bluuebunny/crossref_metadata_2025 huggingface dataset:
Differential clearance and induction of host responses by various administered or released lipopolysaccharides
Authors: Hasunuma Ryoichi , Morita Hiroyuki , Tanaka Shigenori , Ryll Roland , Freudenberg Marina A. , Galanos Chris , Kumazawa Yoshio
Published: June 2007

The clearance and activity of different types of lipopolysaccharide (LPS) released during infection with Gram-negative bacteria were investigated. When highly purified preparations differing in their specific endotoxin activity were administered intravenously to mice, the clearance of rough (R)-form LPS preparations from Salmonella minnesota and Escherichia coli was much faster than that of a smooth (S)-form LPS preparation from Salmonella abortus equi, but slower than that of lipooligosaccharides (LOS) preparations from Bordetella pertussis and Helicobacter pylori. After intraperitoneal infection with 107 and 108 CFU E. coli O111:B4, relatively high levels of LPS were detected dose-dependently in the plasma of infected mice and persisted for a long time. In addition, plasma sCD14 levels in infected mice were higher than in LPS-administered mice. These results indicate that continuously higher levels of plasma LPS followed by stronger host responses occur during infection and suggest that these differences between LPS-administered and infected mice should be taken into consideration when analyzing host responses induced by LPS.
LBP, CD14, TLR4 and the murine innate immune response to a peritoneal Salmonella infection
Authors: Bernheiden Martin , Heinrich Jan-Michael , Minigo Gabriela , Schütt Christine , Stelter Felix , Freeman Mason , Golenbock Douglas , Jack Robert S.
Published: June 2007

In mice, defense against an intraperitoneal Salmonella infection depends on a vigorous innate immune response. Mutations which lead to an inadequate early response to the pathogen thus identify genes involved in innate immunity. The best studied host resistance factor, NRAMP-1, is an endosomal membrane protein whose loss leads to an inability of the animals to hold the infection in check. However, innate defense against Salmonella is not restricted to mechanisms which directly attack the pathogen within macrophages. Here we have examined the contribution of the LBP, CD14 and TLR4 gene products to innate defense against Salmonella. To this end, we have generated mice which carry a wild-type allele of NRAMP-1, but which are deficient for the LBP, CD14 or TLR4 genes. Loss of any of these genes leads to a susceptibility to Salmonella as dramatic as that seen in animals lacking functional NRAMP-1 protein. This indicates that LBP, CD14 and TLR4 are all critical elements required in the proper induction of this innate defense system.
Investigation into the interaction of recombinant human serum albumin with Re-lipopolysaccharide and lipid A
Authors: Jürgens Gudrun , Müller Mareike , Garidel Patrick , Koch Michel H.J. , Nakakubo Hiroshi , Blume Alfred , Brandenburg Klaus
Published: June 2007

The interaction of bacterial endotoxins, deep rough mutant lipopolysaccharide LPS Re and the `endotoxic principle' lipid A, with recombinant human serum albumin (rHSA) was investigated with a variety of physical techniques and biological assays. With Fourier-transform infrared spectroscopy and differential scanning calorimetry, the influence of albumin on the acyl chain melting behavior of the endotoxins was measured. Also, the effect on the functional groups of the endotoxins, in particular with respect to their orientation, was studied, including competition experiments with polymyxin B. Furthermore, the influence of endotoxin binding to rHSA on the protein's secondary structure was investigated. The results indicate a non-electrostatic binding with no change of the backbone orientation of LPS and only a slight change of the secondary structure of rHSA. Correspondingly, the amount of charge neutralization of the endotoxins due to rHSA measured by the electrophoretic mobility exhibited only a slight reduction of the surface potential. From these measurements and isothermal titration calorimetry, the lipid:protein binding stoichiometry was estimated to [LPS]:[rHSA], 10:1 molar. The determination of the aggregate structure of the endotoxins by X-ray small-angle scattering exhibited a complex change of a cubic into a non-lamellar structure. No influence of rHSA on endotoxin intercalation into phospholipid liposomes induced by lipopolysaccharide-binding protein could be detected by fluorescence resonance energy transfer. Finally, the LPS-induced cytokine production of human mononuclear cells was only slightly increased at high molar rHSA excess, while the coagulation of amebocyte lysate in the Limulus test yielded a complex change due to rHSA binding of LPS.
Review: Endotoxin in the environment — exposure and effects
Authors: Rylander Ragnar
Published: June 2007

This review deals with endotoxin in the environment and its relation to disease among exposed persons. Data are presented on levels of endotoxin in different environments with maximum values of several μg/m3. The cellular reactions of importance for inhalation exposure effects are attachment to lipopolysaccharide binding protein, CD14 cell surface protein and TLR-4 receptors. The internalisation of endotoxin in macrophages and endothelial cells results in local production of inflammatory cytokines with subsequent migration of inflammatory cells into the lung and the penetration of cytokines into the blood. These events orchestrate clinical effects in terms of toxic pneumonitis, airways' inflammation and systemic symptoms. Inhalation challenges with pure endotoxin and field studies confirm the relation between these effects and exposure to dusts containing endotoxin. It is possible that polymorphism in genes determining endotoxin reactivity, particularly TLR-4, influences the risk for disease after environmental exposures. Some data suggest that the inflammation caused by inhaled endotoxin may decrease the risk for atopic sensitisation among children and lung cancer among workers exposed to organic dust. Additional research is needed to clarify the role of other environmental agents that are present in connection with endotoxin, particularly (1→3)-β-D-glucan from mold cell walls.
Does high mobility group 1 protein function as a late mediator for LPS- or TNF-induced shock in galactosamine-sensitized mice?
Authors: Hasunuma Ryoichi , Maruyama Hiroko , Takimoto Hiroaki , Ryll Roland , Tanaka Shigenori , Kumazawa Yoshio
Published: June 2007

