Lipopolysaccharide Neutralization Research Articles

Antimicrobial and Chemoattractant Activity, Lipopolysaccharide Neutralization, Cytotoxicity, and Inhibition by Serum of Analogs of Human Cathelicidin LL-37

Antimicrobial peptides have been evaluated in vitro and in vivo as alternatives to conventional antibiotics. Apart from being antimicrobial, the native human cathelicidin-derived peptide LL-37 (amino acids [aa] 104 to 140 of the human cathelicidin antimicrobial peptide) also binds and neutralizes bacterial lipopolysaccharide (LPS) and might therefore have beneficial effects in the treatment of septic shock. However, clinical trials have been hampered by indications of toxic effects of LL-37 on mammalian cells and evidence that its antimicrobial effects are inhibited by serum. For the present study, LL-37 was compared to two less hydrophobic fragments obtained by N-terminal truncation, named 106 (aa 106 to 140) and 110 (aa 110 to 140), and to a previously described more hydrophobic variant, the 18-mer LLKKK, concerning antimicrobial properties, lipopolysaccharide neutralization, toxicity against human erythrocytes and cultured vascular smooth muscle cells, chemotactic activity, and inhibition by serum. LL-37, fragments 106 and 110, and the 18-mer LLKKK inhibited the growth of Escherichia coli, Pseudomonas aeruginosa, Staphylococcus aureus, and Candida albicans in a radial diffusion assay, inhibited lipopolysaccharide-induced vascular nitric oxide production, and attracted neutrophil granulocytes similarly. While fragments 106 and 110 caused less hemolysis and DNA fragmentation in cultured cells than did LL-37, the 18-mer LLKKK induced severe hemolysis. The antibacterial effect of fragments 106 and 110 was not affected by serum, while the effect of LL-37 was reduced. We concluded that the removal of N-terminal hydrophobic amino acids from LL-37 decreases its cytotoxicity as well as its inhibition by serum without negatively affecting its antimicrobial or LPS-neutralizing action. Such LL-37-derived peptides may thus be beneficial for the treatment of patients with sepsis.

The screening and isolation of an effective anti-endotoxin monomer from Radix Paeoniae Rubra using affinity biosensor technology

Lipopolysaccharide (LPS) is a known trigger in the pathogenesis of sepsis, lipid A being the toxic component. One of several adjuvant therapeutic approaches for severe sepsis is currently focusing on the neutralization of LPS. In order to obtain the components from traditional Chinese herbs that can neutralize the endotoxin, aqueous extractions were tested using affinity biosensor technology. From amongst 42 herbs, eight were found to possess lipid A-binding abilities. Radix Paeoniae Rubras had the highest lipid A-binding ability; therefore an aqueous extraction from this plant was investigated further. After preparation using standard methods, including silica gel chromatography and HPLC, we obtained 1, 2, 3, 4, 6-β- d-pentagalloylglucose (PGG), with lipid A-binding ability. It was found that in vitro, PGG directly bound to lipid A, with a Kd of 32 μM, and that it neutralized the endotoxin both in the Limulus Amebocyte Lysate (LAL) assay and in a TNF-α release experiment, in a dose-dependent manner. In in vivo experiments, PGG was found to protect mice from a lethal challenge by LPS, and significantly decreased the plasma endotoxin level both in endotoxemic mice and rats, the reduction of the endotoxin level in rats being tightly associated with the TNF-α level. In conclusion, we demonstrate the effectiveness of affinity biosensor technology in discovering useful agents amongst traditional Chinese herbs and using this approach we found a new anti-endotoxin agent.

