Lipopolysaccharide Uptake Research Articles

Nitric oxide mediates lipopolysaccharide-induced alteration of mitochondrial function in cultured hepatocytes and isolated perfused liver

The influence of endogenous nitric oxide, which is generated by stimulation with lipopolysaccharide, on the mitochondrial energization of rat hepatocytes was investigated in vitro and ex vivo. Using a fluorescence microscope equipped with a silicon intensifier target camera, we visualized fluorescence of rhodamine-123, a mitochondrial energization-sensitive fluorescence probe, in individual hepatocytes and measured the fluorescence intensity with a digital imaging processor. Although addition of Kupffer cells or lipopolysaccharide in a range of 0.1 to 1.0 microgram/ml caused no significant alteration in the fluorescence in hepatocytes, Kupffer cells plus 1.0 microgram/ml lipopolysaccharide reduced fluorescence intensity in the cocultured hepatocytes. The alteration of rhodamine-123 fluorescence in the hepatocytes induced by lipopolysaccharide-activated Kupffer cells was significantly inhibited by the addition of NG-monomethyl-L-arginine, a selective inhibitor of nitric oxide synthesis. The transportal infusion of lipopolysaccharide also decreased rhodamine-123 fluorescence in perfused rat liver. The decrease was significantly enhanced in the pericentral regions. Autofluorescence of NADH was elicited by continuous infusion of lipopolysaccharide; this reaction was also enhanced in the pericentral regions. We showed the main site of uptake of infused lipopolysaccharide in the hepatic lobule to be in the periportal regions with fluorescein isothiocyanate-labeled lipopolysaccharide. Our results indicate that the inhibition of mitochondrial energization occurs mainly in pericentral regions, which are distant from the lipopolysaccharide uptake site. The continuous administration of NG-monomethyl-L-arginine significantly attenuated the lipopolysaccharide-induced decrease in rhodamine-123 fluorescence and increase of the NADH contents of the hepatic lobule.(ABSTRACT TRUNCATED AT 250 WORDS)

Nitric oxide mediates lipopolysaccharide-induced alteration of mitochondrial function in cultured hepatocytes and isolated perfused liver

The influence of endogenous nitric oxide, which is generated by stimulation with lipopolysaccharide, on the mitochondrial energization of rat hepatocytes was investigated in vitro and ex vivo. Using a fluorescence microscope equipped with a silicon intensifier target camera, we visualized fluorescence of rhodamine-123, a mitochondrial energization–sensitive fluorescence probe, in individual hepatocytes and measured the fluorescence intensity with a digital imaging processor. Although addition of Kupffer cells or lipopolysaccharide in a range of 0.1 to 1.0 νg/ml caused no significant alteration in the fluorescence in hepatocytes, Kupffer cells plus 1.0 μg/ml lipopolysaccharide reduced fluorescence intensity in the cocultured hepatocytes. The alteration of rhodamine-123 fluorescence in the hepatocytes induced by lipopolysaccharide-activated Kupffer cells was significantly inhibited by the addition of NG-monomethyl-L-arginine, a selective inhibitor of nitric oxide synthesis. The transportal infusion of lipopolysaccharide also decreased rhodamine-123 fluorescence in perfused rat liver. The decrease was significantly enhanced in the pericentral regions. Autofluorescence of NADH was elicited by continuous infusion of lipopolysaccharide; this reaction was also enhanced in the pericentral regions. We showed the main site of uptake of infused lipopolysaccharide in the hepatic lobule to be in the periportal regions with fluorescein isothiocyanate–labeled lipopolysaccharide. Our results indicate that the inhibition of mitochondrial energization occurs mainly in pericentral regions, which are distant from the lipopolysaccharide uptake site. The continuous administration of NG-monomethyl-L-arginine significantly attenuated the lipopolysaccharide-induced decrease in rhodamine-123 fluorescence and increase of the NADH contents of the hepatic lobule. These results suggest that nitric oxide mediates the lipopolysaccharide-activated, Kupffer cell–induced inhibition of mitochondrial electron transport in hepatocytes. (HEPATOLOGY 1993;18:380–388).

