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.     

The antibiotic polymyxin B modulates P2X7 receptor function

The natural peptide polymyxin B (PMB) is a well-known and potent antibiotic that binds and neutralizes bacterial endotoxin (LPS), thus preventing its noxious effects among LPS-mediated endotoxin shock in animal models. We have investigated the effect of PMB on responses mediated by the P2X(7)R in HEK293 and K562 cells transfected with P2X(7) cDNA and in mouse and human macrophages. In addition, in view of the potential exploitation of P2X(7)-directed agonists in antitumor therapy, we also investigated the effect of PMB in B lymphocytes from patients affected by chronic lymphocytic leukemia. PMB, at an optimal concentration dependent on the given cell type, greatly potentiated the effect of nucleotide-mediated P2X(7) stimulation. In particular, ATP-mediated Ca(2+) influx, plasma membrane permeabilization, and cytotoxicity were enhanced to an extent that, in the presence of PMB, cells were killed by otherwise ineffective nucleotide concentrations. The synergistic effect due to the combined application of ATP and PMB was prevented by incubation with the irreversible P2X blocker oxidized ATP (oATP), but not with the reversible antagonist 1-(N,O-bis(1,5-isoquinolinesulfonyl)-N-methyl-l-tyrosyl)-4-phenilpiperazine (KN-62). Cells lacking P2X(7) were fully insensitive to the combined stimulation with PMB and ATP. Furthermore, PMB at the concentrations used had no untoward effects on cell viability. These results point to PMB as a useful tool for the modulation of P2X(7)R function and suggest that care should be used in the evaluation of ATP-stimulated immune cell responses in the presence of PMB as they may not solely be affected by removal of contaminating LPS.     

Thermodynamic analysis of the lipopolysaccharide-dependent resistance of gram-negative bacteria against polymyxin B

Cationic antimicrobial cationic peptides (CAMP) have been found in recent years to play a decisive role in hosts' defense against microbial infection. They have also been investigated as a new therapeutic tool, necessary in particular due to the increasing resistance of microbiological populations to antibiotics. The structural basis of the activity of CAMPs has only partly been elucidated and may comprise quite different mechanism at the site of the bacterial cell membranes or in their cytoplasm. Polymyxin B (PMB) is a CAMP which is effective in particular against Gram-negative bacteria and has been well studied with the aim to understand its interaction with the outer membrane or isolated membrane components such as lipopolysaccharide (LPS) and to define the mechanism by which the peptides kill bacteria or neutralize LPS. Since PMB resistance of bacteria is a long-known phenomenon and is attributed to structural changes in the LPS moiety of the respective bacteria, we have performed a thermodynamic and biophysical analysis to get insights into the mechanisms of various LPS/PMB interactions in comparison to LPS from sensitive strains. In isothermal titration calorimetric (ITC) experiments considerable differences of PMB binding to sensitive and resistant LPS were found. For sensitive LPS the endothermic enthalpy change in the gel phase of the hydrocarbon chains converts into an exothermic reaction in the liquid crystalline phase. In contrast, for resistant LPS the binding enthalpy change remains endothermic in both phases. As infrared data show, these differences can be explained by steric changes in the headgroup region of the respective LPS.     

Polymyxin B binds to anandamide and inhibits its cytotoxic effect

Anandamide (ANA), an endogenous cannabinoid, can be generated by activated macrophages during endotoxin shock and is thought to be a paracrine contributor to hypotension. We discovered that ANA in saline/ethanol solution and in serum was efficiently adsorbed in a polymyxin B (PMB)-immobilized beads column and eluted with ethanol. We confirmed the direct binding of PMB to ANA by using surface plasmon resonance. The adsorption of ANA by PMB may abolish the diverse effects of ANA such as hypotension, immunosuppression, and cytotoxicity, and may suggest a new therapeutic strategy for endotoxin shock.     