The role of tumour necrosis factor in the kinetics of lipopolysaccharide-mediated neutrophil priming in whole blood

Neutrophils can be primed by bacterial lipopolysaccharide (LPS) for an enhanced oxidative burst, which is a key element in the pathogenesis of Gram-negative sepsis. Some serum proteins (e.g. lipopolysaccharide-binding protein) avidly bind LPS and markedly enhance receptor binding and cellular activation while other serum factors (lipoproteins, bactericidal/permeability-increasing protein) neutralize LPS and prevent neutrophil activation. In this paper we examined the kinetics of this priming reaction in whole blood. To study the balance between neutrophil activation and LPS neutralization a sensitive chemiluminescence assay was used in a whole blood system. LPS was able to prime neutrophils for enhanced oxidative burst in whole blood with an optimum incubation time of 25 min. However, LPS was neutralized very rapidly with a t(1/2) of 10 min. After 20 min a second priming factor was already generated, which was shown to be monocyte-derived tumour necrosis factor (TNF).

Opposite relationship between circulating soluble CD14 concentration and endothelial function in diabetic and nondiabetic subjects

Recent prospective studies indicate endothelial dysfunction and increased risk for cardiovascular events in patients with serological evidence of multiple infections. Soluble CD14 (sCD 14) plays a key role in the neutralization of lipopolysaccharide (LPS), a well-established bacterial product inducing endothelial dysfunction. Insulin resistance was recently identified as a significant factor influencing circulating sCD 14 concentration. Thus, we investigated the association of circulating sCD14 and endothelial dysfunction in subjects with well-established insulin resistance (patients with type 2 diabetes, n = 40) compared to control non-diabetic subjects (n = 100). To further explore the underlying mechanisms, we also analysed C-reactive protein and circulating NO2-/NO3- and cyclic GMP in the diabetic group. Serum sCD 14 concentration (ELISA) was found to be differently associated with endothelium-dependent vasodilatation (EDVD, high-resolution ultrasound) in diabetic and non-diabetic subjects. In nondiabetic subjects, serum sCD14 and C-reactive protein correlated negatively with EDVD (r = -0.21, p = 0.03, and r = -0.21, p = 0.03, respectively). In a partial correlation analysis, these associations remained significant after controlling for age and weight (sCD 14 and EDVD, r = -0.23, p = 0.023; C-reactive protein and EDVD, r = -0.21, p = 0.03; sCD14 and C-reactive protein, r = 0.30, p = 0.002). In contrast, sCD 14 was positively associated with EDVD in type 2 diabetic patients (r = 0.37, p = 0.019,). Interestingly, sCD14 was also associated with NO2-/NO3- in this group (r = 0.62, p = 0.001, n = 22). EDVD also correlated with cyclic GMP (r = 0.47, p = 0.03, n = 22). In summary, circulating sCD 14 is associated with endothelial function. While in non-diabetic subjects sCD14 behaves as an acute phase reactant, its role in type 2 diabetic patients should be further clarified. These findings need to be confirmed in further studies with larger number of patients.

Polymyxin B: An ode to an old antidote for endotoxic shock

Endotoxic shock, a syndrome characterized by deranged hemodynamics, coagulation abnormalities, and multiple system organ failure is caused by the release into the circulation of lipopolysaccharide (LPS), the structurally diverse component of Gram-negative bacterial outer membranes, and is responsible for 60% mortality in humans. Polymyxin B (PMB), a cyclic, cationic peptide antibiotic, neutralizes endotoxin but induces severe side effects in the process. The potent endotoxin neutralizing ability of PMB, however, offers possibilities for designing non-toxic therapeutic agents for combating endotoxicosis. Amongst the numerous approaches for combating endotoxic shock, peptide mediated neutralization of LPS seems to be the most attractive one. The precise mode of binding of PMB to LPS and the structural features involved therein have been elucidated only recently using a variety of biophysical approaches. These suggest that efficient neutralization of endotoxin by PMB is not achieved by mere binding to LPS but requires its sequestration from the membrane. Incorporation of this feature into the design of endotoxin neutralizing peptides should lead to the development of effective antidotes for endotoxic shock.

