Release Of Lipopolysaccharide Research Articles

Inflammatory cytokines mediate C-C (monocyte chemotactic protein 1) and C-X-C (interleukin 8) chemokine expression in human pleural fibroblasts.

Current knowledge implicates pleural mesothelial cells as mainly responsible for inflammatory responses in the pleural space. However, a vast body of recent evidence underscores the important role of fibroblasts in the process of inflammation in several types of tissues. We hypothesize that HPFBs (human pleural fibroblasts) play an important role in pleural responses and also when activated by bacterial endotoxin LPS (lipopolysaccharide), IL-1 beta (interleukin-1 beta), or TNF-alpha (tumor necrosis factor-alpha) release of C-C and C-X-C chemokines-specifically, MCP-1 and IL-8. Our results show that pleural fluid-isolated human fibroblasts release IL-8 and MCP-1 upon stimulation with IL-1 beta, TNF-alpha, and LPS in both a concentration- and time-dependent manner. RT-PCR (reverse-transcriptase-polymerase chain reaction) studies have also confirmed IL-8- and MCP-1-specific mRNA expression in activated pleural fibroblasts. On the time-dependent response curve, IL-8 was found in maximum concentrations at 144 hr, whereas MCP-1 continued to increase even after 196 hr following stimulation. IL-1 beta induced the maximum release of IL-8 (800-fold) and MCP-1 (164-fold), as compared to the controls. TNF-alpha induced a 95-fold increase in IL-8 and an 84-fold increase in MCP-1 levels, as compared to the controls. Collectively, our results show that human pleural fibroblasts contribute to the inflammatory cascade in the pleural space.

Effect of pH on endotoxin affinity for metal-ceramic alloys

Statement of Problem. Crevicular pH may modify bacterial endotoxin affinity for high-noble metal-ceramic alloys. Purpose.Porphyromonas gingivalis lipopolysaccharide (LPS) affinity for 3 metal-ceramic alloys at 3 different pH levels was compared in vitro by measuring adsorption to and release from the alloy surface. Material and Methods. Metallographically polished disks were fabricated from Pd-Ag-Sn, Au-Pd-Ag-Sn-In, and Au-Pd-In-Ga alloys. Clean disks were placed individually into 1 mL at pH 6.5, 7.0, or 7.5 phosphate-buffered saline solution containing 0.9 endotoxin units per square millimeter tritiated LPS (n = 3 disks per alloy-pH group). The disks were incubated for 24 hours at 37°C before being transferred to LPS-free buffer and incubated, again for 24 hours at 37°C, to evaluate elution. This transfer continued at 24-hour intervals up to 96 hours total elution incubation. Lipopolysaccharide adsorption to and elution from disks was determined through liquid scintillation spectrometry. Adsorption data were evaluated with a 2-way analysis of variance (α=.05) and the post hoc Tukey honestly significant difference test. Results. Lipopolysaccharide adsorption values ranged from 0.48 ± 0.04 EU/mm2 for the Au-Pd-Ag-Sn-In alloy at pH 7.5 to 0.75 ± 0.04 EU/mm2 for the Pd-Ag-Sn alloy at pH 6.5. Alloy type (P=.0001) and environmental pH (P=.0001) significantly influenced adsorption. Adsorption to the Pd-Ag-Sn and Au-Pd-In-Ga alloys at pH 6.5, 7.0, and 7.5 were similar and decreased with increasing pH. In contrast, adsorption to the Au-Pd-Ag-Sn-In alloy was significantly less than to other alloys at pH 6.5 but did not differ at other pH levels. Lipopolysaccharide release from the alloy surface could not be detected. Conclusion.P. gingivalis LPS affinity for metal-ceramic alloys was modified by environmental pH. The degree of LPS adsorption depended on the composition and surface chemistry of each alloy. (J Prosthet Dent 2001;86:644-9.)

