R-form LPS, the master key to the activation ofTLR4/MD-2-positive cells

Lipopolysaccharide (endotoxin, LPS) is a major recognition marker for the detection of gram-negative bacteria by the host and a powerful initiator of the inflammatory response to infection. Using S- and R-form LPS from wild-type and R-mutants of Salmonella and E. coli, we show that R-form LPS readily activates mouse cells expressing the signaling receptor Toll-like receptor 4/myeloid differentiation protein 2 (TLR4/MD-2), while the S-form requires further the help of the LPS-binding proteins CD14 and LBP, which limits its activating capacity. Therefore, the R-form LPS under physiological conditions recruits a larger spectrum of cells in endotoxic reactions than S-form LPS. We also show that soluble CD14 at high concentrations enables CD14-negative cells to respond to S-form LPS. The presented in vitro data are corroborated by an in vivo study measuring TNF-alpha levels in response to injection of R- and S-form LPS in mice. Since the R-form LPS constitutes ubiquitously part of the total LPS present in all wild-type bacteria its contribution to the innate immune response and pathophysiology of infection is much higher than anticipated during the last half century.     

Induction of human granulocyte chemiluminescence by bacterial lipopolysaccharides

Bacterial lipopolysaccharides (LPS) have been reported to influence the oxidative response of human polymorphonuclear leukocytes (PMN). However, results sometimes conflict. In the present study, we demonstrated that activation of human PMN by LPS depends on the class (smooth [S] or rough [R]) to which the LPS belongs. Lucigenin-dependent chemiluminescence was used to assay oxygen radical production. Twenty different S- and R-form LPS and free lipid A were tested in concentrations of 0.01 to 100 micrograms/ml. S-form LPS activated PMN only at maximal concentrations and to a low extent. R-form LPS and free lipid A were potent inducers of granulocyte chemiluminescence even at a concentration of 0.1 microgram/ml. The results indicated that R-form LPS are very effective in inducing granulocyte chemiluminescence, whereas true S-form preparations are inactive. It is not known at present whether this higher activity is due to a more lipophilic character of R-form LPS or whether the presence of the O polysaccharide in S-form LPS exerts an inhibitory effect on their action on granulocytes.     

Modification of the silver staining technique to detect lipopolysaccharide in polyacrylamide gels.

A silver staining method used routinely for detecting bacterial lipopolysaccharide (LPS) in sodium dodecyl sulfate-polyacrylamide gels (C. Tsai and E. Frasch, Anal. Biochem. 119:115-119, 1982) appeared to be inappropriate for visualizing certain LPS preparations. It did not stain S-form fractions of polyagglutinable Pseudomonas aeruginosa LPS or several partly deacylated (alkali-treated) S-form LPS after sodium dodecyl sulfate-polyacrylamide gel electrophoresis. However, these LPS preparations could be detected by anti-LPS sera after electroblotting onto nitrocellulose, thereby confirming their integrity and presence in the polyacrylamide gel. This is because LPS fractions containing a low number of fatty acids are washed out of the gel during the initial fixing step (40% ethanol-4% acetic acid, overnight). By omitting this fixing step, which was originally developed for detecting proteins, and by increasing the LPS oxidation time (from 5 to 20 min), we restored the ability to detect LPS fractions that otherwise would not be stained. These modifications did not affect the detection of other S- and R-form LPSs. Thus, differences in the number of fatty acids present in polyagglutinable P. aeruginosa LPS may result in a selective loss of fatty acid-deficient S-form LPS in these apparent R-form LPS preparations. This modified procedure provides a fast, simple, and sensitive way to analyze LPS in polyacrylamide gels despite the number of acyl groups present.     

Enhancement of endotoxin lethality and generation of anaphylactoid reactions by lipopolysaccharides in muramyl-dipeptide-treated mice.