Lipopolysaccharide (LPS) partial structures inhibit responses to LPS in a human macrophage cell line without inhibiting LPS uptake by a CD14-mediated pathway.

Lipopolysaccharides (LPS) that lack acyloxyacyl groups can antagonize responses to LPS in human cells. Although the site and mechanism of inhibition are not known, it has been proposed that these inhibitory molecules compete with LPS for a common cellular target such as a cell-surface binding receptor. In the present study, we used an in vitro model system to test this hypothesis and to evaluate the role of CD14 in cellular responses to LPS. Cells of the THP-1 human monocyte-macrophage cell line were exposed to 1,25 dihydroxyvitamin D3 to induce adherence to plastic and expression of CD14, a binding receptor for LPS complexed with LPS-binding protein (LBP). The uptake of picograms of [3H]LPS (agonist) and enzymatically deacylated LPS [3H]dLPS (antagonist) was measured by exposing the cells to the radiolabeled ligands for short incubation periods. The amounts of cell-associated LPS and dLPS were then correlated with cellular responses by measuring the induction of nuclear NF-kappa B binding activity and the production of cell-associated interleukin (IL)-1 beta. We found that similar amounts of [3H]LPS or [3H]dLPS were taken up by the cells. The rate of cellular accumulation of the ligands was greatly enhanced by LBP and blocked by a monoclonal antibody to CD14 (mAb 60b), yet no cellular responses were induced by dLPS or dLPS-LBP complexes. In contrast, LPS stimulated marked increases of NF-kappa B binding activity and IL-1 beta. These responses were enhanced by LBP and inhibited by mAb 60b. dLPS and its synthetic lipid A counterpart, LA-14-PP (also known as lipid Ia, lipid IVa, or compound 406) strongly inhibited LPS-induced NF-kappa B and IL-1 beta, yet neither antagonist inhibited the uptake of LPS via CD14. dLPS did not inhibit NF-kappa B responses to tumor necrosis factor (TNF) alpha or phorbol ester. Our results indicate that (a) both stimulatory and nonstimulatory ligands can bind to CD14 in the presence of LBP; (b) the mechanism of inhibition by dLPS is LPS-specific, yet does not involve blockade of LPS binding to CD14; and (c) in keeping with previous results of others, large concentrations of LPS can stimulate the cells in the absence of detectable binding to CD14. The findings indicate that the site of dLPS inhibition is distal to CD14 binding in the LPS signal pathway in THP-1 cells, and suggest that molecules other than CD14 are important in LPS signaling.

Uptake, distribution and fate of bacterial lipopolysaccharides in monocytes and macrophages: an ultrastructural and functional correlation.

Bacterial lipopolysaccharides (LPS), which are important components of the cell wall of gram-negative bacteria, induce a number of host responses both beneficial and harmful. The present review elucidates the uptake, distribution and functions of LPS in mononuclear phagocytes in an attempt to gain an insight into the mechanisms which control the pathogenesis of LPS mediated septic shock. The unique feature of LPS bilayer structure, the tagged LPS and antibodies to LPS provide means for studying binding, uptake, fate and subcellular distribution of LPS in tissues and cells. LPS bind to monocytes and macrophages by specific interaction via receptors such as scavenger receptors, CD14 and CD18 and by non-specific interactions, and enter the cells via receptor-mediated endocytosis, absorptive pinocytosis, phagocytosis, and diffusion. The ingested LPS are localized in pinocytic vesicles, phagocytic vacuoles, cytoplasm, mitochondria, rough endoplasmic reticulum, Golgi apparatus, and nucleus. The interactions of LPS with monocytes and macrophages trigger a broad spectrum of cellular responses, including production of important bioactive factors or mediators, such as IL-1, TNF, interferons, prostaglandins, and macrophage-derived growth factor, which are implicated in the pathogenesis of septic shock and wound healing. However, there is no conclusive evidence indicating that production of the mediators can only be induced through specific interactions.