Targeting of Scavenger Receptor Class B Type I by Synthetic Amphipathic α-Helical-containing Peptides Blocks Lipopolysaccharide (LPS) Uptake and LPS-induced Pro-inflammatory Cytokine Responses in THP-1 Monocyte Cells

Human scavenger receptor class B type I, CLA-1, mediates lipopolysaccharide (LPS) binding and internalization (Vishnyakova, T. G., Bocharov, A. V., Baranova, I. N., Chen, Z., Remaley, A. T., Csako, G., Eggerman, T. L., and Patterson, A. P. (2003) J. Biol. Chem. 278, 22771-22780). Because one of the recognition motifs in SR-B1 ligands is the anionic amphipathic alpha-helix, we analyzed the effects of model amphipathic alpha-helical-containing peptides on LPS uptake and LPS-stimulated cytokine production. The L-37pA model peptide, containing two class A amphipathic helices, bound with high affinity (K(d) = 0.94 microg/ml) to CLA-1-expressing HeLa cells with a 10-fold increased capacity when compared with mock transfected HeLa cells. Both LPS and L-37pA colocalized with anti-CLA-1 antibody and directly bound CLA-1 as determined by cross-linking. SR-BI/CLA-1 ligands such as HDL, apoA-I, and L-37pA efficiently competed against iodinated L-37pA. Bacterial LPS, lipoteichoic acid, and hsp60 also competed against iodinated L-37pA. Model peptides blocked uptake of iodinated LPS in both mock transfected and CLA-1-overexpressing HeLa cells. Bound and internalized Alexa-L-37pA and BODIPY-LPS colocalized at the cell surface and perinuclear compartment. Both ligands were predominantly transported to the Golgi complex, colocalizing with the Golgi markers bovine serum albumin-ceramide, anti-Golgin97 antibody, and cholera toxin subunit B. A 100-fold excess of L-37pA nearly eliminated BODIPY-LPS binding and internalization. L-37pA and its d-amino acid analogue, D-37pA peptide were similarly effective in blocking LPS, Gram-positive bacterial wall component lipoteichoic acid and bacterial heat shock protein Gro-EL-stimulated cytokine secretion in THP-1 cells. In the same culture media used for the cytokine stimulation study, neither L-37pA nor D-37pA affected the Limulus amebocyte lysate activity of LPS, indicating that LPS uptake and cytokine stimulation were blocked independently of LPS neutralization. These results demonstrate that amphipathic helices of exchangeable apolipoproteins may represent a general host defense mechanism against inflammation.

Cyclic antimicrobial peptides based on Limulus anti-lipopolysaccharide factor for neutralization of lipopolysaccharide

Bacterial endotoxin (lipopolysaccharide, LPS) is responsible for the septic shock syndrom. As potential therapeutic agents cyclic cationic antimicrobial peptides of different length, based on the Limulus anti-lipopolysaccharide factor (LALF), were synthesized, and their interaction with LPS was characterized physico-chemically and related to results in biological assays. All peptides inhibited the LPS-induced cytokine production in human mononuclear cells and the Limulus amebocyte lysate in a concentration-dependent way, with the peptide comprising the complete LPS-binding loop of the LALF (cLALF22) being the most effective. The peptides were neither cytotoxic nor hemolytic, except a slight effect of cLALF22. The peptides were able to displace Ca 2+ cations from a LPS monolayer, with cLALF22 being again most effective in accordance with results from isothermal titration calorimetry, in which saturation of binding was observed at an equimolar [cLALF22]:[LPS] ratio, and at a ratio 2–2.5 for the other peptides. For cLALF22, zeta ( ξ) potential experiments exhibited a complete compensation of the negative charges of LPS, whereas for the other peptides a residual negative potential of −20 to −40 mV was found. X-ray diffraction experiments showed that the mixed unilamellar/cubic inverted aggregate structure of the lipid A part of LPS was converted into a multilamellar one. The gel to liquid crystalline phase transition of the acyl chains of LPS was changed upon cLALF22 binding, leading to a clear fluidization, which was not observed or only to a lesser degree for the other peptides. The affinity of the peptides for LPS led to a reduced binding of lipopolysaccharide-binding protein (LBP) to target membranes and hence to an inhibition of cytokine induction in human mononuclear cells.