Characterizing the effect of polymyxin B antibiotics to lipopolysaccharide on Escherichia coli surface using atomic force microscopy

Lipopolysaccharide (LPS) on gram‐negative bacterial outer membranes is the first target for antimicrobial agents, due to their spatial proximity to outer environments of microorganisms. To develop antibacterial compounds with high specificity for LPS binding, the understanding of the molecular nature and their mode of recognition is of key importance. In this study, atomic force microscopy (AFM) and single molecular force spectroscopy were used to characterize the effects of antibiotic polymyxin B (PMB) to the bacterial membrane at the nanoscale. Isolated LPS layer and the intact bacterial membrane were examined with respect to morphological changes at different concentrations of PMB. Our results revealed that 3 hours of 10 μg/mL of PMB exposure caused the highest roughness changes on intact bacterial surfaces, arising from the direct binding of PMB to LPS on the bacterial membrane. Single molecular force spectroscopy was used to probe specific interaction forces between the isolated LPS layer and PMB coupled to the AFM tip. A short range interaction regime mediated by electrostatic forces was visible. Unbinding forces between isolated LPS and PMB were about 30 pN at a retraction velocity of 500 nm/s. We further investigated the effects of the polycationic peptide PMB on bacterial outer membranes and monitored its influences on the deterioration of the bacterial membrane structure. Polymyxin B binding led to rougher appearances and wrinkles on the outer membranes surface, which may finally lead to lethal membrane damage of bacteria. Our studies indicate the potential of AFM for applications in pathogen recognition and nano‐resolution approaches in microbiology.     

The functional association of polymyxin B with bacterial lipopolysaccharide is stereospecific: studies on polymyxin B nonapeptide

The Gram-negative bacterial endotoxin lipopolysaccharide (LPS) is a major inducer of sepsis. The natural cyclic peptide polymyxin B (PMB) is a potent antimicrobial agent, albeit highly toxic, by virtue of its capacity to neutralize the devastating effects of LPS. However, the exact mode of association between PMB and LPS is not clear. In this study, we have synthesized polymyxin B nonapeptide, the LPS-binding cyclic domain of PMB, and its enantiomeric analogue and studied several parameters related to their interaction with LPS and their capacity to sensitize Gram-negative bacteria toward hydrophobic antibiotics. The results suggest that whereas the binding of the two enantiomeric peptides to E. coli and to E. coli LPS is rather similar, functional association with the bacterial cell is stereospecific. Thus, the L-enantiomer is capable of synergism with the hydrophobic antimicrobial drugs novobiocin and erythromycin, whereas the D-enantiomer is devoid of such activity. The potential of understanding and consequently utilizing the PMB-LPS association for novel, nontoxic PMB-derived drugs is discussed.     

Polymyxins as Novel and Safe Mucosal Adjuvants to Induce Humoral Immune Responses in Mice

There is currently an urgent need to develop safe and effective adjuvants for enhancing vaccine-induced antigen-specific immune responses. We demonstrate here that intranasal immunization with clinically used polypeptide antibiotics, polymyxin B (PMB) and colistin (CL), along with ovalbumin (OVA), increases OVA-specific humoral immune responses in a dose-dependently manner at both mucosal and systemic compartments. Enhanced immunity by boosting was found to persist during 8 months of observation. Moreover, mice intranasally immunized with OVA plus various doses of PMB or CL showed neither inflammatory responses in the nasal cavity and olfactory bulbs nor renal damages, compared to those given OVA alone. These data suggest that polymyxins may serve as novel and safe mucosal adjuvants to induce humoral immune responses. The polymyxin adjuvanticity was found to be independent of endotoxins liberated by its bactericidal activity, as indicated by similar enhancing effects of PMB in lipopolysaccharide (LPS)-hyporesponsive and LPS-susceptible mice. However, despite the presence of preexisting anti-PMB antibodies, we observed no reduction in the adjuvant function of polymyxins when they were given intranasally. Furthermore, the titers of OVA-specific Abs in mice intranasally immunized with OVA plus PMB or CL were significantly higher than those in mice administered with polymyxin analogues, such as polymyxin B nonapeptide and colistin methanesulfonate. The levels of released β-hexosaminidase and histamine in mast cell culture supernatants stimulated by PMB or CL were also significantly higher than those stimulated by their analogues. These results suggest that both the hydrophobic carbon chain and hydrophilic cationic cyclic peptide contribute to the mucosal adjuvanticity of PMB and CL.     