Ultrastructural and Immunocytochemical Study of the Uptake and Distribution of Bacterial Lipopolysaccharide in Human Monocytes

Interaction of bacterial lipopolysaccharide (LPS) with monocytes stimulates production of a variety of mediators that are involved in the pathogenesis of septic shock and wound repair. We report here the mechanisms of LPS uptake and intracellular distribution of LPS in human monocytes. Ficoll-Hypaque-purified peripheral mononuclear cells (PBMC) were exposed to LPS from rough Escherichia coli (J5) or to biotin-conjugated LPS (biotin-LPS) from smooth E. coli (0111:B4), or to fluorescein isothiocyanate-conjugated LPS of E. coli (055:B5) at 37 degrees C for various times and processed for electron microscopy, immunocytochemistry, and flow cytometry. Monocytes were identified by the presence of numerous cytoplasmic peroxidase-positive granules or by monoclonal antibodies against monocyte. LPS micelles were identified by their specific bilayer structure, staining of horseradish peroxidase reaction product, or colloidal gold using biotin-LPS or a monoclonal antibody to LPS. Binding of LPS to cell surface was observed 5 min after incubation with LPS. Intracellular localization of LPS micelles was found 30 min following exposure to LPS. Prolonged incubation with LPS increased intracellular LPS. Intracellularly, LPS micelles were found in large membrane-bound vacuoles, in small vesicles, and in the cytoplasm and nucleus. They were also observed in association with the cytomembrane of various organelles. The overall results indicate that LPS may be taken up by monocytes by direct passive diffusion through ruptures of plasma membrane, pinocytosis, and phagocytosis, involving specific and/or nonspecific binding, and suggest that peripheral blood monocytes play an important role in clearance of LPS; that LPS may have broad effects on cell functions; and that the nonspecific binding to various cytomembranes may be destructive to cell organelles and cells in general.

The role of bile acids in the development of endotoxemia during obstructive jaundice in the rat

The role of bile acids in the development of endotoxemia during obstructive jaundice was studied in rats. Endotoxin was not found in portal and peripheral plasma of control rats. The bile ducts of seven rats were ligated. On day 7 following bile duct ligation, six animals showed portal endotoxemia and five peripheral. Oral administration of sodium ursodeoxycholate reduced this frequency to 1/7 for portal plasma and 0/7 in the case of peripheral plasma. Subsequently the influence of a bile salt mixture (85% taurocholate, 15% taurodeoxycholate) on the binding and uptake of Salmonella abortus equi lipopolysaccharide by cultured rat Kupffer cells was studied. In control preparations, the percentage cell-associated lipopolysaccharide increased with time and reached a plateau after about 2 h of incubation at 37 degrees C. In the presence of 0.3, 0.6 and 1 mumol bile salts/ml the cell-associated lipopolysaccharide was about 5%, 13% and 29% lower, respectively, of that in control cultures. Tauroursodeoxycholate (1 mM) did not inhibit the lipopolysaccharide uptake by cultured rat Kupffer cells. Based on these observations, it is likely that both phenomena, i.e., (a) the low amount of bile acids in the intestines and (b) the high serum bile acid level, account for the high frequency of endotoxemia in the peripheral blood during obstructive jaundice.

The fate of lipopolysaccharide in cultured rat Kupffer cells.

Cultured rat Kupffer cells were incubated in presence of biologically tritiated Salmonella abortus equi lipopolysaccharide. Uptake of lipopolysaccharide increased rapidly during the first 2 h of incubation and then levelled off. Within the first h of incubation 10(6) Kupffer cells were able to ingest up to 18 micrograms lipopolysaccharide. Kupffer cells metabolised lipopolysaccharide and released lipopolysaccharide-related substances, but neither the cell-associated lipopolysaccharide nor the released lipopolysaccharide products were detoxified, as measured by the mouse lethality test.

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.

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.

Incorporation of LPS in liposomes diminishes its ability to induce tumoricidal activity and tumor necrosis factor secretion in murine macrophages.