Human lipoproteins have divergent neutralizing effects on E. coli LPS, N. meningitidis LPS, and complete Gram-negative bacteria

The use of lipoproteins has been suggested as a treatment for Gram-negative sepsis because they inhibit lipopolysaccharide (LPS)-mediated cytokine production. However, little is known about the neutralizing effects of lipoproteins on cytokine production by meningococcal LPS or whole Gram-negative bacteria. We assessed the neutralizing effect of LDLs, HDLs, and VLDLs on LPS- or whole bacteria-induced cytokines in human mononuclear cells. A strong inhibition of Escherichia coli LPS-induced interleukin-1beta (IL-1beta), tumor necrosis factor-alpha, and IL-10 by LDL and HDL was seen, whereas VLDL had a less pronounced effect. In contrast, Neisseria meningitidis LPS, in similar concentrations, was neutralized much less effectively than E. coli LPS. Effective neutralization of meningococcal LPS required a longer interaction time, a lower concentration of LPS, or higher concentrations of lipoproteins. The difference in neutralization was independent of the saccharide tail, suggesting that the lipid A moiety accounted for the difference. Minimal neutralizing effects of the lipoproteins were observed on whole E. coli or N. meningitidis bacteria under all conditions tested. These results indicate that efficient neutralization of LPS depends on the type of LPS, but a sufficiently long interaction time, a low LPS concentration, or high lipoprotein concentration also inhibited cytokines by the less efficiently neutralized N. meningitidis LPS. Irrespective of these differences, whole bacteria showed no neutralization by lipoproteins.

Intercellular adhesion molecule-1 mediates cellular cross-talk between parenchymal and immune cells after lipopolysaccharide neutralization.

The mechanisms by which parenchymal cells interact with immune cells, particularly after removal of LPS, remain unknown. Lung explants from rats, mice deficient in the TNF gene, or human lung epithelial A549 cells were treated with LPS and washed, before naive alveolar macrophages, bone marrow monocytes, or PBMC, respectively, were added to the cultures. When the immune cells were cocultured with LPS-challenged explants or A549 cells, TNF production was greatly enhanced. This was not affected by neutralization of LPS with polymyxin B. The LPS-induced increase in the expression of ICAM-1 on A549 cells correlated with TNF production by PBMC. The cellular cross talk leading to the TNF response was blunted by an anti-ICAM-1 Ab and an ICAM-1 antisense oligonucleotide. In A549 cells, a persistent decrease in the concentration of intracellular cAMP was associated with colocalization of LPS into Toll-like receptor 4 and the Golgi apparatus, resulting in increased ICAM-1 expression. Inhibition of LPS internalization by cytochalasin D or treatment with dibutyryl cAMP attenuated ICAM-1 expression and TNF production by PBMC. In conclusion, lung epithelial cells are not bystanders, but possess memory of LPS through the expression of ICAM-1 that interacts with and activates leukocytes. This may provide an explanation for the failure of anti-LPS therapies in sepsis trials.

Acute inflammation and infection maintain circulating phospholipid levels and enhance lipopolysaccharide binding to plasma lipoproteins

Circulating lipoproteins are thought to play an important role in the detoxification of lipopolysaccharide (LPS) by binding the bioactive lipid A portion of LPS to the lipoprotein surface. It has been assumed that hypocholesterolemia contributes to inflammation during critical illness by impairing LPS neutralization. We tested whether critical illness impaired LPS binding to lipoproteins and found, to the contrary, that LPS binding was enhanced and that LPS binding to the lipoprotein classes correlated with their phospholipid content. Whereas low serum cholesterol was almost entirely due to the loss of esterified cholesterol (a lipoprotein core component), phospholipids (the major lipoprotein surface lipid) were maintained at near normal levels and were increased in a hypertriglyceridemic subset of septic patients. The levels of phospholipids found in the LDL and VLDL fractions varied inversely with those in the HDL fraction, and LPS bound predominantly to lipoproteins in the LDL and VLDL fractions when HDL levels were low. Lipoproteins isolated from the serum of septic patients neutralized the bioactivity of the LPS that had bound to them. Our results show that the host response to acute inflammation and infection tends to maintain lipoprotein phospholipid levels and that, despite hypocholesterolemia and reduced HDL levels, circulating lipoproteins maintain their ability to bind and neutralize an important bacterial agonist, LPS.

