SDZ MRL 953, A Novel Synthetic Lipid a Analogue, Induces Tolerance to the Lethal Effects of Endotoxin and Enhances Nonspecific Immunity

Despite the availability of potent antimicrobials, shock due to Gram-negative rod sepsis remains an important clinical concern in immunosuppressed patients. Current strategies for treating septic shock by removal of endotoxin (lipopolysaccharides [LPS]) or putative mediators have only been partially effective. An attractive alternative appears to be the induction of hyporesponsiveness to LPS by synthetic lipid A analogues. SDZ MRL 953, a novel prototype of lipid A analogues. was examined for its ability to induce early-phase tolerance against experimental endotoxemia in mice following single or multiple administrations 2, 24, or 72 h before challenge. The analogue protected mice against a lethal dose of LPS or infections in a time- and dose-dependent manner. Maximum effects were observed in animals pretreated with SDZ MRL 953 on three consecutive days before the LPS challenge or microbial inoculation. To examine the mechanisms involved, peritoneal macrophages from the tolerant mice were monitored ex v1vo for the release of tumour necrosis factor (TNF) and killing of an isolate of Escherichia coli. It was found that macrophages from endotoxin-tolerant mice produced only a fraction of the TNF released by cells from control groups in response to LPS. However, the killing of E coli by the macrophages was enhanced. In conclusion, SDZ MRL 953 may have a prophylactic potential in reducing the risk of endotoxic shock in traumatized or in myelosuppressed patients.

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Suppression of Murine Endotoxin Response by E5531, a Novel Synthetic Lipid A Antagonist

Antimicrobial Agents and Chemotherapy ◽

10.1128/aac.42.11.2824 ◽

1998 ◽

Vol 42 (11) ◽

pp. 2824-2829 ◽

Cited By ~ 22

Author(s):

Seiichi Kobayashi ◽

Tsutomu Kawata ◽

Akifumi Kimura ◽

Kaname Miyamoto ◽

Koichi Katayama ◽

...

Keyword(s):

Inflammatory Response ◽

Lipid A ◽

Cell Surface Receptor ◽

Dependent Manner ◽

Necrosis Factor Alpha ◽

In Vivo Models ◽

Lps Challenge ◽

Tnf Α

ABSTRACT As a consequence of blood-borne bacterial sepsis, endotoxin or lipopolysaccharide (LPS) from the cell walls of gram-negative bacteria can trigger an acute inflammatory response, leading to a series of pathological events and often resulting in death. To block this inflammatory response to endotoxin, a novel lipid A analogue, E5531, was designed and synthesized as an LPS antagonist, and its biological properties were examined in vitro and in vivo. In murine peritoneal macrophages, E5531 inhibited the release of tumor necrosis factor alpha (TNF-α) by Escherichia coli LPS with a 50% inhibitory concentration (IC50) of 2.2 nM, while E5531 elicited no significant increases in TNF-α on its own. In support of a mechanism consistent with antagonism of binding to a cell surface receptor for LPS, E5531 inhibited equilibrium binding of radioiodinated LPS ([125I]2-(r-azidosalicylamido)-1, 3′-dithiopropionate-LPS) to mouse macrophages with an IC50 of 0.50 μM. E5531 inhibited LPS-induced increases in TNF-α in vivo when it was coinjected with LPS into C57BL/6 mice primed with Mycobacterium bovis bacillus Calmette-Guérin (BCG). In this model, the efficacy of E5531 was inversely correlated to the LPS challenge dose, consistent with a competitive antagonist-like mechanism of action. Blockade of the inflammatory response by E5531 could further be demonstrated in other in vivo models: E5531 protected BCG-primed mice from LPS-induced lethality in a dose-dependent manner and suppressed LPS-induced hepatic injury in Propionibacterium acnes-primed or galactosamine-sensitized mice. These results argue that the novel synthetic lipid A analogue E5531 can antagonize the action of LPS in in vitro and suppress the pathological effects of LPS in vivo in mice.

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Rat Mannose-Binding Protein A Binds CD14

Infection and Immunity ◽

10.1128/iai.69.3.1587-1592.2001 ◽

2001 ◽

Vol 69 (3) ◽

pp. 1587-1592 ◽

Cited By ~ 23

Author(s):

Hirofumi Chiba ◽

Hitomi Sano ◽

Daisuke Iwaki ◽

Seiji Murakami ◽

Hiroaki Mitsuzawa ◽

...