Intravenous injection of muramyl dipeptide (MDP) and Salmonella lipopolysaccharides (LPS) enhanced lethal toxicity of the LPS in C57BL/6 mice. This was true for S (smooth)- and R (rough)-form LPS and free lipid A. Enhancement of toxicity was maximum when the LPS was administered 4 h after MDP, at which time the lethal doses for 50% of mice of S- and R-form LPS and free lipid A were between 1 and 10 micrograms, compared with more than 100 micrograms in normal animals. This sensitization was absent in endotoxin-resistant C3H/HeJ mice. Lethality usually commenced 15 h after LPS injection and was complete after 72 h. Higher doses of some S-form LPS (100 micrograms or more) administered 4 h after MDP led to a strong anaphylactoid reaction within 10 to 20 min of injection, with lethal outcomes in less than 1 h after LPS administration. This early anaphylactoid reaction was observed for various mouse strains, including LPS-resistant C3H/HeJ mice, but it was very weak or completely absent with R-form LPS or free lipid A even in concentrations of up to 1,000 micrograms. A strong anaphylactoid reaction comparable to that seen with S-form LPS was also obtained, after MDP treatment, with an LPS of low toxicity prepared from Bacteroides gingivalis. It is noteworthy that oral administration of MDP also contributed to the anaphylactoid reaction and enhanced the late-phase lethality of LPS. The present findings strongly suggest that the early- and late-phase reactions induced by MDP and LPS are caused by different mechanisms.     

Lipid A-induced tolerance and hyperreactivity to hypothermia in mice.

Mice responded to lipopolysaccharide (LPS) with a dose-dependent, monophasic hypothermia reaching a maximum at 2 h postinjection. Degraded polysaccharide was not active; free lipid A, however, induced a similar pattern of hypothermia, indicating that the hypothermic principle of LPS was embedded within the lipid A component. The hypothermic response of mice to LPS was modified by prior exposure of the host to LPS. This altered reactivity was manifested by refractory periods (early and late tolerance), in which animals no longer responded with hypothermia, or a hyperreactive phase (hypersensitivity), in which hypothermic responses were greatly augmented upon LPS challenge. Thus, tolerance observed 24 h after a single injection of LPS (early tolerance) was followed, on further LPS challenge, by an enhanced hypothermic responses reaching a maximum on day 4. Further daily exposure of the animals to LPS eliminated hyperreactivity and led to the establishment of a late tolerance maximally expressed on day 8. Hyperreactivity could also be evoked on day 4 after a single injection of LPS. Mice pretreated with Salmonella S- and R-form LPS or free lipid A (Salmonella) demonstrated tolerance and hyperreactivity to both homologous and heterologous challenge. In addition, complete cross-tolerance was observed with S-form LPS derived from Shigella. It was concluded that the differential effects of LPS on host responses (tolerance and hyperreactivity) were due to lipid A.     

Production and characterisation of mouse monoclonal antibodies reactive with the lipopolysaccharide core of Pseudomonas aeruginosa.

Monoclonal antibodies (MAbs) to the core antigen region of lipopolysaccharide (LPS) of Pseudomonas aeruginosa were produced from mice immunised with whole cells of heat-killed rough mutants of Pseudomonas aeruginosa expressing partial or complete core LPS. MAbs were screened in an enzyme-linked immunosorbent assay (ELISA) against three different antigen cocktails: S-form LPS from three P. aeruginosa strains, R-form LPS from six P. aeruginosa strains and, as a negative control, R-form LPS from Salmonella typhimurium and Escherichia coli. Selected MAbs were subsequently screened against a range of extracted LPS and whole cells from both reference strains and clinical isolates of P. aeruginosa. The antibodies were also screened in ELISA against whole-cell antigens from other Pseudomonas spp. as well as strains of Haemophilus influenzae, Neisseria subflava and Staphylococcus aureus. Five MAbs reacting with the core component of P. aeruginosa LPS were finally selected. Two of these, MAbs 360.7 and 304.1.4, were particularly reactive in immunoblots against unsubstituted core LPS, including that from O-antigenic serotypes of P. aeruginosa. The MAbs also reacted with some of the other Pseudomonas spp., but not with P. cepacia or Xanthomonas (Pseudomonas) maltophilia. Cross-reactivity with whole cells from other bacterial species was minimal or not observed. Reactivity of MAbs with some Staph. aureus strains was observed, and binding to the protein A component was implicated. The reactivity of the MAbs was investigated further by flow cytometry and immunogold electronmicroscopy. The suitability of the MAbs for an immunological assay for detection of P. aeruginosa in respiratory secretions from CF patients is discussed.     