The bovine neutrophil: Structure and function in blood and milk

Migration of polymorphonuclear neutrophil leukocytes (PMN) into the mammary gland provide the first line of defense against invading mastitis pathogens. Bacteria release potent toxins that activate white blood cells and epithelial cells in the mammary gland to secrete cytokines that recruit PMN that function as phagocytes at the site of infection. While freshly migrated PMN are active phagocytes, continued exposure of PMN to inhibitory factors in milk such as fat globules and casein, leads to altered PMN morphology and reduced phagocytosis. In the course of phagocytosing and destroying invading pathogens, PMN release chemicals that not only kill the pathogens but that also cause injury to the delicate lining of the mammary gland. This will result in permanent scarring and reduced numbers of milk secretory cells. The life span of PMN is limited by the onset of apoptosis. To minimize damage to mammary tissue, PMN undergo a specialized process of programmed cell death known as apoptosis. Macrophages quickly engulf and phagocytose apoptotic PMN, thereby minimizing the release of PMN granular contents that are damaging to tissue. The PMN possess an array of cell surface receptors that allow them to adhere and migrate through endothelium and to recognize and phagocytose bacteria. One receptor found on phagocytes that is receiving considerable attention in the control of infections by Gram-negative bacteria is CD14. Binding of lipopolysaccharide (LPS) to membrane bound CD14 causes release of tumor necrosis factor-alpha and sepsis. Binding of LPS to soluble CD14 shed from CD14-bearing cells results in neutralization of LPS and rapid recruitment of PMN to the site of infection. Recent advances in the fields of genomics and proteomics should greatly enhance our understanding of the PMN role in controlling intramammary infections in ruminants. Further, manipulation of PMN, through either recombinant proteins such as soluble CD14 that enhance PMN response or agents that mediate PMN apoptosis, may serve as novel therapeutics for the treatment of mastitis.

Effects of an aqueous extract of cocoa on nitric oxide production of macrophages activated by lipopolysaccharide and interferon-γ

Objective Macrophages are the primary targets of bacterial lipopolysaccharide (LPS). The effects of cocoa extract on production of nitric oxide (NO) by murine J774.1 macrophages activated by LPS and interferon-γ (IFN-γ) were examined. Methods Cocoa was suspended in heated water and centrifuged, and the supernatant was then filtered. Nitrite was measured as a quantitative indicator of NO by spectrophotometry. LPS (1.0 mg/mL) and IFN-γ (100 U/mL) were added to cultured macrophages with 0.05% cocoa extract, 0.25% cocoa extract, or pure water. NO synthesis by macrophages was significantly inhibited by cocoa extract ( P < 0.01). Results The inhibitory effect increased with concentration of the extract ( P < 0.01). IFN-γ (100 U/mL) and, later, LPS (100 μg/mL) were added, together with 2.0% cocoa or pure water, to cultured macrophages. An inhibitory effect on NO production was observed on addition of only IFN-γ, but more significant effects were obtained with addition of LPS ( P < 0.01) and addition of both was most effective ( P < 0.01). Conclusions These data suggested that cocoa extract contains a suppressor of NO production in murine macrophages activated by LPS and IFN-γ. This effect does not appear to be caused merely by neutralization of LPS.

