pI 4.74), transforming protein RhoA (21.8 kDa, pI 5.83), nucleolin (76.6 kDa, pI 4.69), and T-complex protein 1 zeta subunit (58 kDa, pI 6.63) were down-regulated, and three of which, nucleophosmin (32.6 kDa, pI 4.62, two spots) and proteosome subunit alpha type-1 (29.5 kDa, pI 6.00), were up-regulated. The suppression of the proteolytic degradation of nucleophosmin was associated with LPS-induced RAW 264.7 cell activation. Cleaved caspase-3 decreased, thus it might be involved in proteolysis of nucleophosmin in LPS-induced macrophage activation. Our study also demonstrated that there was no change of the expression of nucleophosmin at the mRNA level.

Animals↗
PubMed2000

Interferon-gamma- and lipopolysaccharide-induced tumor necrosis factor-alpha is required for nitric oxide production: tumor necrosis factor-alpha and nitric oxide are independently involved in the killing of Mycobacterium microti in interferon-gamma- and lipopolysaccharide-treated J774A.1 cells.

A comparative study was done using J774A.1 and J774A.1-derived transfected cells (J774A.1 C.1) containing antisense tumor necrosis factor alpha (TNF-alpha) plasmid to determine the role of endogenous TNF-alpha on nitric oxide production as well as on the growth of Mycobacterium microti in interferon gamma (IFN-gamma)- and lipopolysaccharide (LPS)-treated cells. On stimulation with IFN-gamma and LPS a higher level of NO was observed in J774A.1 cells compared to J774A.1 C.1 which indicated that endogenous TNF-alpha is required for the production of NO. Comparing the effect of IFN-gamma and LPS on the intracellular growth of M. microti, the growth-reducing activity was higher in J774A.1 cells than in J774A.1 C.1 cells and was not completely abrogated in the presence of the nitric oxide inhibitor NG-methyl-L-arginine (L-NMA). J774A.1 C.1 cells infected with M. microti produced a significant amount of NO when exogenous TNF-alpha was added along with IFN-gamma and LPS and the concentration of intracellular bacteria decreased almost to that in IFN-gamma and LPS treated parental J774A.1 cells. Addition of exogenous TNF-alpha even in the presence of L-NMA in J774A.1 C.1 cells could also partially restore intracellular growth inhibition of M. microti caused by IFN-gamma and LPS. TNF-alpha is probably required for the production of NO in J774A.1 cells by IFN-gamma and LPS but TNF-alpha and NO are independently involved in the killing of intracellular M. microti with IFN-gamma and LPS.

Animals↗
PubMed1983

Lipopolysaccharides from Pseudomonas maltophilia: composition of the lipopolysaccharide and structure of the side-chain polysaccharide from strain N.C.I.B. 9204.

Lipopolysaccharide was extracted from defatted cell-walls of Pseudomonas maltophilia N.C.I.B. 9204. The major fatty acid components were 9-methyldecanoic acid, 2-hydroxy-9-methyldecanoic acid, 3-hydroxy-9-methyldecanoic acid, 3-hydroxy-dodecanoic acid, and 3-hydroxy-11-methyldodecanoic acid. Monosaccharide components of the phosphorylated core-oligosaccharide were D-glucose, D-mannose, D-galacturonic acid, 2-amino-2-deoxyglucose, and a 3-deoxyoctulosonic acid. The putative O-specific polysaccharide was composed mainly of 2-amino-2-deoxy-D-glucose, D-arabinose, and 6-deoxy-L-talose, but also contained an O-acetyl group and small proportions of rhamnose and 6-deoxy-3-O-methyltalose. Degradative and n.m.r. (1H and 13C) studies showed that the polymer had a branched trisaccharide repeating-unit with the following structure; the O-acetyl group was tentatively assigned to C-2 of the 6-deoxytalopyranosyl residue. (Formula: see text).

Carbohydrate Conformation↗
PubMed1992

Structures of the oligosaccharides obtained from the core regions of the lipopolysaccharides of Bradyrhizobium japonicum 61A101c and its symbiotically defective lipopolysaccharide mutant, JS314.