Chitosan disrupts the barrier properties of the outer membrane of Gram-negative bacteria

The mode of antimicrobial action of chitosan (polymeric β-1,4- N-acetylglucosamine) on Gram-negative bacteria was studied with special emphasis on its ability to bind to and weaken the barrier function of the outer membrane (OM). Chitosan (250 ppm) at pH 5.3 induced significant uptake of the hydrophobic probe 1- N-phenylnaphthylamine (NPN) in Escherichia coli, Pseudomonas aeruginosa and Salmonella typhimurium. The effect was reduced ( E. coli, salmonellae) or abolished ( P. aeruginosa) by MgCl 2. No NPN uptake was observed during exposure of the salmonellae to chitosan at pH 7.2. Chitosan also sensitized P. aeruginosa and the salmonellae to the lytic effect of sodium dodecyl sulfate (SDS); such sensitization was not blocked by MgCl 2 and was reversible by washing chitosan-treated cells prior to SDS exposure. Chemical and electrophoretic analyses of cell-free supernatants of chitosan-treated cell suspensions showed that interaction of chitosan with E. coli and the salmonellae involved no release of lipopolysaccharide (LPS) or other membrane lipids. However, chitosan rendered E. coli more sensitive to the inhibitory action of dyes and bile acids used in selective media. Highly cationic mutants of S. typhimurium were more resistant to chitosan than the parent strains. Electron microscopy showed that chitosan caused extensive cell surface alterations and covered the OM with vesicular structures. Chitosan thus appeared to bind to the outer membrane, explaining the loss of the barrier function. This property makes chitosan a potentially useful indirect antimicrobial for food protection.

Temperature shock, injury and transient sensitivity to nisin in Gram negatives.

The effect of thermal stresses on survival, injury and nisin sensitivity was investigated in Salmonella Enteritidis PT4, PT7 and Pseudomonas aeruginosa. Heating at 55 degrees C, rapid chilling to 0.5 degrees C or freezing at -20 degrees C produced transient sensitivity to nisin. Cells were only sensitive if nisin was present during stress. Resistance recovered rapidly afterwards, though some cells displayed residual injury. Injury was assessed by SDS sensitivity, hydrophobicity changes, lipopolysaccharide release and NPN uptake. LPS release and hydrophobicity were not always associated with transient nisin sensitivity. Uptake of NPN correlated better but persisted longer after treatment. Thermal shocks produce transient injury to the outer membrane, allowing nisin access. After treatment, the permeability barrier is rapidly restored by a process apparently involving reorganization rather than biosynthetic repair. Inclusion of nisin during food treatments that impose sub-lethal stress on Gram negatives could increase process lethality, enhancing microbiological safety and stability.

The preparation of the chronic hyper-endotoxemia experimental animal model by means of a drug delivery system

A drug delivery system (DDS) consisting of lipopolysaccharide (LPS) as a drug and 2-hydroxyethyl methacrylate (HEMA)-diethylene glycol dimethacrylate (2G) or –polyethylene glycol dimethacrylate (4G, 9G) copolymer was prepared, and used for the efficient preparation of an experimental animal model of chronic hyper-endotoxemia. The release profiles of LPS in the in-vitro test were greatly influenced by the composition of HEMA-2G, 4G, 9G in the copolymer. It was found that LPS release from the DDS continued gradually and constantly throughout 2 weeks. In the in-vivo experiment with rats, the DDS maintained a high blood concentration level of LPS for 3 days. These results strongly suggest the possibility of convenient and reproducible preparation of a chronic hyper-endotoxemia animal model.

Application of a drug delivery system in a novel rat model of chronic hyperendotoxemia

There has not been an ideal reproducible small-animal model of chronic hyperendotoxemia to date. Our drug delivery system (DDS) is a new technology that can deliver a drug conveniently to a target organ at an optional rate. 2-Hydroxyethyl methacrylate (HEMA) was used as a carrier of lipopolysaccharide (LPS), and diethylene glycol and polyethylene glycol dimethacrylates (2G, 4G, 9G) were used as cross-linking agents. A mixed solution of HEMA and di(poly)ethylene glycol dimethacrylate was charged into a glass tube with or without LPS and polymerized by ultraviolet irradiation. This polymer was cut into DDS tablets of the same size with or without LPS. A mixture with HEMA:4G = 1:3 was the most suitable composition to release a constant concentration of LPS. We also developed a novel rat model of chronic hyperendotoxemia. Four DDS tablets, each containing 15 mg LPS, were implanted into the abdominal cavity of rats in the LPS group. The control group was implanted with four DDS tablets without LPS. Plasma levels of LPS in the study group were maintained at more than 2,000 pg/ml for 72 h after implantation. Weight gain was lower and body temperature was higher in the LPS group than in the control group. Plasma levels of inter leukin (IL)-6 in the LPS group were higher than in the control group only during the initial 12 h after implantation of DDS tablets. The white blood cell count at 24 h and platelet counts at 24, 48, and 72 h in the LPS group were lower than those in the control group. These results indicate that chronic hyperendotoxemia was maintained for 72 h by continuous release of LPS from the DDS. Moreover, the intensity of endotoxemia could be varied by varying the number of DDS tablets. It is concluded that our new rat model using LPS-DDS will be applicable and useful as a model of chronic hyperendotoxemia.