Anti-Inflammatory Role for Intracellular Dimeric Immunoglobulin A by Neutralization of Lipopolysaccharide in Epithelial Cells

Intestinal epithelial cells (IEC) play a central role in innate and acquired mucosal immunity. They ensure early signaling to trigger an inflammatory response against pathogens. Moreover, IEC mediate transcytosis of dimeric IgA (dIgA), through the polymeric-immunoglobulin receptor (pIgR), to provide secretory IgA, the major protective Ig in mucosal secretions. Using an in vitro model of polarized IEC, we describe an additional anti-inflammatory mechanism of dIgA-mediated protection against intracellular bacterial components involved in the proinflammatory activation of IEC. Specific dIgA colocalizes to lipopolysaccharide (LPS) in the apical recycling endosome compartment, preventing LPS-induced NF-κB translocation and subsequent proinflammatory response. Thus, intracellular neutralization by dIgA limits the acute local inflammation induced by proinflammatory pathogen-associated molecular patterns such as LPS.

CD14 Monocyte Receptor, Involved in the Inflammatory Cascade, and Insulin Sensitivity

Soluble CD14 (sCD14), detectable at high concentrations constitutively present in the circulation, plays a key role in the neutralization of lipopolysaccharide, one of the most potent biologic response modifiers currently recognized and involved in the regulation of the inflammatory cascade. We tested whether circulating sCD14 was linked to inflammatory parameters and to insulin resistance in apparently healthy subjects. Serum sCD14 concentration did not significantly correlate with body mass index (BMI), waist to hip ratio, systolic or diastolic blood pressure, serum glucose, fasting insulin, fasting insulin resistance index [homeostasis model assessment (HOMA)], or serum uric acid among 123 subjects. The association between sCD14 and fasting insulin (r = -0.19, P = 0.08) and between sCD14 and HOMA (r = -0.21, P = 0.06) tended toward statistical significance among men. Unexpectedly, sCD14 correlated positively with fasting triglycerides (TG) in all the subjects (r = 0.22, P = 0.014), and this association was most significant in men (r = 0.34, P = 0.002). After controlling for TG, the relationships between sCD14 levels and fasting insulin (r = -0.25, P = 0.029), HOMA (r = -0.28, P = 0.014), and uric acid (r = -0.30, P = 0.006) were significant in men. Among nonsmoking men (n = 44), sCD14 significantly correlated with waist diameter (r = -0.30, P = 0.03), diastolic blood pressure (r = -0.34, P = 0.022), and HOMA (r = -0.30, P = 0.03). In a multiple linear regression analysis, BMI (P < 0.00001), TG (P = 0.003), and sCD14 (P = 0.04) (but not age, sex, waist, or smoking status) independently contributed to 26% of HOMA variance. A polymorphism of the CD14 gene, a C-to-T transition at bp -159 from the major transcription start site, seems to play a significant role in regulating serum sCD14 levels. In a subsample of 33 healthy subjects, carriers of the T allele were similar (in age, sex, BMI, fat mass, waist to hip ratio, blood pressure, and fasting glucose and insulin levels) to C/C homozygotes. In the former, integrated area under the curve for serum glucose concentrations after an oral glucose tolerance test was significantly lower (P = 0.02), and insulin sensitivity (SI) index (minimal model analysis) significantly higher (P = 0.036), than in C/C homozygotes. Among 32 type 2 diabetic subjects, carriers of the T allele also showed a significantly higher SI index (P = 0.03) and had significantly lower C-reactive protein (P = 0.03) and lower circulating soluble intercellular adhesion molecule-1 concentrations (P = 0.01) than did C/C homozygotes. To our knowledge, this is the first study to suggest an effect of a genetic polymorphism on both SI (healthy subjects and type 2 diabetic patients) and endothelial dysfunction (sICAM-1 levels) in type 2 diabetes mellitus.

Effects of human cathelicidin antimicrobial peptide LL-37 on lipopolysaccharide-induced nitric oxide release from rat aorta in vitro.