LDL apheresis as an alternate method for plasma LPS purification in healthy volunteers and dyslipidemic and septic patients

Lipopolysaccharide (LPS) is a key player for innate immunity activation. It is therefore a prime target for sepsis treatment, as antibiotics are not sufficient to improve outcome during septic shock. An extracorporeal removal method by polymyxin (PMX) B direct hemoperfusion (PMX-DHP) is used in Japan, but recent trials failed to show a significant lowering of circulating LPS levels after PMX-DHP therapy. PMX-DHP has a direct effect on LPS molecules. However, LPS is not present in a free form in the circulation, as it is mainly carried by lipoproteins, including LDLs. Lipoproteins are critical for physiological LPS clearance, as LPSs are carried by LDLs to the liver for elimination. We hypothesized that LDL apheresis could be an alternate method for LPS removal. First, we demonstrated in vitro that LDL apheresis microbeads are almost as efficient as PMX beads to reduce LPS concentration in LPS-spiked human plasma, whereas it is not active in PBS. We found that PMX was also adsorbing lipoproteins, although less specifically. Then, we found that endogenous LPS of patients treated by LDL apheresis for familial hypercholesterolemia is also removed during their LDL apheresis sessions, with both electrostatic-based devices and filtration devices. Finally, LPS circulating in the plasma of septic shock and severe sepsis patients with gram-negative bacteremia was also removed in vitro by LDL adsorption. Overall, these results underline the importance of lipoproteins for LPS clearance, making them a prime target to study and treat endotoxemia-related conditions.     

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.     

A comparison of reactivating and therapeutic efficacy of the oxime K203 and its fluorinated analog (KR-22836) with currently available oximes (obidoxime,trimedoxime, HI-6) against tabun in rats and mice

The potency of newly developed bispyridinium compound K203 and its fluorinated analog KR-22836 in reactivating tabun-inhibited acetylcholinesterase and reducing tabun-induced lethal toxic effects was compared with commonly used oximes (obidoxime, trimedoxime, the oxime HI-6) using in vivo methods. Studies determining the percentage of reactivation of tabun-inhibited blood and tissue acetylcholinesterase in rats showed that the reactivating efficacy of K203 is higher than the reactivating efficacy of its fluorinated analog KR-22836 as well as currently available oximes studied. The therapeutic efficacy of the oxime K203 and its fluorinated analog corresponds to their potency to reactivate tabun-inhibited acetylcholinesterase. According to the results, the oxime K203 is more suitable than KR-22836 for the replacement of commonly used oximes for the antidotal treatment of acute tabun poisoning due to its relatively high potency to counteract the acute toxicity of tabun.     

The role of bactericidal/permeability-increasing protein as a natural inhibitor of bacterial endotoxin.

Systemic release of endotoxin (LPS) after Gram-negative infection initiates a cascade of host cytokines that are thought to be the direct cause of shock, multisystem organ failure, and death. Endogenous LPS-binding proteins may play a role in regulating LPS toxicity in vivo. The human neutrophil granule protein bactericidal/permeability-increasing protein (BPI) shares sequence homology and immunocrossreactivity with an acute phase lipopolysaccharide binding protein (LBP) which has been shown to bind to LPS and accelerate LPS activation of neutrophils and macrophages. Although structurally similar, LBP and BPI are apparently functionally antagonistic. We previously showed that BPI inhibits LPS-mediated neutrophil activation in vitro. Here we demonstrate that BPI binds to LPS near the lipid A domain, and formation of the LPS-BPI complex abrogates detrimental host responses to LPS. For example, BPI blocks LPS-stimulated TNF release in vitro and in vivo, and LPS complexed to BPI is not pyrogenic in rabbits. Results demonstrating that BPI is released by stimulated human neutrophils further support the idea that BPI functions extracellularly in vivo to neutralize endotoxin. Taken together, these data argue that BPI neutralizes the toxic effects of LPS in vivo, and that BPI may represent a new therapeutic approach to the treatment of endotoxic shock.     