Complement-mediated lipopolysaccharide release and outer membrane damage in Escherichia coli J5: requirement for C9.

Lipopolysaccharides (LPS) are major antigenic components of the outer membrane of Gram-negative bacteria and can stimulate activation of the complement system. Such activation leads to formation of the complement membrane attack complex (MAC) on the cell walls, LPS release and, in serum-sensitive strains, to cell death. In this study, Escherichia coli J5 strains, which incorporate exogenous galactose exclusively into LPS, were used to generate target strains with different LPS chemotypes, and the LPS of the strains was labelled with tritium (3H-LPS). The ability of normal human serum (NHS) and human complement-deficient sera to release LPS was subsequently monitored. NHS-induced release of 64-95.7% of 3H-LPS within 30 min; overall, no significant difference was observed between release of LPS from E. coli J5 strains with different LPS chemotypes. In functional assays, maximum LPS release had occurred by 30 min and before maximum bacterial killing. Electron microscopy revealed NHS-induced outer-membrane disruption in the form of blebs at 15 min; at this time-point the inner membrane remained intact. Background LPS release and no bactericidal activity were detected in heat-inactivated serum or human sera deficient in C6, C7 or C8. The C9-deficient (C9D) serum had low bactericidal activity and failed to induce LPS release; however, addition of purified human C9 reconstituted its ability to release LPS. This study demonstrated the need for functional C9 molecules for LPS-releasing activities in serum-sensitive E. coli J5 strains.

Ajoene, a natural product with non-steroidal anti-inflammatory drug (NSAID)-like properties?

The inducible isoform of cyclooxygenase (COX-2) is implicated in the pathogenesis of various inflammatory diseases as well as in carcinogenesis, especially of gastrointestinal tumors. Epidemiological as well as experimental data support a role for constituents of allium vegetables, such as garlic and onions, in the prevention of gastrointestinal cancer. Therefore, the aim of the present study was to examine whether the garlic-derived natural product ajoene interferes with the COX-2 pathway by using lipopolysaccharide (LPS)-activated RAW 264.7 cells as in vitro model. Ajoene was shown to dose-dependently inhibit the release of LPS (1 μg/mL)-induced prostaglandin E 2 in RAW 264.7 macrophages ( ic 50 value: 2.4 μM). This effect was found to be due to an inhibition of COX-2 enzyme activity by ajoene ( ic 50 value: 3.4 μM). Ajoene did not reduce COX-2 expression, but rather increased LPS-induced COX-2 protein and mRNA expression compared to LPS-stimulated cells only. In the absence of LPS, however, ajoene was unable to induce COX-2. The non-steroidal anti-inflammatory drug indomethacin was shown to act similarly in LPS-activated RAW 264.7 cells. These data suggest that ajoene works by a mechanism of action similar to that attributed to non-steroidal anti-inflammatory drugs. This finding may add a novel aspect to the biological profile of the garlic-derived natural product ajoene which might be important for understanding the usefulness of garlic for chemoprevention of gastrointestinal carcinomas.

塩化ナトリウム存在下における大腸菌の増殖抑制に及ぼす加熱処理の影響

Antibacterial action of heat treatment on Escherichia coli IFO3301 (E. coli) in the presence of sodium chloride was investigated. Sodium chloride (0.39 M) more inhibited the growth of E. coli than any other reagents under heating at 55°C. The combined effect of sodium chloride and heating was facilitated by an addition of organic acid with longer chain of carbon atoms. The cell-surface hydrophobicity of E. coli increased with sodium chloride under heating. Release of lipopolysaccharide from the cell membrane was enhanced by heating and was promoted with the addition of sodium chloride. Heat treatment increased leakage of UV-absorbing substances from E. coli, while sodium chloride did not enhance leakage of UV-absorbing substances from the cell under heating.