Lipopolysaccharides (LPS), released by Gram-negative bacteria, cause vascular expression of inducible nitric oxide synthase (iNOS) leading to nitric oxide (NO) production and septic shock. Human cathelicidin antimicrobial peptide (LL-37) can bind and neutralize LPS. We wanted to study whether LL-37 affects LPS or interleukin-1beta (IL-1beta)-induced production, release and function of NO in intact rat aorta rings and cultured rat aorta smooth muscle cells. Isolated segments of thoracic aorta and cultured cells were incubated in the presence of LPS, LL-37, LPS + IL-37, IL-1beta, IL-1beta + IL-37 or in medium alone. Smooth muscle contraction in response to phenylephrine and accumulation of the sdegradation products of NO, nitrate and nitrite, were measured on aorta segments. Levels of iNOS were assessed by Western blot and cytotoxic effects were detected by measurement of DNA fragmentation in cultured cells. Number of viable cells were determined after Trypan blue treatment. Both LPS and IL-1beta reduced contractility in response to phenylephrine and increased NO production as well as iNOS expression. LL-37 inhibited the LPS depression of vascular contractility induced only by LPS. LL-37 reduced both the LPS- and IL-1beta-induced NO production and iNOS expression. LL-37 at high concentrations induced DNA fragmentation and decreased the number of living cells. IL-37 reduces NO production induced by LPS and IL-1beta. The reduction does not seem to result only from neutralization of LPS but also from a cytotoxic effect, possibly via induction of apoptosis.

Modulation of the hydrophobic domain of polymyxin B nonapeptide: effect on outer-membrane permeabilization and lipopolysaccharide neutralization.

Polymyxin B nonapeptide (PMBN), a cationic cyclic peptide derived from the antibacterial peptide polymyxin B, is capable of specifically increasing the permeability of the outer membrane (OM) of Gram-negative bacteria toward hydrophobic antibiotics. In this study, we evaluated the contribution of the hydrophobic segment of PMBN (i.e., D-Phe(5)-Leu(6)) to this activity. Accordingly, we synthesized four analogs of PMBN by replacing D-Phe(5) with either with D-Trp or D-Tyr and Leu(6) with Phe or Ala and evaluated their ability to bind cell-free lipopolysaccharide (LPS) and increase bacterial OM permeability. Compared with PMBN, [D-Tyr(5)]PMBN and [Ala(6)]PMBN possessed reduced LPS affinity (IC(50) = 2.5, 25, and 12 microM, respectively) and significantly reduced OM permeability and LPS neutralization activity. [Phe(6)]PMBN exhibited rather similar affinity to cell-free LPS (IC(50) = 5 microM) and the same OM permeability capacity as PMBN. However, [D-Trp(5)]PMBN, despite its similar affinity to cell-free LPS (IC(50) = 4 microM), had moderately reduced OM permeability capacity. These results demonstrate the significant role of the PMBN hydrophobic segment in promoting biological activity.

Identification of LPS-Binding Peptide Fragment of MD-2, a Toll-Receptor Accessory Protein

Members of the toll-like receptor family are crucial in recognition of microbial pathogens as part of innate immune response. MD-2, an accessory protein to TLR4, present on the extracellular side of the membrane is needed to initiate the signal transduction. We have identified a 15 amino acid region of human MD-2 that contains several features of other lipopolysaccharide (LPS) binding proteins and peptides. In vitro LPS neutralization by this peptide was observed and confirmed by 2D transferred NOESY NMR experiments. NMR experiments have also shown binding of the MD-2 peptide to lipoteichoic acid (LTA) but not to peptidoglycan. Furthermore this peptide inhibited growth of gram-negative and to a lower extent of some gram-positive bacteria. Our results indicate that this region of MD-2 might be responsible for binding of LPS and confirms the role of MD-2 as an accessory protein in LPS signaling bestowing the Toll receptors their specificity.

Adrenaline suppression of the macrophage nitric oxide response to lipopolysaccharide is associated with differential regulation of tumour necrosis factor-alpha and interleukin-10.