Polymyxin B neutralizes bacteria-released endotoxin and improves the quality of boar sperm during liquid storage and cryopreservation

Gram negative bacteria are the predominant type detected in boar semen. Since these bacteria release lipopolysaccharide (LPS), and because polymyxin B (PMB) neutralizes LPS activity, the objective was to improve techniques for long-term storage of boar sperm by testing the beneficial effects of PMB. In the present study, LPS bound directly to the head region of sperm, decreased sperm motility, and induced sperm apoptosis. The addition of 100 μg/mL PMB suppressed LPS binding activity and blocked the negative effects of LPS on sperm quality. Additionally, when PMB treatment was combined with penicillin G (PenG), sperm motility was increased after 10 d of liquid storage or in frozen-thawed sperm (P<0.05). When the PMB- and PenG-treated sperm was used for artificial insemination, the conception rate was increased relative to that of artificial insemination with sperm treated by PenG alone in both liquid (62 vs. 81%) and cryopreserved forms (50 vs. 80%, P < 0.05). We concluded that PMB suppressed LPS-induced low sperm motility and apoptosis via the reduction of LPS binding to Toll-like receptors (TLRs). Thus, in order to enhance sperm quality for artificial insemination, a combined treatment with PMB and PenG immediately after ejaculation seemed appropriate to maintain sperm motility and function during both liquid storage and cryopreservation.     

Polymyxin B neutralizes bacteria-released endotoxin and improves the quality of boar sperm during liquid storage and cryopreservation

Gram negative bacteria are the predominant type detected in boar semen. Since these bacteria release lipopolysaccharide (LPS), and because polymyxin B (PMB) neutralizes LPS activity, the objective was to improve techniques for long-term storage of boar sperm by testing the beneficial effects of PMB. In the present study, LPS bound directly to the head region of sperm, decreased sperm motility, and induced sperm apoptosis. The addition of 100 μg/mL PMB suppressed LPS binding activity and blocked the negative effects of LPS on sperm quality. Additionally, when PMB treatment was combined with penicillin G (PenG), sperm motility was increased after 10 d of liquid storage or in frozen-thawed sperm (P<0.05). When the PMB- and PenG-treated sperm was used for artificial insemination, the conception rate was increased relative to that of artificial insemination with sperm treated by PenG alone in both liquid (62 vs. 81%) and cryopreserved forms (50 vs. 80%, P < 0.05). We concluded that PMB suppressed LPS-induced low sperm motility and apoptosis via the reduction of LPS binding to Toll-like receptors (TLRs). Thus, in order to enhance sperm quality for artificial insemination, a combined treatment with PMB and PenG immediately after ejaculation seemed appropriate to maintain sperm motility and function during both liquid storage and cryopreservation.     

Endotoxin downregulates peroxisome proliferator-activated receptor-gamma via the increase in TNF-alpha release

The nuclear receptor peroxisome proliferator-activated receptor-gamma (PPAR-gamma) is anti-inflammatory in a cell-based system and in animal models of endotoxemia. We have shown that PPAR-gamma gene expression is downregulated in macrophages after lipopolysaccharide (LPS) stimulation. However, it remains unknown whether hepatic PPAR-gamma is altered in sepsis and, if so, whether LPS directly downregulates PPAR-gamma. To study this, rats were subjected to sepsis by cecal ligation and puncture (CLP). Hepatic tissues were harvested at 5, 10, and 20 h after CLP. PPAR-gamma gene expression and protein levels were determined by RT-PCR and Western blot analysis, respectively. The results showed that PPAR-gamma gene expression decreased at 10 and 20 h and that its proteins levels were reduced at 20 h after CLP. PPAR-gamma levels were also decreased in animals that were administered LPS. To determine the direct effects of LPS on PPAR-gamma downregulation, LPS binding agent polymyxin B (PMB) was administered intramuscularly after CLP. The administration of PMB significantly reduced plasma levels of endotoxin, but it did not prevent the downregulation of PPAR-gamma expression. We found that circulating levels of TNF-alpha still remained significantly elevated in PMB-treated septic animals. We, therefore, hypothesize that the decrease of PPAR-gamma expression is TNF-alpha dependent. To investigate this, Kupffer cells (KCs) were isolated from normal rats and stimulated with LPS or TNF-alpha. TNF-alpha significantly attenuated PPAR-gamma gene expression in KCs. Although LPS decreased PPAR-gamma in KCs, the downregulatory effect of LPS was blocked by the addition of TNF-alpha-neutralizing antibodies. Furthermore, the administration of TNF-alpha-neutralizing antibodies to animals before the onset of sepsis prevented the downregulation of PPAR-gamma in sepsis. We, therefore, conclude that LPS downregulates PPAR-gamma expression during sepsis via an increase in TNF-alpha release.     