Influence of In-Vivo Endotoxin Liberation on Anti-Anaerobic Antimicrobial Efficacy

The ability of cefoxitin, clindamycin, imipenem, meropenem, metronidazole and piperacillin-tazobactam to cause Gram-negative anaerobic bacteria to release endotoxin and the influence of such liberated endotoxin on antibiotic efficacy were investigated in In-Vivo experiments in animal models. Experimental infections in various animal models (mice, hamster and infant rats) with cultures of wild and reference strains of Bacteroides fragilis group and Fusobacterium spp. were carried out by injecting these animals with different inocula (106, 107 and 108 cfu/ml) of the bacterial suspension. Appropriate doses of the test antibiotics were then injected and the plasma lipopolysaccharide (endotoxin) release measured by the Limulus Amoebocyte Lysate (LAL) Assay. Evidence of worsening of the outcome of the infections post-therapy was assessed, including histopathological changes in the internal organs. Infection with generalized septicemia was established with F. nuclea-tum in the mice and hamster models while with the B. fragilis group, infections only led to intra-abdominal abscess formation. Plasma endotoxin release was higher in animals infected with F. nucleatum than B. fragilis and was unrelated to the bacterial inoculum. Imipenem, meropenem and cefoxitin, in that order, induced the highest levels of endotoxin activities in the animal model, particularly following F. nucleatum infection. Histological examination of the internal organs of various animals showed variation in the pattern of histopathological changes; grades 3-4 inflammatory changes in the liver were observed in the Fusobacterium-infected animals that were treated with the car-bapenems and cefoxitin. Therapy with the other antibiotics did not exacerbate anaerobic sepsis. In this study, bacteremia did not lead to massive endotoxin release and antibiotic therapy appeared not to have negatively influenced the outcome of most of the Gram-negative anaerobic infections, except for infections caused by Fusobacterium spp. However, it is conceivable that if the gastrointestinal tract is the source of the endotoxin in patients with systemic inflammatory response syndrome, then the obligate anaerobes like Bacteroides and Fusobacterium species, which are members of the gut flora, may play a major role in the unfavorable outcome of antibiotic therapy in some of these infections.

Role of porin ofShigella dysenteriaetype 1 in modulation of lipopolysaccharide mediated nitric oxide and interleukin-1 release by murine peritoneal macrophages

The ability of Shigella dysenteriae type 1 porin to induce the release of nitric oxide (NO) and interleukin-1 (IL-1) from peritoneal macrophages of mouse and to regulate lipopolysaccharide (LPS) and gamma interferon (IFN-gamma) mediated release of the two proinflammatory mediators was investigated. Porin released nitrite when added to macrophage cultures. A maximum of 3.2-fold nitrite release by macrophages was observed with 100 ng ml(-1) of porin. The nitrite release of LPS was enhanced significantly by lower concentrations of porin, whereas the effect of IFN-gamma was enhanced by porin at higher concentrations. Polysaccharide (PS) moiety of LPS stimulated the nitrite release of elicited macrophages by 1.6-fold compared to untreated control. It also enhanced the stimulatory effect of 1 and 10 ng ml(-1) of porin by 1.3-fold. Lipid A (LPA) moiety of LPS did not release nitrite, nor did it increase the porin mediated nitrite production. Porin treated 24 h old macrophage culture supernatants were applied for ConA activated thymocyte proliferation as a measure for determination of IL-1 release. Sixty percent depletion of thymocyte proliferation was observed when the porin treated macrophage supernatants were absorbed with anti-IL-1 antibody. A maximum of 5.5-fold increase of thymocyte proliferation over control was found with 1 and 10 ng ml(-1) of porin. One or 10 ng ml(-1) of porin and LPS augmented the thymocyte growth, 1.5-fold beyond that obtained by porin and 1.8-/1. 7-fold more than that obtained by LPS, alone. Similarly, porin and IFN-gamma co-stimulated the cell growth also. PS enhanced the thymocyte proliferation by 5-fold. It also enhanced the thymocyte growth by co-stimulating 1.4-fold the effect observed by 1 or 10 ng ml(-1) of porin alone. LPA could not participate in the cell proliferating activity nor did it enhance the stimulatory effect of porin. Therefore, both nitrite release and thymocyte proliferation by LPS could be substituted by PS only. The tight association of the two bacterial outer membrane components, porin and LPS, could be a necessary co-signal for boosting the release of the two proinflammatory mediators, namely NO and IL-1, which may be associated with the inflammatory response of the colon during Shigella invasion.