We investigated the effect of lipopolysaccharide (LPS) incorporated into phospholipid vesicles (liposomes) on the induction of macrophage-mediated tumor cytotoxicity and tumor necrosis factor (TNF) secretion. The incorporation of Salmonella minnesota rough (Re)-LPS into multilamellar or small unilamellar vesicles (liposomes) resulted in an 100- to 1,000-fold reduction in its potency to activate both the macrophage cell line RAW 264.7 and murine thioglycolate elicited peritoneal macrophages to become cytotoxic for L929 and P815 tumor cells. Liposomal LPS was also a 100- to 1,000-fold less potent inducer of TNF secretion from RAW 264.7 cells. Cytokines secreted by the activated macrophages contributed to the cytotoxic effect on the L929 cells but not the P815 cell line. Human recombinant TNF was not cytotoxic for either cell line but was cytostatic for the L929 cell line. Morphological examination of the cells after uptake of fluorescent, free, and liposomal LPS revealed that both forms were internalized by the endocytic pathway. This, together with the considerably reduced potency of liposomal LPS to induce tumor cytotoxicity and TNF secretion, suggests that the interaction of the hydrophobic part of the lipid A moiety of LPS with the macrophage plasma membrane is needed to optimally activate these cells. Incorporation of LPS into liposomes effectively abrogates this interaction.

Cellular and subcellular distribution of injected lipopolysaccharide in rat liver and its inactivation by bile salts

The cellular and subcellular distribution of biologically tritiated Salmonella abortus equi lipopolysaccharide (LPS) was studied at different time intervals after intravenous injection in rats. At 1 min after injection of LPS via the portal vein label was present over Kupffer cell phagosomes. Between 30 min and 7 days after injection, silver grains were mainly associated with phagosomes and lysosomes and occasionally with the membrane of Kupffer cells. A few parenchymal cells were labeled at 5 min in their mitochondria, cell membrane and the periphery of the cell. Radioactivity was also present in the rough endoplasmic reticulum (from 15 min), fat droplets and the nucleus (from 3 h) up to 7 days. Sinusoidal endothelial and fat-storing cells were never labeled. In conclusion, both Kupffer cells and parenchymal cells play a role in the uptake of LPS by the liver. The uptake and processing of endotoxin is rapid, since label is found early after administration and radioactivity is detected in the bile within 1 h. This radioactivity represents non-detoxified LPS, since it is lethal for galactosamine-sensitised mice after extraction with hot phenol/water. However, in the presence of bile salts, the LPS is non-lethal and not capable of clotting the limulus amebocyte lysate. LPS injection causes bile flow reduction within 45 min.

Effects of specific antibodies, hormones, and lipoproteins on bacterial lipopolysaccharides injected into the rat.

When gram-negative lipopolysaccharides (LPS) are injected intravenously into experimental animals, approximately one-third of the LPS bind to high-density lipoproteins (HDL) in the plasma and are slowly taken up into tissues by HDL-mediated mechanisms. Molecules of LPS in the plasma that do not bind to HDL are taken up more rapidly by tissues that are rich in phagocytic cells (e.g., liver, spleen). In these experiments we evaluated the effects of several potential host factors on the binding of LPS to HDL and on the tissue uptake of LPS injected intravenously into rats. Antibodies to LPS inhibited LPS binding to HDL and increased the uptake of the injected LPS (as well as the LPS in preformed LPS-HDL complexes) by the liver and spleen. High levels of circulating HDL decreased the uptake of injected LPS and LPS-HDL complexes by the adrenal gland, presumably by occupying tissue receptors for HDL. Pretreating rats with dexamethasone unexpectedly decreased the uptake of injected LPS-HDL complexes by the adrenal gland and increased uptake of LPS by the gland, a result suggesting that this drug has opposing effects on the uptake of HDL-bound and -unbound LPS by the adrenal gland. The host factors studied appear to influence the fate of injected LPS by acting at different sites: IgG antibody to LPS blocks the binding of LPS to HDL in the plasma, whereas all of the factors studied have effects that modulate the uptake of LPS or LPS-HDL complexes or both by the cells of specific tissues.