Binding of Salmonella typhimurium lipopolysaccharides to rat high-density lipoproteins.

These studies were undertaken to investigate the binding of gram-negative bacterial lipopolysaccharides (LPS) to high-density lipoproteins (HDL) of rat plasma. Purified Salmonella typhimurium LPS, intrinsically labeled with [3H]-galactose, bound rapidly in vitro to isolated rat HDL. Maximal binding of LPS to HDL occurred when LPS and HDL were incubated with lipoprotein-free plasma (rho greater than 1.21 g/ml). Since LPS, when purified, form large aggregates, we tested the hypothesis that disaggregation of LPS enhances LPS-HDL binding. We found that calcium chloride (1 mM), an agent which prevents LPS disaggregation, inhibited binding of LPS to HDL by interfering with the modification of LPS by lipoprotein-free plasma. Conversely, sodium deoxycholate (0.15 g/dl), which disaggregates LPS, greatly increased binding of LPS to HDL in the absence of lipoprotein-free plasma. Analysis of labeled LPS by sodium deodecyl sulfate-polyacrylamide gel electrophoresis showed only minor differences in the sizes of LPS molecules before and after binding to HDL, suggesting that chemical modification of LPS is not required for binding. The results provide evidence that disaggregation increases the binding of LPS to HDL.     

Deacylation of bacterial lipopolysaccharide in rat hepatocytes in vitro

The possible role of liver parenchymal cells in the uptake and degradation of bacterial lipopolysaccharide (LPS) was investigated in vitro by employing radiolabelled LPS as substrate. Hepatocytes obtained from Wistar rats by collagenase treatment were found to take up LPS only when it was not linked to the polysaccharide of O-antigen. The amount of LPS taken up increased with time and after 48 h incubation it increased in a dose-dependent manner up to at least 30 micrograms. When incubated with LPS radiolabelled exclusively in the fatty-acid moiety, cultured hepatocytes released lipophilic materials into the culture medium. These were identified as beta-hydroxytetradecanoic acid and triglyceride, in the ratio of 7:I. These results indicate that the R-form of LPS which lacks the O-antigen polysaccharide is taken up and deacylated in hepatocytes, and the derived fatty acids are released into the culture medium either in the free form or after conversion to triglyceride.     

Deacylation of bacterial lipopolysaccharide in rat hepatocytes in vitro.

The possible role of liver parenchymal cells in the uptake and degradation of bacterial lipopolysaccharide (LPS) was investigated in vitro by employing radiolabelled LPS as substrate. Hepatocytes obtained from Wistar rats by collagenase treatment were found to take up LPS only when it was not linked to the polysaccharide of O-antigen. The amount of LPS taken up increased with time and after 48 h incubation it increased in a dose-dependent manner up to at least 30 micrograms. When incubated with LPS radiolabelled exclusively in the fatty-acid moiety, cultured hepatocytes released lipophilic materials into the culture medium. These were identified as beta-hydroxytetradecanoic acid and triglyceride, in the ratio of 7:I. These results indicate that the R-form of LPS which lacks the O-antigen polysaccharide is taken up and deacylated in hepatocytes, and the derived fatty acids are released into the culture medium either in the free form or after conversion to triglyceride.     

Local skin response in mice induced by a single intradermal injection of bacterial lipopolysaccharide and lipid A.