Polymyxin B-horseradish peroxidase conjugates as tools in endotoxin research.

The peptide antibiotic Polymyxin B (PMB) binds to bacterial endotoxin (lipopolysaccharide, LPS). We prepared covalent conjugates of PMB and horseradish peroxidase (HRP) by periodation of HRP-linked oligosaccharides followed by direct condensation with PMB. In addition we prepared monoclonal antibodies (Mabs) to PMB. The PMB-HRP conjugates and anti-PMB Mabs were used to study in ELISA the binding of PMB to LPS from Escherichia coli, Klebsiella pneumoniae, and Pseudomonas aeruginosa. In addition, PMB-HRP was used to quantify lipid A in ELISA, and to stain gram-negative bacteria histochemically. For the study of PMB-LPS interaction, PMB-HRP proved to be superior to the anti-PMB Mabs. PMB-HRP conjugates are useful general probes to detect or measure lipid A and LPS of various species using very simple methods and to stain bacteria, and they may obviate the need for many specific antisera. Thus, PMB-HRP conjugates are useful probes for endotoxin research.     

Prevention of endotoxin-induced lethality, but not of liver apoptosis in poly(ADP-ribose) polymerase-deficient mice.

Activation of poly-(ADP-ribose) polymerase (PARP) is often associated with cytotoxicity, but its precise role in shock-induced lethality and in different modes of tissue injury is still unknown. We took advantage of the existence of mice with a targeted deletion of the PARP gene (PARP-/-) to examine the differential sensitivity of wild-type (wt) and PARP-/- mice toward endotoxin (LPS)-induced lethality and different forms of liver damage. All PARP-/- animals survived high-dose (20 mg/kg) LPS-mediated shock, which killed 60% of wt animals. Moreover, LPS-induced necrotic liver damage was significantly reduced. In contrast, when apoptotic liver damage was induced via injection of low concentrations of LPS (30 microgram/kg) into D-galactosamine-sensitized mice, or via activation of hepatic cell death receptors, PARP-/- animals were not protected. We conclude that PARP is involved in systemic LPS toxicity, while it plays a minor role in apoptotic liver damage mediated by TNF or CD95.     

Lipopolysaccharide (LPS) labeled with Alexa 488 hydrazide as a novel probe for LPS binding studies

BACKGROUND: Lipopolysaccharide (LPS) comprises the outer cell wall of all gram-negative bacteria. It consists of an oligosaccharide core and lipid A. All LPS-induced biological responses are lipid A-dependent. Once released, LPS triggers a host systemic inflammatory response that leads to septic shock. Binding studies have helped to reveal some of the molecular interactions behind septic shock. Such studies have employed methods of labeling bacterial LPS with either radiochemicals or fluorescent dyes. Poor labeling of the LPS has resulted in the use of high concentrations of LPS in order to detect its binding. METHODS: In this study, we have devised a new methodology for labeling LPS, using hydrazide and galactose oxidase in order to oxidize galactose residues to aldehyde groups in the oligosaccharide core of the LPS. RESULTS: We have managed to generate a conjugate that is highly fluorescent (LPS-to-Alexa 488 labeling ratio of 1:5) and biologically active. CONCLUSIONS: For the first time, this probe has enabled us to detect LPS binding even at pg/ml concentrations. Using this methodology, any Alexa-hydrazide dye can be conjugated to LPS, providing us with novel probes for imaging studies.     

N-acetylcysteine and glutathione as inhibitors of tumor necrosis factor production.