Keyword(s):

Lipid A ◽

Binding Protein ◽

Protein A ◽

Cellular Responses ◽

Dependent Manner ◽

Soluble Cd14 ◽

Concentration Dependent Manner ◽

E Coli ◽

Mannose Binding Protein ◽

Mannose Binding

ABSTRACT Lipopolysaccharide (LPS) has been known to induce inflammation by interacting with CD14, which serves as a receptor for LPS. Mannose-binding protein (MBP) belongs to the collectin subgroup of the C-type lectin superfamily, along with surfactant proteins SP-A and SP-D. We have recently demonstrated that SP-A modulates LPS-induced cellular responses by interaction with CD14 (H. Sano, H. Sohma, T. Muta, S. Nomura, D. R. Voelker, and Y. Kuroki, J. Immunol. 163:387–395, 2000) and that SP-D also interacts with CD14 (H. Sano, H. Chiba, D. Iwaki, H. Sohma, D. R. Voelker, and Y. Kuroki, J. Biol. Chem. 275:22442–22451, 2000). In this study, we examined whether MBP, a collectin highly homologous to SP-A and SP-D, could bind CD14. Recombinant rat MBP-A bound recombinant human soluble CD14 in a concentration-dependent manner. Its binding was not inhibited in the presence of excess mannose or EDTA. MBP-A bound deglycosylated CD14 treated with N-glycosidase F, neuraminidase, and O-glycosidase, indicating that MBP-A interacts with the peptide portion of CD14. Since LPS was also a ligand for the collectins, we compared the characteristics of binding of MBP-A to LPS with those of binding to CD14. MBP-A bound to lipid A fromSalmonella enterica serovar Minnesota and rough LPS (S. enterica serovar Minnesota Re595 and Escherichia coli J5, Rc), but not to smooth LPS (E. coli O26:B6 and O111:B4). Unlike CD14 binding, EDTA and excess mannose attenuated the binding of MBP-A to rough LPS. From these results, we conclude that CD14 is a novel ligand for MBP-A and that MBP-A utilizes a different mechanism for CD14 recognition from that for LPS.

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The STK Receptor Tyrosine Kinase Regulates the Response of Primary Macrophages to LPS in a Ligand-Independent Manner.

Blood ◽

10.1182/blood.v104.11.3436.3436 ◽

2004 ◽

Vol 104 (11) ◽

pp. 3436-3436

Author(s):

Pamela Correll ◽

Qingping Liu Liu

Keyword(s):

Tyrosine Kinase ◽

Receptor Tyrosine Kinase ◽

Macrophage Activation ◽

Endotoxic Shock ◽

Peritoneal Macrophages ◽

Dependent Manner ◽

Wild Type ◽

Independent Manner ◽

Retroviral Transduction

Abstract We have shown previously that activation of the STK/RON receptor tyrosine kinase expressed on tissue resident macrophages, by it’s ligand macrophage stimulating protein (MSP), results in the inhibition of NFkB activation, inducible nitric oxide synthase (iNOS) expression and TNFa production, as well as the induction of arginase expression, suggesting a role for this receptor in the regulation of classical vs. alternative macrophage activation. Furthermore, mice with a targeted deletion in this receptor exhibit increased sensitivity to endotoxic shock and DTH responses. More recently, we have demonstrated that MSP stimulation of primary peritoneal macrophages inhibits the production of IL-12. In order to map the domains of STK responsible for the inhibition of classical macrophage activation by MSP, we generated mutant forms of the receptor and expressed wild-type and mutant receptors in primary bone marrow derived macrophages by retroviral transduction. Expression of wild-type STK in these primary cells resulted in the ligand-independent reduction in IL-12p40 production in response to LPS stimulation, which was further inhibited by MSP treatment. This is consistent with the lack of a requirement for MSP in regulating responses to endotoxin in vivo. Surprisingly, a kinase dead receptor, which fails to signal in 293T cells, was fully functional in this assay, suggesting that the kinase activity of the receptor is not required for the inhibition of IL-12p40 under these conditions. However, the docking site tyrosines in the c-terminal tail of the receptor are essential for the inhibition of IL-12p40 by STK, suggesting that STK may be phosphorylated by an another kinase in this system. STK/RON has been shown to associate both physically and functionally with a number of other cell-surface receptors including EpoR, IL-3R bc, EGFR, MET as well as a number of integrins and cadherins. We have shown previously that STK regulates the activity of the aMb2 integrin (CR3) in peritoneal macrophages in a PI3K, PKCz-dependent manner. Here we show that STK also physically associates with CR3, as well as CD14, in RAW264.7 cells in the absence of ligand. Both CR3 and CD14 are capable of directly binding to LPS. Thus, we speculate that STK may exist as part of a receptor complex in macrophages and that signalling through STK might be induced directly by LPS. This would provide a means by which STK could temper the response of tissue-resident macrophages to LPS thereby preventing damage to host tissues.