The role of high mobility group-1 protein (HMG-1) in LPS- and TNF-a-induced lethal shock in galactosamine (GalN)-sensitized mice was investigated. No detectable HMG-1 levels were observed by immunoblotting analysis in plasma from untreated or GalN-sensitized BALB/c mice 5 h after LPS injection, although significant levels of HMG-1 were detected in plasma 6 h after the challenge. All GalN-sensitized BALB/c but not BALB/lpsd mice succumbed by 6 h after LPS injection. When GalN-sensitized mice were injected with TNF-α, the presence of HMG-1 was seen at 5.5 h in plasma of BALB/c mice and at 6 h in BALB/lpsd mice, although almost all GalN-sensitized BALB/c mice died by 6 h after challenge. The time-dependent phenomenon correlated with elevated serum aspartate aminotransferase (AST) levels and the appearance of apoptotic cells in livers. Administration of pooled plasma, equivalent to approximately 200 μg recombinant murine HMG-1, taken from mice on the verge of near death, did not result in induction of lethal shock in GalN-sensitized mice. Taken together with the late appearance of HMG-1 in moribund mice, these data suggest that HMG-1 does not decisively contribute to lethality in the GalN sensitization model.
Structural and biological characterization of highly purified hepta-acyl lipid A present in the lipopolysaccharide of the Salmonella enterica sv. Minnesota Re deep rough mutant strain R595
Authors: Janusch Holger , Brecker Lothar , Lindner Buko , Alexander Christian , Gronow Sabine , Heine Holger , Ulmer Artur J. , Rietschel Ernst Th. , Zähringer Ulrich
Published: June 2007

One major component of the Salmonella enterica sv. Minnesota Re deep rough mutant (strain R595) lipopolysaccharide is hepta-acyl lipid A (LAhepta). In a recent publication [Tanamoto K-I, Azumi S. Salmonella-type heptaacylated lipid A is inactive and acts as an antagonist of lipopolysaccharide action on human line cells. J Immunol 2000; 164: 3149—3156] the corresponding synthetic hepta-acyl lipid A (compound 516) was reported to be agonistically inactive but to rather suppress pro-inflammatory activation by the endotoxichexa-acyl lipid A (LAhexa, compound 506) and S-form LPS from Escherichia coli in the human macrophage-like cell lines THP-1 and U937. These results, however, were in contrast to previous findings with human mononuclear cells (hMNC) isolated from peripheral blood, in which compound 516 was found to be an agonist, expressing low, but significant,cytokine-inducing activity as compared to LAhexa. We have investigated the structure of natural LA hepta from the S. enterica sv. Minnesota Re deep rough mutant strain (R595) by TLC immunoblot, MALDI-TOF mass spectrometry and NMR spectroscopy. Using these techniques, the structural identity between LAhepta and the synthetic compound 516 was confirmed. In corroboration of previous findings with studies employing compound 516, purified LA hepta was found to induce the production of TNF- , IL-1 and IL-6 in hMNC, thus displaying moderate agonistic activity. Furthermore, we showed that LAhepta agonistically activated nuclear translocation of NF- B in THP-1 cells, thus clearly ruling out the possibility that LAhepta is an antagonist and that its biological activity is influenced by the type of human myeloid cells used for testing endotoxicity(hMNC versus THP-1 cells).
Lipopolysaccharide—cell interaction and induced cellular activation in whole blood of septic patients
Authors: Salomao Reinaldo , Brunialti Milena K.C. , Kallás Esper G. , Martins Paulo S. , Rigato Otelo , Freudenberg Marina
Published: June 2007

We used biotinylatedLPS (LPSb) and flow cytometry to study LPS—monocyte interaction and LPS-induced cellular activation in whole blood from septic patients (SP). Expression of surface activation markers was evaluated on monocytes (HLA-DR) and T lymphocytes (CD69 and CD95), and intracellular TNF-α on monocytes. Saturating curve and kinetics of LPSb detection on monocytes were similar in SP and healthy volunteers (HV). LPSb bound to monocytes was detected after 5 min of incubation in both groups, with a more pronounced decay in SP. Monocytes from SP had a lower expression of HLA-DR as compared to HV, both constitutive and upon LPS stimulation. The proportion of monocytes producing TNF-α after LPS stimulus was higher in HV than SP (mean ± SD = 25.2 ± 14.2% and 2.2 ± 2.6%, respectively, P < 0.001). LPS-induced CD69 on T CD8+ and CD8— lymphocytes was similar for patients and controls. Expression of CD95 on T lymphocytes was higher in SP as compared to HV on T CD8+ cells (GMFI, mean ± SD = 22.3 ± 14.6 and 8.6 ± 5.0, respectively, P = 0.01) and CD8— cells (GMFI, mean ± SD = 28.3 ± 7.7 and 14 ± 4.3 respectively, P < 0.001). Thus, monocytes and lymphocytes seem to respond differently to LPS in septic patients. Monocyte hyporesponsiveness appears not to be related to a decreased binding capacity of LPS, but rather to an impaired signal transduction.
Direct hemoperfusion with polymyxin B-immobilized fiber improves shock and hypoxemia during endotoxemia in anesthetized sheep
Authors: Yamamoto Hiroshi , Koizumi Tomonobu , Kaneki Toshimichi , Fujimoto Keisaku , Kubo Keishi , Honda Takayuki
Published: June 2007