TNF is a major mediator in the pathogenesis of endotoxic shock, and its inhibition has a protective effect in various animal models of sepsis or endotoxin (lipopolysaccharide, LPS) toxicity. LPS treatment also induces an oxidative damage mediated by increased production of reactive oxygen intermediates. N-Acetylcysteine (NAC) is an antioxidant and a precursor of the synthesis of glutathione (GSH) and was reported to protect against LPS toxicity and LPS-induced pulmonary edema. In this study we investigated the effect of NAC on TNF production and LPS lethality in mice. The results indicated that oral administration of NAC protects against LPS toxicity and inhibits the increase in serum TNF levels in LPS-treated mice. The inhibition was not confined to the released form of TNF, since NAC also inhibited LPS-induced spleen-associated TNF. On the other hand, the inhibitor of GSH synthesis, DL-buthionine-(SR)-sulfoximine (BSO), had the opposite effect of potentiating LPS-induced TNF production, and this was associated with a decrease in liver GSH levels. Repletion of liver GSH with NAC reversed this effect. NAC was also active in inhibiting TNF production and hepatotoxicity in mice treated with LPS in association with a sensitizing dose of Actinomycin D. These data indicate that GSH can be an endogenous modulator of TNF production in vivo. On the other hand, NAC pretreatment did not inhibit other effects of LPS, particularly induction of serum IL-6, spleen IL-1 alpha, and corticosterone, in the same experimental model, suggesting that the observed effect could be specific for TNF.     

Inhibition of the amino‐acid transporter LAT1 demonstrates anti‐neoplastic activity in medulloblastoma

Most cases of medulloblastoma (MB) occur in young children. While the overall survival rate can be relatively high, current treatments combining surgery, chemo‐ and radiotherapy are very destructive for patient development and quality of life. Moreover, aggressive forms and recurrences of MB cannot be controlled by classical therapies. Therefore, new therapeutic approaches yielding good efficacy and low toxicity for healthy tissues are required to improve patient outcome. Cancer cells sustain their proliferation by optimizing their nutrient uptake capacities. The L‐type amino acid transporter 1 (LAT1) is an essential amino acid carrier overexpressed in aggressive human cancers that was described as a potential therapeutic target. In this study, we investigated the therapeutic potential of JPH203, a LAT1‐specific pharmacological inhibitor, on two independent MB cell lines belonging to subgroups 3 (HD‐MB03) and Shh (DAOY). We show that while displaying low toxicity towards normal cerebral cells, JPH203 disrupts AA homeostasis, mTORC1 activity, proliferation and survival in MB cells. Moreover, we demonstrate that a long‐term treatment with JPH203 does not lead to resistance in MB cells. Therefore, this study suggests that targeting LAT1 with JPH203 is a promising therapeutic approach for MB treatment.     

Septic shock is associated with receptor for advanced glycation end products ligation of LPS

Septic shock is a severe systemic response to bacterial infection. Receptor for advanced glycation end products (RAGE) plays a role in immune reactions to recognize specific molecular patterns as pathogen recognition receptors. However, the interaction between LPS, the bioactive component of bacterial cell walls, and RAGE is unclear. In this study, we found direct LPS binding to RAGE by a surface plasmon resonance assay, a plate competition assay, and flow cytometry. LPS increased TNF-α secretion from peritoneal macrophages and an NF-κB promoter-driven luciferase activity through RAGE. Blood neutrophils and monocytes expressed RAGE, and TLR2 was counterregulated in RAGE(-/-) mice. After LPS injection, RAGE(+/+) mice showed a higher mortality, higher serum levels of IL-6, TNF-α, high mobility group box 1, and endothelin-1, and severe lung and liver pathologies compared with RAGE(-/-) mice without significant differences in plasma LPS level. Administration of soluble RAGE significantly reduced the LPS-induced cytokine release and tissue damage and improved the LPS-induced lethality even in RAGE(-/-) as well as RAGE(+/+) mice. The results thus suggest that RAGE can associate with LPS and that RAGE system can regulate inflammatory responses. Soluble RAGE would be a therapeutic tool for LPS-induced septic shock.