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Roquefort Cheese Proteins InhibitChlamydia pneumoniaePropagation and LPS-Induced Leukocyte Migration

The Scientific World JOURNAL ◽

10.1155/2013/140591 ◽

2013 ◽

Vol 2013 ◽

pp. 1-6 ◽

Cited By ~ 3

Author(s):

Ivan M. Petyaev ◽

Naylia A. Zigangirova ◽

Natalie V. Kobets ◽

Valery Tsibezov ◽

Lydia N. Kapotina ◽

...

Keyword(s):

Peritoneal Macrophages ◽

Relative Increase ◽

Dependent Manner ◽

Key Factors ◽

Protein Extract ◽

E Coli ◽

Clinical Complications ◽

Peritoneal Leukocytes ◽

Low Prevalence ◽

Dose Dependent

Inflammation in atherosclerosis, which could be associated with some subclinical infections such asC. pneumoniae, is one of the key factors responsible for the development of clinical complications of this disease. We report that a proprietary protein extract isolated from Roquefort cheese inhibits the propagation ofC. pneumoniaein a human HL cell line in a dose-dependent manner, as revealed by the immunofluorescence analysis. These changes were accompanied by a significant reduction in the infective progeny formation over the protein extract range of 0.12–0.5 μg/mL. Moreover, short term feeding of mice with Roquefort cheese (twice, 10 mg per mouse with an interval of 24 hours) led to the inhibition of the migration of peritoneal leukocytes caused by intraperitoneal injection ofE. colilipopolysaccharide. These changes were complemented by a reduction in neutrophil count and a relative increase in peritoneal macrophages, suggesting that ingestion of Roquefort could promote regenerative processes at the site of inflammation. The ability of this protein to inhibit propagation ofChlamydiainfection, as well as the anti-inflammatory and proregenerative effects of Roquefort itself, may contribute to the low prevalence of cardiovascular mortality in France where consumption of fungal fermented cheeses is the highest in the world.

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Myeloperoxidase and Eosinophil Peroxidase Inhibit Endotoxin Activity and Increase Mouse Survival in a Lipopolysaccharide Lethal Dose 90% Model

Journal of Immunology Research ◽

10.1155/2019/4783018 ◽

2019 ◽

Vol 2019 ◽

pp. 1-10

Author(s):

Robert C. Allen ◽

Mary L. Henery ◽

John C. Allen ◽

Roger J. Hawks ◽

Jackson T. Stephens

Keyword(s):

Lipid A ◽

Lethal Dose ◽

High Dose ◽

Dependent Manner ◽

Eosinophil Peroxidase ◽

Endotoxin Activity ◽

Fixed Quantity ◽

Lal Assay

Myeloperoxidase (MPO) and eosinophil peroxidase (EPO) are cationic haloperoxidases with potent microbicidal and detoxifying activities. MPO selectively binds to and kills some Gram-positive bacteria (GPB) and all Gram-negative bacteria (GNB) tested. GNB contain endotoxin, i.e., lipopolysaccharide (LPS) comprising a toxic lipid A component. The possibility that MPO and EPO bind and inhibit the endotoxin of GNB was tested by mixing MPO or EPO with LPS or lipid A and measuring for inhibition of endotoxin activity using the chromogenic Limulus amebocyte lysate (LAL) assay. The endotoxin-inhibiting activities of MPO and EPO were also tested in vivo using an LPS 90% lethal dose (LD90) mouse model studied over a five-day period. Mixing MPO or EPO with a fixed quantity of LPS from Escherichia coli O55:B5 or with diphosphoryl lipid A from E. coli F583 inhibited LAL endotoxin activity in proportion to the natural log of the MPO or EPO concentration. MPO and EPO enzymatic activities were not required for inhibition, and MPO haloperoxidase action did not increase endotoxin inhibition. Both MPO and EPO increased mouse survival in the LPS LD90 model. In conclusion, MPO and EPO nonenzymatically inhibited in vitro endotoxin activity using the LAL assay, and MPO and high-dose EPO significantly increased mouse survival in a LPS LD90 model, and such survival was increased in a dose-dependent manner.

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Evidence for the involvement of interleukin 10 in the differential deactivation of murine peritoneal macrophages by prostaglandin E2.