Dermal inflammation and hemorrhagic necrosis induced by bacterial lipopolysaccharide (LPS) and lipid A were studied in mice. In ddY mice, a single intradermal injection of Salmonella typhimurium S-form LPS and lipid A into the abdominal dermis elicited an edematous change due to an increase in local vascular permeability 12 h postinjection, followed by hemorrhagic necrosis from 24 to 72 h. This skin reaction was also induced in a dose-dependent manner by S-form LPS, R-mutant LPS, and lipid A of S. typhimurium and Escherichia coli, but not by polysaccharide from Salmonella S-form LPS. The dermal inflammation-inducing activities of LPS and lipid A were roughly in the following order (from highest to lowest): Re-form LPS, Rc-form LPS and lipid A, Ra-form LPS, and S-form LPS. These results suggest that the lipid A portion of the LPS molecule is responsible for the skin reaction. In C3H/HeN mice, Re-form LPS and lipid A induced the same intensity of skin reaction as that in ddY mice. In C3H/HeJ mice, which have a low response to LPS, Re-LPS and lipid A did not induce any hemorrhagic response but showed a distinct edematous change. Although hemorrhagic necrosis and edematous changes could be explained by quantitative differences in skin lesions, the other possible explanation is that hemorrhagic necrosis and the increase in local vascular permeability are induced by different mechanisms, only one of which depends on the regulation of the lps gene.     

Biological activities of lipopolysaccharides of Proteus spp. and their interactions with polymyxin B and an 18-kDa cationic antimicrobial protein (CAP18)-derived peptide

The saccharide constituents of lipopolysaccharides (LPS) of Proteus spp. vary with the strain and contain unique components about which little is known. The biological activities of LPS and lipid A from S- and R-forms of 10 Proteus strains were examined. LPS from all S-form Proteus strains was lethal to D-(+)-galactosamine (GalN)-loaded, LPS-responsive, C3H/HeN mice, but not to LPS-hypo-responsive C3H/HeJ mice. P. vulgaris 025 LPS evoked strong anaphylactoid reactions in N-acetylmuramyl-L-alanyl-D-isoglutamine (MDP)-primed C3H/HeJ mice. LPS from S- and R-form Proteus strains induced production of nitric oxide (NO) and tumour necrosis factor (TNF) by macrophages isolated from C3H/HeN but not C3H/HeJ mice. Lipid A from Proteus strains also induced NO and TNF production, although lipid A was less potent than LPS. The effects of LPS were mainly dependent on CD14; LPS-induced NO and TNF production in CD14+ J774.1 cells was significantly greater than in CD14-J7.DEF.3 cells. All LPS from Proteus strains, and especially from P. vulgaris 025, exhibited higher anti-complementary activity than LPS from Escherichia coli or Pseudomonas aeruginosa. Polymyxin B inactivated proteus LPS in a dose-dependent manner, but these LPS preparations were more resistant to polymyxin B than E. coli LPS. CAP18(109-135), a granulocyte-derived peptide, inhibited proteus LPS endotoxicity only when the LPS:CAP18(109-135) ratio was appropriate, which suggests that CAP18(109-135) acts through a different mechanism than polymyxin B. The results indicate that LPS from Proteus spp. are potently endotoxic, but that the toxicity is different from that of LPS from E. coli or Salmonella spp. and even varies among different Proteus strains. The variation in biological activities among proteus LPS may be due to unique components within the respective LPS.     

Time course of cellular distribution of endotoxin in liver,lungs and kidneys of rats.

The time course of distribution of 2 endotoxic lipopolysaccharides (LPS), S. abortus equi (S form) and S. minnesota R 595 (R form, Re), in liver, lungs and kidneys was studied by the immunoperoxidase method in rats. After its uptake in the liver, both LPS were first detectable in Kupffer cells and granulocytes, the R form also in hepatocytes. A redistribution of the S-form LPS from Kupffer cells to hepatocytes was observed on Day 3 after injection. The detectability of both LPS was lost between Days 5 and 9 after injection. In the lungs both LPS were detectable later than in the liver. Here the LPS were also found in alveolar and bronchiolar macrophages, which shows that they can also be eliminated through this organ. The kidneys remained essentially free of LPS, small amounts being detectable here only in the first 24 h.     