Adrenaline is a catecholamine hormone secreted by the adrenal medulla in response to acute stress. Previous studies have shown that adrenaline suppresses the nitric oxide (NO) response of murine macrophages (M phi s) stimulated in vitro with lipopolysaccharide (LPS). We have now extended these studies to examine the effects of adrenaline on the production of tumour necrosis factor alpha (TNF-alpha) and interleukin-10 (IL-10). Our results showed that NO, TNF-alpha and IL-10 were concurrently produced following in vitro LPS (10 micrograms/ml) stimulation of murine peritoneal M phi s. Adrenaline suppressed both NO and TNF-alpha with concomitant up-regulation of the IL-10 response above that seen with LPS alone. In this in vitro model of LPS stimulation we demonstrated that TNF-alpha was required for NO production, as the TNF-alpha neutralizing monoclonal antibody, TN3.19.12, abolished the response; in contrast, IL-10 suppressed NO. In order to determine any functional consequence of adrenaline-mediated IL-10 augmentation on NO production, M phi s were stimulated with LPS and specific neutralizing anti-IL-10 antibodies were added to the cultures. The LPS NO response was suppressed to 43% of the control value by adrenaline (10(-8) M) and an irrelevant control antibody had no effect on the adrenaline-mediated inhibition of NO, but anti-IL-10 treatment restored the NO response to levels similar to those observed with LPS alone. Furthermore, we demonstrated that exogenous TNF-alpha, at a dose range of 1.9-50 ng per ml, also restored the nitrite response to LPS in the presence of adrenaline. Together, the observations that neutralization of IL-10 and addition of TNF-alpha abrogate adrenaline's inhibition of NO, suggest that this hormone suppresses NO partly through up-regulation of IL-10 which, in turn, may suppress TNF-alpha that is required for NO production. Finally, we also observed that the M phi-activating cytokine, interferon-gamma (IFN-gamma), attenuated the inhibitory effect of adrenaline on the LPS NO response.

Dual role of lipopolysaccharide (LPS)-binding protein in neutralization of LPS and enhancement of LPS-induced activation of mononuclear cells.

The lipopolysaccharide (LPS)-binding protein (LBP) has a concentration-dependent dual role in the pathogenesis of gram-negative sepsis: low concentrations of LBP enhance the LPS-induced activation of mononuclear cells (MNC), whereas the acute-phase rise in LBP concentrations inhibits LPS-induced cellular stimulation. In stimulation experiments, we have found that LBP mediates the LPS-induced cytokine release from MNC even under serum-free conditions. In biophysical experiments we demonstrated that LBP binds and intercalates into lipid membranes, amplified by negative charges of the latter, and that intercalated LBP can mediate the CD14-independent intercalation of LPS into membranes in a lipid-specific and temperature-dependent manner. In contrast, prior complexation of LBP and LPS inhibited binding of these complexes to membranes due to different binding of LBP to LPS or phospholipids. This results in a neutralization of LPS and, therefore, to a reduced production of tumor necrosis factor by MNC. We propose that LBP is not only present as a soluble protein in the serum but may also be incorporated as a transmembrane protein in the cytoplasmic membrane of MNC and that the interaction of LPS with membrane-associated LBP may be an important step in LBP-mediated activation of MNC, whereas LBP-LPS complexation in the serum leads to a neutralization of LPS.

N-terminal modifications of Polymyxin B nonapeptide and their effect on antibacterial activity

Polymyxin B (PMB) is a potent antibacterial lipopeptide composed of a positively charged cyclic peptide ring and a fatty acid containing tail. Polymyxin B nonapeptide (PMBN), the deacylated amino derivative of polymyxin B, is much less bactericidal but able to permeabilize the outer membrane of Gram-negative bacteria and to neutralize the toxic effects of lipopolysaccharide (LPS). In this study, we synthesized and evaluated the antibacterial and LPS neutralizing activities of four PMBN analogs modified at their N-terminal. Our results suggest that oligoalanyl substitutions of PMBN do not effect most of PMBN activities. However, a hydrophobic aromatic substitution generated a PMB-like molecule with high antibacterial activity and significant reduced toxicity.