This study evaluates the effect of direct hemoperfusion (DHP) using polymyxin B-immobilized fibers (PMX-F) as an extracorporeal blood filter on systemic hypotension and lung injury during endotoxemia. Sheep were anesthetized, intubated, mechanically ventilated with 50% oxygen and connected to the DHP system between the right femoral artery and left jugular vein. Group 1 ( n = 6) sheep were infused with 10 μg/kg Escherichia coli endotoxin over a 30 min period. At the same time, sheep underwent DHP with PMX-F (Toraymyxin®: PMX-20R) for 2 h at a flow rate of 60 ml/h. Group 2 ( n = 6) sheep were infused with the same dose of endotoxin and treated with a sham column, in the same manner as those in group 1. DHP with PMX-F significantly improved and restored systemic pressure and arterial oxygen tension in group 1 sheep, although these values never returned to the baseline levels of group 2 sheep. Pulmonary hypertension and leukocytopenia were observed after endotoxin infusion in both groups, but there were no significant differences between these values. DHP with PMX-F significantly decreased the elevation of plasma nitric oxide products. The treatment with PMX-F improves shock and deteriorated oxygenation during endotoxemia, probably through the suppression of nitric oxide production.
Essential role of MD-2 in B-cell responses to lipopolysaccharide and Toll-like receptor 4 distribution
Authors: Miyake Kensuke , Nagai Yoshinori , Akashi Sachiko , Nagafuku Masakazu , Ogata Masato , Kosugi Atsushi
Published: June 2007

Toll-like receptor 4 (TLR4) mediates lipopolysaccharide (LPS) signaling in a variety of cell types. MD-2 is associated with the extracellular domain of TLR4 and augments TLR4-dependent LPS responses in vitro. Moreover, mice lacking MD-2 (MD-2— /—) do not respond to LPS, survive endotoxin shock, and are susceptible to Salmonella typhimurium infection. Here, we further show that B cells lacking MD-2 do not up-regulate CD23 in response to LPS. TLR4 predominantly resides in the Golgi apparatus without MD-2. MD-2 is essential for LPS responses in vivo.
Lipoprotein-bound LPS induces cytokine tolerance in hepatocytes
Authors: Harris Hobart W. , Kasravi F. Behzad
Published: June 2007

Bacterial endotoxin (LPS) elicits dramatic responses in the host including elevated plasma lipid levels due to the increased synthesis and secretion of triglyceride (TG)-rich lipoproteins by the liver. We postulate that this cytokine-induced hyperlipoproteinemia, clinically termed the `lipemia of sepsis', represents an innate, non-adaptive host immune response to infection. Data in support of this hypothesis include the capacity of TG-rich lipoproteins (VLDL and chylomicrons, CM) to bind and neutralize LPS. Herein, we present evidence that CM-bound LPS attenuates the hepatocellular response to pro-inflammatory cytokines. Primary rodent hepatocytes pretreated with CM—LPS complexes for 2 h demonstrated a near 70% reduction in cytokine-induced NO production as compared to non-pretreated control cells ( P ≥ 0.04). Whereas hepatocytes were maximally tolerant to cytokine stimulation 6 h after CM—LPS pretreatment, the cells spontaneously regained cytokine responsiveness within 40 h. The induction of cytokine tolerance in hepatocytes follows the internalization of CM—LPS complexes and is a process regulated by the LDL receptor. CM—LPS complexes failed to induce cytokine tolerance in hepatocytes wherein lipoprotein receptor activity was inhibited with high dose receptor associated protein (30 μg/ml). Similarly, CM-bound LPS did not induce tolerance in hepatocytes from ldlr—/— mice. Thus, the biochemical or genetic inhibition of LDL receptor activity effectively prevented the CM-mediated induction of the cytokine tolerant phenotype. In conclusion, the lipemia of sepsis likely represents a mechanism by which the host combats sporadic, non-life-threatening episodes of endotoxemia. Also, it may indicate a negative regulatory mechanism for the hepatic response to sepsis, serving to effectively down-regulate the acute phase response. A better understanding of how TG-rich lipoproteins modulate the host response to LPS could yield novel biological insights with important clinical implications, including the development of lipid-based therapies for bacterial infections.
Synergistic and antagonistic interactions between LPS and superantigens
Authors: Dalpke Alexander H. , Heeg Klaus
Published: June 2007

Superantigens trigger polyclonal activation of T lymphocytes with cytokine release that eventually may lead to lethal cytokine syndrome (toxic shock). In contrast, bacterial components that are recognized by Toll-like receptors ( e.g. LPS or CpG DNA) primarily target macrophages and dendritic cells. We have analyzed whether superantigens and TLR ligands interact with each other. We found that superantigens synergize with LPS in an IFN-γ-dependent pathway. More important, we found compelling evidence that superantigens prime the innate immune cell system to a subsequent challenge with endotoxin. This sensitization was critically dependent on T-cell derived IFN-γ. When we analyzed the underlying molecular mechanisms, we additionally found that TLR stimulation enhanced IFN-γ-mediated cellular responses. Moreover, TLR ligands induced proteins of the SOCS family thus shutting off IFN-γ-mediated cellular activation. Since IFN-γ is synthesized by T cells after superantigen triggering, these results show that superantigen and TLR pathways are interconnected and regulate each other. They further show that the outcome of this interaction may include activation as well as down-regulation of the respective response pattern.
Review: Towards antibacterial strategies: studies on the mechanisms of interaction between antibacterial peptides and model membranes
Authors: Wiese Andre , Gutsmann Thomas , Seydel Ulrich
Published: June 2007