Priming of polymorphonuclear granulocytes by lipopolysaccharides and its complexes with lipopolysaccharide binding protein and high density lipoprotein

Human peripheral blood neutrophils are primed, or enabled to respond to formyl peptide, by prior exposure to bacterial lipopolysaccharide (LPS). The activity of LPS and the size of its aggregates are altered by plasma constituents such as high density lipoprotein (HDL) and the recently discovered acute phase reactant lipopolysaccharide binding protein (LBP) Tobias et al.: J. Exp. Med. 164,777, 1986]. The ability of LPS, LPS-LBP, and LPS-HDL complexes to activate a number of cellular responses have been compared. LPS-LBP and LPS-HDL were prepared using LBP and HDL from rabbit serum. LPS from Salmonella minnesota Re595 and its LPS-LBP and LPS-HDL complexes differed in their ability to prime PMN O2- production in response to formyl peptide (f-Nle-Leu-Phe-Nle-Tyr-Leu [FNLPNTL]). Human PMN prepared under conditions in which O2- production is minimal (less than 1 nmol O2-/10(6) PMN/10 min) after exposure to 10(-7) M FNLPNTL can be primed with 0.1-100 ng/ml LPS in a dose- and time-dependent manner to produce up to 12 nmol O2-/10(6) PMN/10 min. LBP complexation accelerated the priming induced by LPS, whereas HDL complexation retarded it. Priming was accompanied by a parallel two- to threefold increase in formyl peptide receptor number as determined by FACS analysis of fluoresceinated FNLPNTL binding and SDS-PAGE autoradiographic analysis of photoaffinity ligand binding. Thus binding of LPS to plasma proteins changes the response of the PMS to LPS and may represent one way in which the response of the PMN is regulated during infection. Since LBP concentrations change during an acute phase response, complexation of LPS with LBP is a mechanism that may regulate neutrophil responses in vivo during inflammation.     

Lactoferrin-Lipid A-Lipopolysaccharide Interaction: Inhibition by Anti-Human Lactoferrin Monoclonal Antibody AGM 10.14

Lactoferrin (LF) is a glycoprotein that exerts both bacteriostatic and bactericidal activities. The interaction of LF with lipopolysaccharide (LPS) of gram-negative bacteria seems to play a crucial role in the bactericidal effect. In this study, we evaluated, by means of an enzyme-linked immunosorbent assay, the binding of biotinylated LF to the S (smooth) and R (rough) (Ra, Rb, Rc, Rd1, Rd2, and Re) forms of LPS and different lipid A preparations. In addition, the effects of two monoclonal antibodies (AGM 10.14, an immunoglobulin G1 [IgG1] antibody, and AGM 2.29, an IgG2b antibody), directed against spatially distant epitopes of human LF, on the LF-lipid A or LF-LPS interaction were evaluated. The results showed that biotinylated LF specifically binds to solid-phase lipid A, as this interaction was prevented in a dose-dependent fashion by either soluble uncoupled LF or lipid A. The binding of LF to S-form LPS was markedly weaker than that to lipid A. Moreover, the rate of LF binding to R-form LPS was inversely related to core length. The results suggest that the polysaccharide O chain as well as oligosaccharide core structures may interfere with the LF-lipid A interaction. In addition, we found that soluble lipid A also inhibited LF binding to immobilized LPS, demonstrating that, in the whole LPS structure, the lipid A region contains the major determinant recognized by LF. AGM 10.14 inhibited LF binding to lipid A and LPS in a dose-dependent fashion, indicating that this monoclonal antibody recognizes an epitope involved in the binding of LF to lipid A or some epitope in its close vicinity. In contrast, AGM 2.29, even in a molar excess, did not prevent the binding of LF to lipid A or LPS. Therefore, AGM 10.14 may represent a useful tool for neutralizing selectively the binding of LF to lipid A. In addition, the use of such a monoclonal antibody could allow better elucidation of the consequences of the LF-lipid A interaction.     