Role of porin of Shigella dysenteriae type 1 in modulation of lipopolysaccharide mediated nitric oxide and interleukin-1 release by murine peritoneal macrophages

The ability of Shigella dysenteriae type 1 porin to induce the release of nitric oxide (NO) and interleukin-1 (IL-1) from peritoneal macrophages of mouse and to regulate lipopolysaccharide (LPS) and gamma interferon (IFN-γ) mediated release of the two proinflammatory mediators was investigated. Porin released nitrite when added to macrophage cultures. A maximum of 3.2-fold nitrite release by macrophages was observed with 100 ng ml −1 of porin. The nitrite release of LPS was enhanced significantly by lower concentrations of porin, whereas the effect of IFN-γ was enhanced by porin at higher concentrations. Polysaccharide (PS) moiety of LPS stimulated the nitrite release of elicited macrophages by 1.6-fold compared to untreated control. It also enhanced the stimulatory effect of 1 and 10 ng ml −1 of porin by 1.3-fold. Lipid A (LPA) moiety of LPS did not release nitrite, nor did it increase the porin mediated nitrite production. Porin treated 24 h old macrophage culture supernatants were applied for ConA activated thymocyte proliferation as a measure for determination of IL-1 release. Sixty percent depletion of thymocyte proliferation was observed when the porin treated macrophage supernatants were absorbed with anti-IL-1 antibody. A maximum of 5.5-fold increase of thymocyte proliferation over control was found with 1 and 10 ng ml −1 of porin. One or 10 ng ml −1 of porin and LPS augmented the thymocyte growth, 1.5-fold beyond that obtained by porin and 1.8-/1.7-fold more than that obtained by LPS, alone. Similarly, porin and IFN-γ co-stimulated the cell growth also. PS enhanced the thymocyte proliferation by 5-fold. It also enhanced the thymocyte growth by co-stimulating 1.4-fold the effect observed by 1 or 10 ng ml −1 of porin alone. LPA could not participate in the cell proliferating activity nor did it enhance the stimulatory effect of porin. Therefore, both nitrite release and thymocyte proliferation by LPS could be substituted by PS only. The tight association of the two bacterial outer membrane components, porin and LPS, could be a necessary co-signal for boosting the release of the two proinflammatory mediators, namely NO and IL-1, which may be associated with the inflammatory response of the colon during Shigella invasion.

Bactericidal and endotoxin neutralizing activity of a peptide derived from Limulus antilipopolysaccharide factor

Background: Release of lipopolysaccharide (endotoxin, LPS) is a critical inciting event in the development of sepsis syndrome due to gram-negative bacteria, and mortality associated with this entity remains ~40%. Limulus anti-LPS factor (LALF) is a naturally occurring horseshoe crab derived protein that, unlike antibiotics, is both bactericidal for gram-negative bacteria and capable of neutralizing LPS. We hypothesized that a peptide derived from the active domain of LALF (LALF #28-54) would exhibit potent biologic activity similar to that of LALF itself and could potentially be useful as a therapeutic agent. Methods: The effects of LALF, synthetic peptide LALF #28-54, polymyxin B (PmB), and a biologically inactive synthetic peptide were examined in several models. In vitro bactericidal activity was determined against Pseudomonas aeruginosa, and LPS-neutralizing capacity was determined via inhibition of LPS-induced tumor necrosis factor–α (TNF-α) secretion by RAW 264.7 cells. In vivo biologic activity was determined via pretreatment following which P aeruginosa endotoxemia or bacteremia was induced; serum TNF-α levels, bacterial clearance, and survival were assessed. Results: LALF and LALF #28–54 exhibited potent in vitro bactericidal and LPS-neutralizing activity comparable to PmB (P <.01). However, although LALF #28-54 diminished systemic TNF-α production and aided bacterial clearance similar to that observed for LALF (P <.01), it did not provide significant protective capacity (P >.1). Conclusions: These data demonstrate that peptide LALF #28-54 retained the LPS-neutralizing and bactericidal biologic activity of LALF but failed to protect during overwhelming P aeruginosa bacteremia, perhaps due to short serum half-life.(Surgery 2000;128:339-44.)

Rhamnolipid-induced removal of lipopolysaccharide from Pseudomonas aeruginosa: effect on cell surface properties and interaction with hydrophobic substrates.