Lipopolysaccharides (LPSs) play a dual role as inflammation-inducing and as membrane-forming molecules. The former role attracts significantly more attention from scientists, possibly because it is more closely related to sepsis and septic shock. This review aims to focus the reader's attention to the other role, the function of LPS as the major constituent of the outer layer of the outer membrane of Gram-negative bacteria, in particular those of enterobacterial strains. In this function, LPS is a necessary component of the cell envelope and guarantees survival of the bacterial organism. At the same time, it represents the first target for attacking molecules which may either be synthesized by the host's innate or adaptive immune system or administered to the human body. The interaction of these molecules with the outer membrane may not only directly cause the death of the bacterial organism, but may also lead to the release of LPS into the circulation. Here, we review membrane model systems and their application for the study of molecular mechanisms of interaction of peptides such as those of the human complement system, the bactericidal/permeability-increasing protein (BPI), cationic antibacterial peptide 18 kDa (CAP18) as an example of cathelicidins, defensins, and polymyxin B (PMB). Emphasis is on electrical measurements with a reconstitution system of the lipid matrix of the outer membrane which was established in the authors' laboratory as a planar asymmetric bilayer with one leaflet being composed solely of LPS and the other of the natural phospholipid mixture. The main conclusion, which can be drawn from these investigations, is that LPS and in general its negative charges are the dominant determinants for specific peptide—membrane interactions. However, the detailed mechanisms of interaction, which finally lead to bacterial killing, may involve further steps and differ for different antibacterial peptides.
C 2-ceramide inhibits LPS-induced nitric oxide production in RAW 264.7 macrophage cells through down-regulating the activation of Akt
Authors: Koide Naoki , Sugiyama Tsuyoshi , Mori Isamu , Mu Mya Mya , Yoshida Tomoaki , Yokochi Takashi
Published: June 2007

The effect of C2-ceramide, a membrane-permeable ceramide analogue, on nitric oxide (NO) production in lipopolysaccharide (LPS)-stimulated RAW 264.7 cells was studied. The non-toxic concentration of C2-ceramide inhibited LPS-induced NO production. It was due to the attenuated expression of the inducible type of NO synthase (iNOS). C2-ceramide did not influence the phosphorylation of a series of mitogen-activated protein (MAP) kinases in response to LPS. On the other hand, C2-ceramide down-regulated the phosphorylation of Akt in LPS-stimulated RAW 264.7 cells, followed by the impairment of nuclear factor (NF)-κB activation. Moreover, the Akt dominant-negative mutant inhibited LPS-induced NO production. C2-ceramide was suggested to inhibit LPS-induced NO production through down-regulating the activation of Akt.
Macrophage migration inhibitory factor (MIF) regulates host responses to endotoxin through modulation of Toll-like receptor 4 (TLR4)
Authors: Roger Thierry , Froidevaux Céline , Martin Christian , Calandra Thierry
Published: June 2007

The cytokine macrophage migration inhibitory factor (MIF) has emerged recently as an important mediator of inflammation and innate immunity. MIF is rapidly released by macrophages after stimulation with microbial products and pro-inflammatory cytokines and, in turn, stimulates the production of pro-inflammatory mediators by immune cells. Immunoneutralization of MIF or deletion of the Mif gene was shown to protect animals from lethal endotoxemia, staphylococcal toxic shock and septic shock in experimental models of bacterial peritonitis. To investigate the function of MIF in innate immunity, we studied the response of macrophages expressing reduced levels of MIF to microbial products. These cells were generated by transduction of an antisense MIF adenovirus or by stable transfection with an antisense MIF plasmid or were obtained from MIFknockout mice. MIF-deficient macrophages were shown to be hyporesponsive to stimulation with LPS and Gram-negative bacteria. The defect was associated with a down-regulation of Toll-like receptor 4 (TLR4), the signal transducing molecule of the LPS receptor complex. Immunoneutralization of extracellular MIF decreased TLR4 expression and responses of macrophages to LPS, indicating that MIF may exert autocrine effects. These findings identify an important role for MIF in innate immunity and provide a rationale for the development of anti-MIF strategy for the treatment of patients with Gram-negative septic shock.
Identification of meningococcal LPS as a major monocyte activator in IL-10 depleted shock plasmas and CSF by blocking the CD14-TLR4 receptor complex
Authors: Bjerre Anna , Brusletto Berit , Øvstebø Reidun , Joø Gun Britt , Kierulf Peter , Brandtzaeg Petter
Published: June 2007

We have examined the in vitro stimulatory effects of lipopolysaccharide (LPS)-containing samples (meningococcal shock plasma, n = 10; non-shock plasma, n = 10; cerebrospinal fluid (CSF), n = 7) before and after immunodepletion of interleukin (IL)-10 in a monocyte target assay. We also studied the stimulatory effects of plasma collected from 3 patients with lethal septicemia caused by Streptococcus pneumoniae without detectable LPS but with 100-fold increased levels of heat-shock protein 70 (HSP70). HSP70 may, like LPS, activate monocytes via the Toll-like receptor 4 (TLR4). The samples were analyzed for LPS, tumor necrosis factor (TNF)-α, IL-10 and HSP70; applied on human monocytes (purity > 95%) before and after IL-10 immunodepletion, in the absence or presence of CD14 blocking mAb (60bca) or the lipid A antagonist, Rhodobacter sphaeroides diphosphoryl lipid A (RsDPLA) which blocks TLR4. Monocyte activation was measured by increased TNF-α secretion and tissue factor (TF) up-regulation by monocyte procoagulant activity (PCA). There was a positive correlation between patientplasma LPS levels ( n = 10) and increases in TNF-α secretion by the monocytes after immunodepletion of IL-10 ( r = 0.82). Pretreatment of the monocytes with mAbCD14 or RsDPLA reduced TNF-α secretion to median 5% and 12%, respectively, of the levels before the receptor complex was blocked. The median levels of HSP70 were 543 ng/ml (range, 468—962 ng/ml) in pneumococcal shock plasma, 81.5 ng/ml (range, 41—331 ng/ml) in meningococcal shock plasma and 24 ng/ml (range, < 0.8—41 ng/ml) in meningococcal non-shock plasma. Pneumococcal septic shock plasmas with significantly higher levels of HSP70 ( P < 0.05) did not induce TNF-α secretion in the monocytes. The results strongly suggest that LPS in meningococcal shock plasma is the major activator of monocytes whereas HSP70 (in plasma concentrations up to 963 ng/ml) does not activate monocytes in this assay.
The role of membrane-bound LBP, endotoxin aggregates, and the MaxiK channel in LPS-induced cell activation
Authors: Müller M. , Scheel O. , Lindner B. , Gutsmann T. , Seydel U.
Published: June 2007