Common lipopolysaccharide specificity: new type of antigen residing in the inner core region of S- and R-form lipopolysaccharides from different families of gram-negative bacteria.

A new antigenic specificity, referred to here as common lipopolysaccharide (LPS) specificity, is described in the LPSs of gram-negative bacteria belonging to various families. The specificity is present in S- and R-form LPS but absent in Re mutants of different enterobacterial genera. By the use of purified LPS and monospecific antibodies obtained by immunoabsorption, the specificity is differentiated from the known core specificities of the genus Salmonella and the lipid A specificity by aid of the passive hemolysis and passive hemolysis inhibition test. In Salmonella minnesota R-form LPS, the specificity may be cryptic (R345, Rb2 mutant) or partly exposed in the intact molecule (R7, Rd1 mutant). The specificity is either demasked or completely exposed after mild acid hydrolysis for a short time, whereas it is destroyed after prolonged hydrolysis. Periodate oxidation, reduction, and hydrolysis under conditions that do not affect the ketosidic linkages of 2-keto-3-deoxyoctulosonic acid destroy the specificity in R4 (Rd2 mutant) LPS, but do not do so in R7 LPS. It is suggested that 2-keto-3-deoxyoctulosonic acid and a following neutral sugar are the compositional requirements for expressing the specificity.     

No significant influence of serum amyloid A1 genotypes on serum lipids or HDL clearance

Serum amyloid A1(SAA1), the major acute phase isotype of SAA protein family, consists of three common allelic variants(SAA1.1, SAA1.3 and SAA1.5) in the Japanese population. We have recently reported that subjects with the SAA1.5 allele have higher plasma SAA concentrations than those without it, a phenomenon probably due to the delayed catabolism of the isotype SAA1.5. Since SAA is present in high density lipoprotein(HDL), this study assessed whether SAA genotype influenced the serum lipid study by altering HDL metabolism. In a total of 279 healthy adults, no difference was noted in their total cholesterol, HDL-cholesterol or triglyceride concentrations among six genotype groups. Plasma clearance of human apolipoprotein AI(apoAI) was studied in mice by giving HDL reconstituted with each recombinant human SAA1 isotype. The apoAI clearance did not differ among each of the SAA1 isotype-conjugated HDLs. Moreover, the changes in content of SAA in HDL also did not alter the apoAI clearance. These results suggest that SAA1 may not play an active role in plasma HDL metabolism.     

Increase in rat plasma antioxidant activity after E. coli lipopolysaccharide administration

It is well recognized that the sensitivity of animals to lipopolysaccharide (LPS) endotoxin varies tremendously. And, it has been recently observed that Sprague-Dawley rats dramatically increase the activity of hepatic endogenous antioxidative enzyme systems after LPS administration. This finding suggests that the relative resistance of rats to LPS may be related to a concomitant increase in the activities of the hepatic antioxidant systems. This study was designed to examine if the above reported hepatic change in rats given LPS could be observed at the systemic level. Male Sprague-Dawley or Wistar rats, weighing 250 - 350 g, were given increasing doses (10 - 100 mg/kg) of LPS i.p. under 1.0% isoflurane anesthesia. Antioxidant capacity (AOC), blood gas analysis, and the cardiovascular parameters of the arterial blood of animals were determined over a 4 hour period following LPS administration. In addition, we studied the effect of pretreatment with the non-specific nitric oxide synthase inhibitor, L-N(G)-Nitroarginine methyl ester hydrochloride (L-NAME), given 50 mg/kg s.c. one and 24 hours before the administration of 20 mg/kg LPS i.p. in Sprague-Dawley rats. Rats given sufficiently high doses of E. coli LPS to produce behavioral effects also showed increased plasma AOCs in the early period after the administration of LPS. Similar changes were noted in Sprague-Dawley and Wistar rat strains, but at different doses that reflect their differential sensitivities to the LPS induced inflammatory response. Also, the resistance of the Sprague-Dawley strain of rats to LPS was not altered by the prior administration of L-NAME, nor was the plasma AOC altered. In conclusion, our study suggests that the rat strains are relatively resistant to develop the toxic signs of LPS in the early period after the administration of LPS, especially in Sprague-Dawley rats. Moreover, endotoxin-induced increases in plasma AOC may contribute to the rats' resistance to LPS intoxication.     