Little is known about the interaction of biosurfactants with bacterial cells. Recent work in the area of biodegradation suggests that there are two mechanisms by which biosurfactants enhance the biodegradation of slightly soluble organic compounds. First, biosurfactants can solubilize hydrophobic compounds within micelle structures, effectively increasing the apparent aqueous solubility of the organic compound and its availability for uptake by a cell. Second, biosurfactants can cause the cell surface to become more hydrophobic, thereby increasing the association of the cell with the slightly soluble substrate. Since the second mechanism requires very low levels of added biosurfactant, it is the more intriguing of the two mechanisms from the perspective of enhancing the biodegradation process. This is because, in practical terms, addition of low levels of biosurfactants will be more cost-effective for bioremediation. To successfully optimize the use of biosurfactants in the bioremediation process, their effect on cell surfaces must be understood. We report here that rhamnolipid biosurfactant causes the cell surface of Pseudomonas spp. to become hydrophobic through release of lipopolysaccharide (LPS). In this study, two Pseudomonas aeruginosa strains were grown on glucose and hexadecane to investigate the chemical and structural changes that occur in the presence of a rhamnolipid biosurfactant. Results showed that rhamnolipids caused an overall loss in cellular fatty acid content. Loss of fatty acids was due to release of LPS from the outer membrane, as demonstrated by 2-keto-3-deoxyoctonic acid and sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis and further confirmed by scanning electron microscopy. The amount of LPS loss was found to be dependent on rhamnolipid concentration, but significant loss occurred even at concentrations less than the critical micelle concentration. We conclude that rhamnolipid-induced LPS release is the probable mechanism of enhanced cell surface hydrophobicity.

Effect of bacteria and antibiotic treatment on whole blood-mediated endothelial permeability in vitro

Abstract Background During Gram-negative sepsis bacterial endotoxin or lipopolysaccharide (LPS) may activate and damage endothelial cells resulting in systemic vascular leakage. Antibiotic treatment of Gram-negative bacteria results in significant release of LPS, inducing production of cytokines by leucocytes in vitro. The biological relevance of this finding is not well understood. A whole blood assay and cultured endothelial monolayers have been used to evaluate the effect of antibiotic treatment of bacteria on endothelial permeability. Methods Heat-inactivated (HI) Escherichia coli (ATCC 25922) was treated with either cefuroxime (CXM), imipenem–cilastin (IPM) or polymyxin B (poly B). The cultures were incubated with heparinized whole blood from healthy donors in a final concentration of 105 colony-forming units ml−1 and incubated at 37°C for 24 h. Plasma was then collected and added to human umbilical vein endothelial cell monolayers cultured on semipermeable Transwell COL membranes. The permeability of the monolayers was determined using fluorescein isothiocyanate-labelled (FITC) albumin and rhodamine-β-isothiocyanate-labelled (RITC) dextran. The production of interleukin (IL) 1β, IL-6, IL-1ra and tumour necrosis factor (TNF) α in whole blood was measured using radioimmunoassay. Results In whole blood, HI bacteria alone induced the production of IL-1β, IL-6, IL-1ra and TNF-α. CXM treatment of HI E. coli before incubation in blood induced significantly more IL-1β, TNF-α and IL-6 than the HI control, while poly B treatment induced less cytokine production. IPM did not significantly alter the production of cytokines. In the permeability assay, whole blood treated with HI bacteria alone induced a permeability increase of 2·3 times control monolayer permeability (P &amp;lt; 0·05). While CXM or IPM treatment resulted in a significant further enhancement of the permeability, there was no significant difference between these antibiotic treatments (permeability increase 2·9 and 2·8 times the control respectively). In contrast, treatment with poly B almost completely abrogated the permeability effect of HI bacteria (1·1 times control; P &amp;gt; 0·05). Conclusion Since neutralization of LPS by poly B reduces the E. coli-induced production of cytokines in whole blood and prevents increased permeability, free LPS may be the driving force of cytokine-mediated endothelial permeability. Treatment of bacteria with CXM or IPM induces a significantly different cytokine production in whole blood. However, in both cases the resulting plasma leads to a similar increase in permeability. Thus, the fact that individual antibiotics can release different amounts of LPS may be irrelevant to endothelial permeability.

Nitric oxide enhances PGI2production by human pulmonary artery smooth muscle cells