We have previously shown in patch-clamp experiments on excised outside-out cytoplasmic membrane patches from human macrophages that the activation of a high-conductance Ca2+ - and voltage-dependent potassium channel, the MaxiK channel, is an early step in LPS-induced transmembrane signal transduction in macrophages. MaxiK can be activated by agonistically active LPS, and activation can be completely inhibited by LPS antagonists ( e.g. synthetic compound 406) and by anti-CD14 antibodies. Furthermore, by inhibiting MaxiK with the specific MaxiK blocker paxilline, we could show that activation of MaxiK is essential for LPS-induced cytokine production. As shown by RT-PCR, blockade of MaxiK by paxilline also inhibits induction of the mRNA of TNF-α and IL-6. This observation together with the fact that all patch-clamp experiments were done on excised outside-out patches reveal that MaxiK activation is an early step in cell activation by endotoxins. Thus, since cells lacking TLR4 on their surface can also not be activated to produce cytokines, these data allow the conclusion that TLR4 and MaxiK are both essential for activation by LPS and may form a co-operative signaling complex. We have also shown that LBP not only exists as a soluble acute-phase serum protein, but is also incorporated as a transmembrane protein (mLBP) in the cytoplasmic membrane of MNC; in this configuration, it is obviously involved in the binding of endotoxin and its transfer to the transmembrane signaling proteins finally triggering cell activation. Complexation of soluble LBP and LPS in the serum prior to binding of LPS to mLBP, in contrast, leads to neutralization of LPS. Here, we provide evidence from fluorescence resonance energy transfer spectroscopy that endotoxin aggregates are intercalated into reconstituted membranes by mLBP. In addition, cell culture assays and patch-clamp experiments demonstrate that endotoxin activates macrophages and the MaxiK channel in the aggregated, but not in the monomeric, state at similar concentrations.
Mycobacterial lipoarabinomannan mediates physical interactions between TLR1 and TLR2 to induce signaling
Authors: Tapping Richard I. , Tobias Peter S.
Published: June 2007

Mycobacteria and their cell wall component lipoarabinomannan (LAM) have recently been established as agonists for TLR2. Our transfection studies with single and pairwise combinations of TLRs 1, 2, 6 and 10 reveal that only TLR1 and TLR2 together mediate strong activation of NF-KB-driven luciferase activity in response to LAM. Co-operative signaling by TLR1 and TLR2 is observed using either non-capped or mannose-capped LAM as a stimulus. Moreover, we have found that phosphatidylinositol mannosides, simple biosynthetic precursors of LAM, also activate cells through the combined actions of TLR1 and TLR2. Co-immunoprecipitation studies show that TLR1 and TLR2 are physically associated, independently of the presence of LAM. To address the mechanism of LAM-induced TLR activation we have used TLR fusion proteins in a protein fragment complementation assay. The results of this assay suggest that LAM alters the physical interaction between the intracellular signaling domains of TLR1 and TLR2. Together, these results identify LAM as an agonist for TLR1 and TLR2 and support the idea that LAM initiates transmembrane signaling by altering the physical association between TLR1 and TLR2.
The role of STAT1 in viral sensitization to LPS
Authors: Durbin Joan , Doughty Leslie , Nguyen Ken , Caligiuri Michael , Van Deusen Jeff , Biron Christine
Published: June 2007

The phenomenon of endotoxin sensitization by virus infection is well documented but not yet well understood. Infection by virtually any viral agent will quickly induce expression of type I interferons (IFN-α/β), and type II IFN-γ production will follow as NK cells and T cells are activated. It has been well established that type II IFN pretreatment can intensify the effects of endotoxin. We have recently demonstrated that type I IFN induction by lymphocytic choriomeningitis virus (LCMV) infection dramatically increases TNF-α production following LPS treatment, and that this sensitization by type I IFN is STAT1 dependent. Taken together these data suggest that the STAT1-mediated, MyD88-independent, arm of the LPS signaling pathway plays an important role in endotoxin toxicity, and that this pathway mediates a major component of virus-enhanced LPS sensitization.
Activated protein C signals through the thrombin receptor PAR1 in endothelial cells
Authors: Riewald Matthias , Petrovan Ramona J. , Donner Aaron , Ruf Wolfram
Published: June 2007