Distribution and localization of endotoxin in the reticulo- endothelial system (RES) and in the main vessels of the rat during shock

The fate of endotoxin was followed with immunohistochemistry and radio-labelled lipopolysaccharide (LPS) in organs of the reticulo-endothelial system (RES), in the great vessels and the thoracic duct of rats during a 14 day period after the injection of a shock-inducing amount of endotoxin. The immunohistochemical detectability of LPS in most tissues increased continuously during the first 48 hours, showing the strongest LPS staining in the liver and adrenal gland. Macrophages were found to be the most important cells of primary LPS uptake in all organs except the adrenal glands, where endotoxin was mainly present in phagocytic vacuoles of the cortical epithelium. The comparison of results obtained with the immunoperoxidase method and radioactivity measurements revealed that at a later stage of the experiment the persisting LPS in liver and spleen looses its antigenic activity. The correlation between the appearance of LPS positive macrophages and histological signs of tissue injury during endotoxin shock is striking.

The Clearance, Tissue Distribution, and Cellular Localization of Intravenously Injected Lipopolysaccharide in Rabbits

Abstract The initial interaction of bacterial lipopolysaccharide (LPS) with the blood and tissues of rabbits was studied by using radioiodinated LPS purified from Escherichia coli O111:B4 and Salmonella minnesota R595. After i.v. injection of 250 µg of 125I-LPS, a rapid clearance phase (t1/2 < 30 min) was observed with substantial uptake of LPS by liver, spleen, and lung during this initial period. With autoradiography, LPS was found to be concentrated in phagocytic vacuoles of hepatic Kupffer cells, splenic macrophages, and leukocytes. LPS remaining in plasma beyond 30 min was converted to a form with a density of less than 1.2 g/cm3, which disappeared from the blood with a half-life of 12 hr. Except for a marked increase in the concentration of O111:B4 LPS in the adrenal glands, tissue-bound LPS concentrations did not change appreciably between 5 and 180 min. When the low density form (ρ < 1.2 g/cm3) of either LPS was injected i.v., the initial rapid clearance phase was absent and approximately one-half of the LPS was cleared from blood during the next 4 hr followed by a slower clearance rate (t1/2 = 15 hr). In contrast to findings with the parent LPS, LPS concentrations in the adrenals at 180 min were 3- to 4-fold increased over those in other tissues. Results from SDS-PAGE of LPS recovered from plasma and liver homogenates indicated that no major cleavage of the glycolipid structure of LPS occurred within 180 min. Thus, i.v. injected LPS is rapidly partitioned into tissuebound LPS and that remaining in plasma, neither of which shows evidence of major degradative change within 180 min. Accumulation of low density LPS in the adrenal may compromise the ability of the host to survive in LPS-induced shock.

Strain Differences in the Immunogenicity of Aggregated Human IgG and the Adjuvant Action of Lipopolysaccharide on the Low-Responder Strain of Mice

Strain differences in the antibody response to human IgG (HGG) were observed when aggregated HGG was injected intravenously. Lipopolysaccharide (LPS) administered subsequently markedly enhanced the antibody response to HGG in low responder C57BL/6 mice as compared with that in high responder DDD, C3H/He or (C57BL/6 X DDD)F1 mice. Aggregate-free preparation of HGG at a dose of 0.5 mg induced immunological tolerance in all strains of mice tested. LPS injected subsequently converted tolerogenic, aggregate-free HGG into immunogen in DDD mice but not in C57BL/6 mice. To determine the correlation between adjuvanticity and mitogenicity of LPS, spleen cells from normal mice were cultured in the presence of LPS and 3H-thymidine uptake was measured. Spleen cells of DDD mice incorporated three times as much 3H-thymidine as those of C57BL/6 mice. There seems no strong correlation between both activities of LPS. The data obtained are discussed in terms of strain differences in the macrophage function for processing the antigen.

In vitro inhibition of leukocyte uptake of radioactive endotoxin by components of normal serum.

SummaryChromium51-labeled enterobacterial lipopolysaccharides were incubated with leukocytes or erythrocytes in the presence or absence of serum (or serum protein components) . Leukocytes exhibit a much greater affinity for endotoxin than erythrocytes. Serum inhibits the rate of uptake of lipopolysaccharide by the blood cells. Each of the serum fractions (albumin, and α1-, α2-, β-, γ-globulins) interfere in the uptake of endotoxin by leukocytes.