To evaluate the effect of exogenous nitric oxide (NO) and endogenous NO on the production of prostacyclin (PGI2) by cultured human pulmonary artery smooth muscle cells (HPASMC) treated with lipopolysaccharide (LPS), interleukin-1β(IL-1β), tumor necrosis factor α (TNFα) or interferon γ (IFNγ), HPASMC were treated with LPS and cytokines together with or without sodium nitroprusside (SNP), NO donor, NG-monomethyl-L-arginine (L-NMMA), NO synthetase inhibitor, and methylene blue (MeB), an inhibitor of the soluble guanylate cyclase. After incubation for 24 h, the postculture media were collected for the assay of nitrite by chemiluminescence method and the assay of PGI2by radioimmunoassay. The incubation of HPASMC with various concentrations of LPS, IL-1βor TNFαfor 24 h caused a significant increase in nitrite release and PGI2production. However, IFNγslightly increased the release of nitrite and had little effect on PGI2production. Although the incubation of these cells for 24 h with SNP did not cause a significant increase in PGI2production, the incubation of HPASMC with SNP and 10 μg/ml LPS, or with SNP and 100 U/ml IL-1βfurther increase PGI2production and this enhancement was closely related to the concentration of SNP. However, stimulatory effect of SNP on PGI2production was not found in TNFα- and IFNγ- treated HPASMC. Addition of L-NMMA to a medium containing LPS or IL-1βreduced nitrite release and attenuated the stimulatory effect of those agents on PGI2production. MeB significantly suppressed the production of PGI2by HPASMC treated with or without LPS or IL-1β. The addition of SNP partly reversed the inhibitory effect of MeB on PGI2production by HPASMC. These experimental results suggest that NO might stimulate PGI2production by HPASMC. Exogenous NO together with endogenous NO induced by LPS or cytokines from smooth muscle cells might synergetically enhance PGI2production by these cells, possibly in clinical disorders such as sepsis and acute respiratory distress syndrome.

Plasma Lipoproteins Promote the Release of Bacterial Lipopolysaccharide from the Monocyte Cell Surface

When bacterial lipopolysaccharide (LPS) enters the bloodstream, it is thought to have two general fates. If LPS binds to circulating leukocytes, it triggers innate host defense mechanisms and often elicits toxic reactions. If instead LPS binds to plasma lipoproteins, its bioactivity is largely neutralized. This study shows that lipoproteins can also take up LPS that has first bound to leukocytes. When monocytes were loaded with [(3)H]LPS and then incubated in plasma, they released over 70% of the cell-associated [(3)H]LPS into lipoproteins (predominantly high density lipoprotein), whereas in serum-free medium the [(3)H]LPS remained tightly associated with the cells. The transfer reaction could be reproduced in the presence of pure native lipoproteins or reconstituted high density lipoprotein. Plasma immunodepletion experiments and experiments using recombinant LPS transfer proteins revealed that soluble CD14 significantly enhances LPS release from the cells, high concentrations of LPS-binding protein have a modest effect, and phospholipid transfer protein is unable to facilitate LPS release. Essentially all of the LPS on the monocyte cell surface can be released. Lipoprotein-mediated LPS release was accompanied by a reduction in several cellular responses to the LPS, suggesting that the movement of LPS from leukocytes into lipoproteins may attenuate host responses to LPS in vivo.

Potentiation of lipopolysaccharide-induced IL-6 release by uridine triphosphate in macrophages: cross-interaction with cyclooxygenase-2-dependent prostaglandin E(2) production.

Our previous study has demonstrated the potentiation by uridine triphosphate (UTP) of nitric oxide (NO) and prostaglandin E(2) (PGE(2)) production in lipopolysaccharide (LPS)-stimulated murine J774 macrophages. In this study, we found that the amount of interleukin-6 (IL-6) release in response to LPS stimulation was greatly enhanced in the presence of UTP. This enhancement exhibited concentration dependence and occurred after 8 h of treatment with LPS. RT-PCR analysis indicated that the steady-state level of IL-6 mRNA induced by LPS was apparently increased upon co-addition of UTP. The potentiation by UTP was inhibited by the treatment with U73122 (a phosphatidylinositol-phospholipase C inhibitor), BAPTA/AM (an intracellular Ca(2+) chelator), KN-93 (a selective inhibitor of calmodulin-dependent protein kinase) or PDTC (a nuclear factor kappaB inhibitor). To understand the cross-regulation among NO, PGE(2) and IL-6, all of which are dramatically induced after LPS stimulation, the effects of L-NAME (a nitric oxide synthase inhibitor), indomethacin (a cyclooxygenase inhibitor), NS-398 (a cycloxygenase-2 inhibitor) and IL-6 antibody were tested. The results revealed the positive regulation between PGE(2) and IL-6 synthesis because NS-398 and indomethacin inhibited LPS plus UTP-induced IL-6 release, and IL-6 antibody attenuated LPS plus UTP-induced PGE(2) release. Taken together these results reinforce the role of UTP as a regulatory element in inflamed sites by demonstrating the capacity of this nucleotide to potentiate LPS-induced release of inflammatory mediators.