The anti-inflammatory effects of activated protein C (APC) have lead to its recent approval for the treatment of sepsis. Although the endothelial cell protein C receptor (EPCR) plays a crucial role in APC's protective roles in septicemia, the precise signaling mechanism of the protease APC remains unclear. In fibroblast overexpression systems, we find that APC activates protease activated receptors (PAR) 1 and 2 in an EPCR-dependent manner. Human endothelial cells (HUVECs) express PAR1, PAR2 and EPCR. Stimulation of HUVECs with either APC, or specific receptor activating peptides for PAR1 or PAR2, show that all three agonists induce a very similar set of early response genes as assessed by high density microarray analysis. Only the transcript for monocyte chemo-attractant protein-1 (MCP-1) was selectively induced by APC and the PAR1 agonist, but not by the PAR2 agonist. APC-mediated MAP kinase phosphorylation and gene induction were inhibited by cleavage blocking antibodies to PAR1, demonstrating that APC signals exclusively through PAR1 in endothelial cells. MCP-1 is protective in animal models of endotoxemia, suggesting that APC may prevent lethality in sepsis by inducing MCP-1 expression through EPCR-dependent activation of endothelial cell PAR1. These data demonstrate unexpected protective functions of the major thrombin receptor PAR1 in endothelial cells.
Immunopathogenesis of Onchocerca volvulus keratitis (river blindness): a novel role for TLR4 and endosymbiotic Wolbachia bacteria
Authors: Hise Amy G. , Gillette-Ferguson Illona , Pearlman Eric
Published: June 2007

Infection with the parasitic nematode Onchocerca volvulus is associated with inflammation of the skin and cornea that can lead to blindness. Corneal damage is thought to occur as a result of the host inflammatory responses to degenerating microfilariae in the eye. We have utilized a murine model of corneal inflammation (keratitis) to investigate the immune and inflammatory responses associated with river blindness. Soluble extracts of O. volvulus, a filarial species that contains the endosymbiont bacteria Wolbachia or Acanthocheilonema viteae (a nematode not naturally infected with the bacteria) were injected into mouse corneas. Inflammatory responses and corneal changes were measured. We demonstrated a major role for endosymbiont Wolbachia bacteria and Toll-like receptor 4 (TLR4) in the pathogenesis of ocular onchocerciasis.
Regulation of interactions of endotoxin with host cells
Authors: Gioannini Theresa L. , Teghanemt Athmane , Zarember Kol A. , Weiss Jerrold P.
Published: June 2007

Potent Toll-like receptor 4 (TLR4)-dependent cell activation by endotoxin requires lipopolysaccharide-binding protein (LBP) and CD14-dependent delivery of endotoxin to cells containing MD-2 and TLR4. We have used metabolically labeled [14C] meningococcal lipooligosaccharide (LOS), purified recombinant endotoxin-binding proteins, and cultured endothelial cells to better define protein: endotoxin intermediates key in cell activation in the absence of functional membrane (m) CD14. Protein:endotoxin complexes or aggregates ( agg) were purified by gel sieving and characterized by immunocapture and bio-assays. Cell activation closely correlated with LBP, albumin and soluble (s) CD14-dependent conversion of endotoxin agg (Mr ≥ 20 × 106) to monomeric (M ~55 × 10 3) endotoxin:sCD14 complexes. Ordered interaction of LBP (+ albumin) and sCD14 with rLOS agg was required for the efficient formation of a bioactive endotoxin:sCD14 complex and potent cell activation. Increasing the ratio of LBP/sCD14 or addition of bactericidal/permeability-increasing protein (BPI) reduced accumulation of endotoxin:sCD14 complexes and instead yielded aggregates of endotoxin (Mr ~1—20 × 106) containing LBP or BPI that were taken up by cells in a CD14- and TLR4-independent manner without inducing pro-inflammatory responses. These findings strongly suggest that host machinery linked to TLR4-dependent cellular activation or TLR4-independent cellular clearance of endotoxin selectively recognizes different protein:endotoxin complexes. At the outset of infection, the low concentrations of LBP present and absence of extracellular BPI favor formation of pro-inflammatory endotoxin:CD14 complexes. The mobilization of LBP and BPI that is triggered by inflammation directs endotoxin for clearance and hence resolution of endotoxin-triggered inflammation.
Neural inhibition of inflammation: the cholinergic anti-inflammatory pathway
Authors: Czura Christopher J. , Friedman Steven G. , Tracey Kevin J.
Published: June 2007

The innate immune system is activated by infection and injury to release pro-inflammatory cytokines, which activate macrophages and neutrophils and modulate specific cellular responses. The magnitude of the cytokine response is critical, because a deficient response may result in secondary infections, while an excessive response may be more injurious than the original insult. We recently described a neural pathway, termed the `cholinergic anti-inflammatory pathway', that reflexively monitors and adjusts the inflammatory response by inhibiting pro-inflammatory cytokine synthesis. Efferent signals in the vagus nerve provide a direct mechanism for neural regulation of the immune response that is rapid, localized, and integrated. Vagus nerve stimulation inhibits the release of TNF, HMGB1, and other cytokines, and protects against endotoxemia and ischemia-reperfusion injury. This newly identified physiological mechanism of maintaining immunological homeostasis suggests that novel therapeutics may effectively modulate inflammatory responses by activating the cholinergic anti-inflammatory pathway.
Structural basis for endotoxic and antagonistic activities: investigation with novel synthetic lipid A analogs
Authors: Kusumoto Shoichi , Fukase Koichi , Fukase Yoshiyuki , Kataoka Mikayo , Yoshizaki Hiroaki , Sato Kenjiro , Oikawa Masato , Suda Yasuo
Published: June 2007