N-acetylcysteine exerts protective effects and prevents lung redox imbalance and peroxynitrite generation in endotoxemic rats

The aim of this study was to determine in endotoxemic rats the effects of N-acetylcysteine on lung redox imbalance and plasma peroxynitrite generation. Eighty male Wistar rats were divided in two sets of five experimental groups. Six hours after vehicle (Control group: isotonic NaCl sterile solution i.p.; n=7), lipopolysaccharide (LPS group: 1 mg/Kg i.p.; n=8), N-acetylcysteine plus LPS (NAC+LPS group, n=8), NAC plus the nitric oxide synthesis inhibitor N(w)-nitro-L-arginine methyl ester plus LPS (NAC+NAME+LPS group; n=8), or NAME plus LPS (NAME+LPS group; n=9), arterial blood and lung samples were taken from each animal under sodium pentobarbital anesthesia. In five additional groups treated as described above, in vivo plasma oxidation of dihydrorhodamine (DRH) 123 to rhodamine (RH)123 was measured as index of peroxynitrite formation. LPS treated rats presented increased plasma lactate, thrombocytopenia and both, decreased reduced thiols and increased lipid peroxidation in lung tissue. Moreover, LPS produced increments in plasma concentration of nitrites/nitrates and DRH 123 oxidation. Pretreatment with NAC prevented all these changes induced by LPS except the increment in plasma concentration of nitrites/nitrates. The protective effects seen in LPS rats pretreated with NAC were not observed in the NAC+NAME+LPS group. In conclusion, the results of this study show that in endotoxemia induced by LPS in rats, NAC produces protective effects on lung redox balance and prevents peroxynitrite anion generation.     

Inhibition of nitric oxide synthase delays the development of tolerance to LPS in rats

The role of nitric oxide (NO) was investigated in endotoxin (lipopolysaccharide, LPS) tolerance in freely moving biotelemetered rats. We monitored changes in febrile response and feeding behavior (food intake, water intake) during the development of tolerance to repeated intraperitoneal injections of LPS (50 microg/kg) along with injections of N(omega)-nitro-L-arginine methyl ester (L-NAME; 50 mg/kg), an inhibitor of NO synthase. Rats were treated with LPS and L-NAME for three consecutive days. On the fourth day, all rats were injected with LPS alone. Control rats were injected with saline along with saline or with L-NAME for four consecutive days. Rats repeatedly injected with LPS became tolerant to pyrogenic and hypophagic/cachexic effects of LPS as early as on the second day of experiment. The treatment with L-NAME prevented the attenuation of febrile response following the second LPS injection. Moreover, the depressive effects of LPS on body weight as well as on water and food intake were prolonged in rats treated with a combination of L-NAME and LPS. Injection of LPS caused a 3.5-fold increase in plasma nitrite within 3 h and nitrite levels remained significantly elevated 6 and 24 h after LPS. Rats injected secondly with LPS did have still 2.5- to 3-fold increase in plasma nitrite levels 3 and 6 h, but not 24 h, after injection. Third injection of LPS did not elevate nitrite level in plasma. Taken together, presented data provide clear evidence that NO formation is involved in mechanisms responsible for development of early-stage tolerance to endotoxin.     

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.