Potentiation of Lipopolysaccharide-Induced IL-6 Release by Uridine Triphosphate in Macrophages: Cross-Interaction with Cyclooxygenase-2-Dependent Prostaglandin E2 Production

Our previous study has demonstrated the potentiation by uridine triphosphate (UTP) of nitric oxide (NO) and prostaglandin E₂ (PGE₂) production in lipopolysaccharide (LPS)-stimulated murine J774 macrophages. In this study, we found that the amount of interleukin-6 (IL-6) release in response to LPS stimulation was greatly enhanced in the presence of UTP. This enhancement exhibited concentration dependence and occurred after 8 h of treatment with LPS. RT-PCR analysis indicated that the steady-state level of IL-6 mRNA induced by LPS was apparently increased upon co-addition of UTP. The potentiation by UTP was inhibited by the treatment with U73122 (a phosphatidylinositol-phospholipase C inhibitor), BAPTA/AM (an intracellular Ca²⁺ chelator), KN-93 (a selective inhibitor of calmodulin-dependent protein kinase) or PDTC (a nuclear factor κB inhibitor). To understand the cross-regulation among NO, PGE₂ and IL-6, all of which are dramatically induced after LPS stimulation, the effects of L-NAME (a nitric oxide synthase inhibitor), indomethacin (a cyclooxygenase inhibitor), NS-398 (a cycloxygenase-2 inhibitor) and IL-6 antibody were tested. The results revealed the positive regulation between PGE₂ and IL-6 synthesis because NS-398 and indomethacin inhibited LPS plus UTP-induced IL-6 release, and IL-6 antibody attenuated LPS plus UTP-induced PGE₂ release. Taken together these results reinforce the role of UTP as a regulatory element in inflamed sites by demonstrating the capacity of this nucleotide to potentiate LPS-induced release of inflammatory mediators.

EFFECTS OF DIESEL EXHAUST PARTICLES (DEP), CARBON BLACK, AND SILICA ON MACROPHAGE RESPONSES TO LIPOPOLYSACCHARIDE: EVIDENCE OF DEP SUPPRESSION OF MACROPHAGE ACTIVITY

The effects of diesel exhaust particle (DEP) exposure on alveolar macrophage (AM) response to ex vivo and in vivo lipopolysaccharide (LPS) challenge were determined by monitoring LPS-stimulated production of interleukin-1 (IL-1) and tumor necrosis factoralpha (TNF-alpha). The roles of the insoluble particulate and the organic compounds of DEP in altering pulmonary responses were evaluated by comparing the DEP-induced pulmonary responses to those of carbon black (CB), a carbonaceous particle with few adsorbed organic compounds, or to silica, a known pneumotoxic dust. Male SpragueDawley rats were exposed to a single intratracheal dose (5 or 35 mg/ kg body weight) of DEP, CB, or silica, or to saline vehicle. Rats were sacrificed 1, 3, or 7 d postexposure. To study the responsiveness to the bacterial product LPS, AM isolated from particleexposed rats were challenged ex vivo with LPS (0.1 mug/ 10 6 AM) and LPS-stimulated cytokine release was monitored. In addition, rats were exposed intratracheally to a single dose of DEP (5 mg/kg) and 3 d later exposed in vivo to 1 mg/kg LPS for 3 h prior to measurement of cytokine production by AM. DEP exposure resulted in neutrophil infiltration and elevated levels of albumin and lactate dehydrogenase (LDH) activity in the bronchoalveolar lavage fluid; these responses were not substantially different from those elicited by CB or silica exposure. AM from DEP-exposed rats showed increased spontaneous production of IL-1, but not TNF-alpha, while the opposite was true for CB or silica. Upon ex vivo challenge with LPS, AM from DEP-exposed rats showed a significant decrease in the secretion of TNF- and, to a lesser extent, IL-1, compared to the sum of the DEP and LPS effects. In contrast, AM from CB- or silica-exposed rats did not show this decreased responsiveness to subsequent LPS challenge. This inhibitory action of DEP on LPS-stimulated AM production of IL-1 and TNF- was further confirmed by the results obtained from rats exposed to both DEP and LPS in vivo. In summary, these results indicate that while DEP, CB, and silica all induce pulmonary inflammatory responses due to particle stimulation, only DEP suppress AM cytokine release in response to LPS stimulation. The contrasting cellular response with respect to DEP and CB exposures may be due to the presence of adsorbed organic compounds on DEP, which may contribute to the increased susceptibility of hosts to pulmonary infections after DEP exposure.