The only core oligosaccharide released from the lipopolysaccharide (LPS) of Bradyrhizobium japonicum 61A101c by prolonged (5 h) mild hydrolysis with acid, and the major core oligosaccharide obtained from its symbiotic and LPS-defective mutant, JS314, was the trisaccharide alpha-D-Man p-(1----4)-alpha-D-Glc p-(1----4)-2,7-anhydro-alpha-Kdof. The 2,7-anhydro-3-deoxy-alpha-D-manno-2- octulofuranosonic acid moiety was probably formed during the prolonged mild hydrolysis with acid. A disaccharide core component, also released by mild acid hydrolysis of the mutant LPS, had the structure 4-O-Me-alpha-D-Man p-(1----5)-Kdo. The Kdo residue in this disaccharide is present as the normal pyranose form and as an anhydro derivative, possibly 4,8-anhydro-3-deoxy-D-manno-2-octulosonic acid, which may have formed also during prolonged mild hydrolysis with acid. Mild acid hydrolysis of the LPS of the parent strain does not produce this disaccharide, but 4-O-Me-Man is found exclusively in the O-chain fraction released from the parent LPS. Additionally, a small amount of O-chain is found in the mutant LPS. The results imply that the O-chain is attached to the remainder of the LPS through the 4-O-Me-Man-Kdo disaccharide component of the core region.

Carbohydrate Conformation↗
PubMed1979

Ultrastructural localisation of lipopolysaccharide-binding sites with peroxidase-conjugated lipopolysaccharides.

The localisation of lipopolysaccharide-binding sites on erythrocytes with peroxidase-coupled LPS is described. LPS was isolated from Fusobacterium nucleatum (Fus MC-8) by phenol-water extraction. The LPS was coupled to horseradish peroxidase by the two-step method of Avrameas and Ternynck (1971). The biological and serological activities of the conjugated LPS were compared with those of the native material. Peroxidase could be coupled to LPS without significant loss of endotoxic or serological activity. The LPS-peroxidase conjugate could be demonstrated on erythrocytes by light and electron microscopy.

Animals↗
PubMed1994

Lipopolysaccharide-lipophorin complex formation in insect hemolymph: a common pathway of lipopolysaccharide detoxification both in insects and in mammals.

The formation of the lipophorin-lipopolysaccharide (LPS) complex in Bombyx mori hemolymph and its role in LPS detoxification were explored. LPS, an antibacterial protein inducer in insects, was injected into B. mori larvae. Analytical density gradient ultracentrifugation revealed that after injection the LPS peak shifts to a zone of lower density with time. The shifted peak was identified as the lipophorin-LPS complex. This complex formation was also achieved in an in vitro mixture of cell-free hemolymph and LPS at 25 degrees C but not at 1 degree C. The lipophorin-LPS complex had a significantly lower capacity to elicit the mRNA of cecropin B, an antibacterial protein. The biological activity of reextracted LPS from the complex was slightly reduced in the Limulus test and no structural modification was observed in sodium dodecylsulfate-polyacrylamide gel electrophoresis (SDS-PAGE). These results suggested that the formation of lipophorin-LPS strikingly reduces the cecropin inducibility of LPS without any structural change in LPS. Similar serum lipoprotein-LPS complex formation and reduction of biological activities of LPS were also observed in mammals. We, therefore, suggest that the formation of the serum lipoprotein-LPS complex is a common pathway to inactivate LPS both in insects and in mammals.

Animals↗
PubMed2005

Removal of lipopolysaccharides from protein-lipopolysaccharide complexes by nonflammable solvents.

During the recovery of recombinant proteins from gram negative bacteria, many of the methods used to extract proteins from cells release lipopolysaccharides (LPS, endotoxin) along with the protein of interest. In many instances, LPS will co-purify with the target protein due to specific or non-specific protein-LPS interactions. We have investigated the ability of alkanediols to effect the separation of LPS from protein-LPS complexes while the complexes are immobilized on ion exchange chromatographic resins. Proteins were complexed with fluorescently labeled LPS and bound to ion exchange resin. Alkanediol washes of the resins were preformed and the proteins eluted. Column eluates were monitored for LPS and protein by fluorescence and UV spectroscopy, respectively. Alkanediols were effective agents for dissociating LPS from protein-LPS complexes. The efficiency of LPS removal increased with increasing alkanediol chain length. The 1,2-alkanediol isomers were more effective than terminal alkanediol isomers in the separation of LPS from protein-LPS complexes, while the separation of LPS from protein-LPS complexes was more efficient on cation exchangers than on anion exchangers. In addition, it was noted during these investigations that the 1,2-alkanediols increased the retention time of the proteins on the ion exchange resins. Alkanediols provide a safer alternative to the use of other organics such as alcohols or acetonitrile for the separation of LPS from protein due to their lower toxicity and decreased inflammability. In addition, they are less costly than many of the detergents that have been used for similar purposes.

Boron Compounds↗