Our early work using homogeneous synthetic preparations demonstrated the presence of a lipid A analog which antagonizes endotoxic activities of LPS and lipid A. The first example was a tetraacylated biosynthetic precursor, now known as precursor Ia or lipid IVa, that contains four 3-hydroxytetradecanoyl moieties linked to the bisphosphorylated disaccharide backbone common to the endotoxic hexa-acyl Escherichia coli lipid A. Various compounds with both endotoxic and antagonistic activities have subsequently been reported from either natural or synthetic sources, but little is known about the factors determining the type of the activities of the respective compounds. To approach this issue, we have synthesized a series of lipid A analogs with various numbers and chain lengths of acyl groups on the backbone. Some were prepared by the aid of a novel affinity separation procedure. The phosphate moieties were also synthetically replaced. Biological tests showed that at least three acyl groups are required for antagonistic activity but one or even both of the phosphates can be replaced with other acidic moieties without losing the activity. The effect of Kdo residues linked to lipid A is also briefly discussed. Molecular dynamics calculations reasonably explain possible conformations required for the biological activity.
Immunoneutralization of procalcitonin as therapy of sepsis
Authors: Becker K.L. , Nylén E.S. , Snider R.H. , Müller B. , White J.C.
Published: June 2007

Prior studies have demonstrated that the prohormone, procalcitonin (ProCT), and its component calcitonin precursors (CTpr) are increased in the serum of septic patients, correlate with the severity of the illness, and persist for relatively long periods of time. Animal studies in septic hamsters have revealed that the administration of ProCT is toxic and that immunoneutralization with IgG that is reactive to this molecule significantly improves survival. A large animal model of a very rapidly lethal polymicrobial sepsis has been developed in the pig in order to measure continuous physiological and metabolic parameters and also to compare the effects in this animal of an immunoneutralization, which is performed late in the course of the disease, to an identical, but early, therapy. Based upon the physiological and metabolic parameters, the late therapy, which was initiated during the fourth hour at a time when pigs were nearly moribund, was found to be as beneficial as early therapy. In both late and early therapy, the only animals to survive at the predetermined time of euthanasia were those which had received immunoneutralization therapy.
Review: D-Galactosamine lethality model: scope and limitations
Authors: Silverstein Richard
Published: June 2007

D-Galactosamine (D-galN) is well established as sensitizing mice and other animals to the lethal effects of TNF, specifically, and by several orders of magnitude. Protection by anti-TNF neutralizing antibody is complete, as is (metabolically-based) protection by uridine. Sensitization occurs regardless of the origin of the released TNF, whether it is released from macrophages and/or T-cells. The same is true for the challenging agent which leads to the release of TNF, whether it is endotoxin, a superantigen, lipoprotein, bacterial DNA, or bacteria, either killed or proliferating. Most studies have utilized endotoxin as the challenging agent, and more than 70 agents have been reported to confer protection against LPS and/or TNF challenge in the model. The model has provided new insight regarding modes of protection, including from dexamethasone, which protects against challenge from LPS but not from challenge by TNF. The D-galN lethality model has also been used to test for synergistic behavior between different bacterial components, and to test for lethality when only small amounts of the challenging agent are available (lipid A chemistry).
Review: Infection, fever, and exogenous and endogenous pyrogens: some concepts have changed
Authors: Dinarello Charles A.
Published: June 2007

For many years, it was thought that bacterial products caused fever via the intermediate production of a host-derived, fever-producing molecule, called endogenous pyrogen (EP). Bacterial products and other fever-producing substances were termed exogenous pyrogens. It was considered highly unlikely that exogenous pyrogens caused fever by acting directly on the hypothalamic thermoregulatory center since there were countless fever-producing microbial products, mostly large molecules, with no common physical structure. In vivo and in vitro, lipopolysaccharides (LPSs) and other microbial products induced EP, subsequently shown to be interleukin-1 (IL-1). The concept of the `endogenous pyrogen' cause of fever gained considerable support when pure, recombinant IL-1 produced fever in humans and in animals at subnanomolar concentrations. Subsequently, recombinant tumor necrosis factor-α (TNF-α), IL-6 and other cytokines were also shown to cause fever and EPs are now termed pyrogenic cytokines. However, the concept was challenged when specific blockade of either IL-1 or TNF activity did not diminish the febrile response to LPS, to other microbial products or to natural infections in animals and in humans. During infection, fever could occur independently of IL-1 or TNF activity. The cytokine-like property of Toll-like receptor (TLR) signal transduction provides an explanation by which any microbial product can cause fever by engaging its specific TLR on the vascular network supplying the thermoregulatory center in the anterior hypothalamus. Since fever induced by IL-1, TNF-α, IL-6 or TLR ligands requires cyclooxygenase-2, production of prostaglandin E2(PGE2) and activation of hypothalamic PGE2receptors provides a unifying mechanism for fever by endogenous and exogenous pyrogens. Thus, fever is the result of either cytokine receptor or TLR triggering; in autoimmune diseases, fever is mostly cytokine mediated whereas both cytokine and TLR account for fever during infection.
p38-MAPK inhibition and endotoxin induced tubular dysfunction in men
Authors: Zijlstra Jan G. , Tulleken Jaap E. , Ligtenberg Jack J.M. , de Boer Peter , van der Werf Tjip S.
Published: June 2007

Background: To evaluate the possibility of preventing endotoxin induced renal damage by p38-MAPK inhibition in a human model.

Design and Methods: Twenty-one healthy young male volunteers received 4 ng/kg Escherichia coli endotoxin as a single dose. Four groups of volunteers received an oral dose of placebo or 350, 700 or 1400 mg RWJ-67657, a p38-MAPK inhibitor, 20 min before endotoxin infusion. Urine samples were collected at set time intervals. The urinary excretion rate of β2-microglobulin and N-acetyl-β-D-glucosaminidase, as indicators of tubular dysfunction was determined.

Results: There was a significant increase of β2-microglobulin and N-acetyl-β-D-glucosaminidase urine excretion rate after endotoxin infusion in the placebo group. p38-MAPK inhibition prevented the increase of markers for tubulopathy.

Conclusions: Endotoxin infusion induces measurable tubular damage. Blocking the p38-MAPK may prevent this damage. The mechanism is unclear, but blocking TNF-α release is a possible explanation.
Cell activation by Toll-like receptors: role of LBP and CD14
Authors: Finberg Robert W. , Re Fabio , Popova Lana , Golenbock Douglas T. , Kurt-Jones Evelyn A.
Published: June 2007

Members of the Toll-like receptor (TLR) family have been shown to be important in the activation of cells by a variety of microbial ligands. TLRs are thought to mediate the `recognition event' that follows an encounter between a mammalian cell and a microbial agent. In the case of the response to bacterial lipopolysaccharide (LPS), it is clear that the ability of these cell surface proteins to initiate the events necessary for activation of cells to produce cytokines is dependent upon `accessory proteins' such as the pattern recognition protein CD14 and the lipopolysaccharide binding protein (LBP). While the role of these proteins in the LPS-specific response is defined, their role in other TLR responses has not been defined, but it is important in understanding these events and, potentially, in designing new therapeutic strategies. Here we report on the role of these proteins in the response to yeast zymosan. The requirements for this response (which unlike the response to LPS is a response to a particulate antigen) and the role of other serum proteins are defined.
Use of murine embryonic fibroblasts to define Toll-like receptor activation and specificity
Authors: Kurt-Jones Evelyn A. , Sandor Frantisek , Ortiz Yasdel , Bowen Glennice N. , Counter Stacy L. , Wang Timothy C. , Finberg Robert W.
Published: June 2007

Toll-like receptors (TLRs) are critically involved in the innate immune response to bacterial, viral and fungal pathogens. We have studied human peripheral blood mononuclear cells, murine embryonic fibroblasts (MEFs) and a panel of human cell lines, including HEK, HeLa, AGS, ECV304 and U373 cells, for expression of TLR-specific mRNAs and for TLR-ligand dependent cytokine secretion. Peripheral blood cells expressed multiple TLRs; however, many studies have shown that blood contains multiple, heterogeneous cell populations with distinct patterns of TLR expression. Cell lines had variable expression of TLRs, and in most cases lacked TLR2 and TLR8 expression and only weakly expressed mRNAs for TLR5, TLR7 and TLR9. In contrast, MEFs expressed high levels of mRNA for TLR1, TLR2, TLR3, TLR4, TLR5, TLR6, TLR7, TLR8 and TLR9. MEFs were highly responsive to TLR-ligand activation and secreted high levels of both IL-6 and MCP-1 in response to TLR ligands. MEFs from mice with targeted deletions of TLR2, TLR4 and MyD88 demonstrated profound defects in their IL-6 response to their specific ligands, consistent with studies of macrophages and tissues from adult knockout animals. MEF cultures are homogenous and amenable to biochemical analysis and should allow rigorous studies of the contribution of individual TLRs to the innate immune response.
CpG oligodeoxynucleotides stimulate IFN-γ-inducible protein-10 production in human B cells
Authors: Vollmer Jörg , Jurk Marion , Samulowitz Ulrike , Lipford Grayson , Forsbach Alexandra , Wüllner Meike , Tluk Sybille , Hartmann Hanna , Kritzler Andrea , Müller Christian , Schetter Christian , Krieg Arthur M.
Published: June 2007

Several classes of CpG oligodeoxynucleotides (ODNs) with different immune stimulatory profiles were recently identified: the A-, B- and C-classes. In this study, we investigated the CpG-dependent stimulation of IFN-γ-inducible protein 10 (IP-10 or CXCL10) in different human immune cell types. CpG ODNs induced IP-10 in monocytes, pDCs and in B cells. Purified B cells as well as RPMI 8226 cells responded to CpG stimulation by IP-10 production. Treatment with exogenous IFN-α2b sensitized PBMCs, purified B cells as well as RPMI 8226 cells to respond more efficiently to stimulation with CpG ODNs by IP-10 production. IP-10 signaling could be directly stimulated via TLR9 in CpG-unresponsive HEK293 cells transfected with human TLR9 and an IP-10 reporter construct. Therefore, CpG-mediated IP-10 production is stimulated through IFN-α in cells that express the IFN-α receptor, a second pathway for IP-10 induction exists in TLR9-expressing B cells and pDCs where IP-10 is stimulated directly upon CpG-mediated TLR9 signaling. Our data provide a better understanding of the mechanisms through which CpG ODNs induce efficient Th1 responses.
Purification and characterization of deacylated and/or palmitoylated lipid A species unique to Salmonella enterica serovar Typhimurium
Authors: Kawasaki Kiyoshi , Ernst Robert K. , Miller Samuel I.
Published: June 2007

The Salmonella enterica serovar Typhimurium virulence gene products PhoP/PhoQ sense host microenvironments to regulate the expression of a lipid A 3- O-deacylase, PagL, and a lipid A palmitoyltransferase, PagP. Therefore, deacylation and/or palmitoylation of lipid A could occur in Salmonellae adapted to host environments. The PhoP/PhoQ-regulated modification of lipid A alters host recognition and signaling, and may play an important role in host defense against Salmonellae infection. Here we report the purification and characterization of modified lipid A species. Deacylated lipid A, deacylated and palmitoylated lipid A, and palmitoylated lipid A species were generated in Escherichia coli cells heterologously expressing salmonellae PagL and/or PagP, and then purified by sequential thin-layer chromatography. The purified lipid A species showed m/z values that correspond to single lipid A species on mass spectrometry analysis. The modified lipid A species showed reduced ability to induce cellular signaling through Toll-like receptor 4, suggesting a specific function of the lipid A modifications in the pathogenesis of salmonellae infection.