Journal of Experimental Medicine ◽

10.1084/jem.180.6.2365 ◽

1994 ◽

Vol 180 (6) ◽

pp. 2365-2370 ◽

Cited By ~ 257

Author(s):

G Strassmann ◽

V Patil-Koota ◽

F Finkelman ◽

M Fong ◽

T Kambayashi

Keyword(s):

Transforming Growth Factor Beta ◽

Transforming Growth Factor ◽

Inhibitory Effect ◽

Peritoneal Macrophages ◽

Mononuclear Phagocytes ◽

Tnf Alpha ◽

Dependent Manner ◽

Lps Challenge ◽

Murine Peritoneal Macrophages

Among other effects, prostaglandins (PG) of the E series are known to inhibit several acute and chronic inflammatory conditions in vivo and proinflammatory cytokine production by activated macrophages in culture. The research presented here demonstrates that the inhibitory effect of PGE2 on tumor necrosis factor alpha (TNF-alpha) and interleukin 6 (IL-6) production by lipopolysaccharide (LPS)-stimulated murine peritoneal macrophages involves IL-10. In a dose-dependent manner, PGE2 inhibits LPS-induced release of TNF-alpha and IL-6, but not of lactate or nitric oxide. The decrease in the level of these cytokines is inversely proportional to the increase in immunoreactive IL-10. This differential inhibitory effect of PGE2 is mimicked by agents that elevate intracellular levels of cAMP, but not cGMP. Neutralizing anti IL-10 antibody but not neutralizing antibodies against other macrophage secretory products (IL-6, leukemia inhibitory factor, and transforming growth factor beta [TGF-beta]), significantly reverse the potent inhibitory effect of PGE2. In vivo, the administration of PGE2 before LPS challenge significantly reduces circulating TNF-alpha and IL-6 levels. Anti-IL-10 antibody substantially enhanced the LPS-induced TNF-alpha and IL-6 levels in mice that received either LPS alone or LPS plus PGE2. These results suggest that the anti-inflammatory effect of PGE2 on mononuclear phagocytes is mediated in part by an autocrine feedback mechanism involving IL-10.

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Lipid A-based affinity biosensor for screening anti-sepsis components from herbs

Bioscience Reports ◽

10.1042/bsr20130103 ◽

2014 ◽

Vol 34 (3) ◽

Cited By ~ 1

Author(s):

Jie Yao ◽

Yiguo Chen ◽

Ning Wang ◽

Dongneng Jiang ◽

Jiang Zheng

Keyword(s):

Lipid A ◽

Biological Activities ◽

Lethal Dose ◽

Chinese Herbs ◽

Dependent Manner ◽

Paeonia Suffruticosa ◽

Effective Measure ◽

Factor Α

LPS (lipopolysaccharide), an outer membrane component of Gram-negative bacteria, plays an important role in the pathogenesis of sepsis and lipid A is known to be essential for its toxicity. Therefore it could be an effective measure to prevent sepsis by neutralizing or destroying LPS. Numerous studies have indicated that many traditional Chinese medicines are natural antagonists of LPS in vitro and in vivo. The goal of this study is to develop a rapid method to screen anti-sepsis components from Chinese herbs by use of a direct lipid A-based affinity biosensor technology based on a resonant mirror. The detergent OG (n-octyl β-D-glucopyranoside) was immobilized on a planar non-derivatized cuvette which provided an alternative surface to bind the terminal hydrophilic group of lipid A. A total of 78 herbs were screened based on the affinity biosensor with a target of lipid A. The aqueous extract of PSA (Paeonia suffruticosa Andr) was found to possess the highest capability of binding lipid A. Therefore an aqueous extraction from this plant was investigated further by our affinity biosensor, polyamide chromatography and IEC–HPLC. Finally, we obtained a component (PSA-I-3) from Paeonia suffruticosa Andr that was evaluated with the affinity biosensor. We also studied the biological activities of PSA-I-3 against sepsis in vitro and in vivo to further confirm the component we screened with the biosensor. In vitro, we found that PSA-I-3 could decrease TNFα (tumour necrosis factor α) release from RAW264.7 cells induced by LPS in a dose-dependent manner. In vivo, it increased remarkably the survival of KM (KunMing) mice by challenging both lethal-dose LPS and heat-killed Escherichia coli compared with control groups. Our results suggest that the constructed affinity biosensor can successfully screen the anti-sepsis component from Chinese herbs.

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Pretreatment of Mice with Clindamycin Improves Survival of Endotoxic Shock by Modulating the Release of Inflammatory Cytokines

Antimicrobial Agents and Chemotherapy ◽

10.1128/aac.45.9.2638-2642.2001 ◽

2001 ◽

Vol 45 (9) ◽

pp. 2638-2642 ◽

Cited By ~ 29

Author(s):

Norio Hirata ◽

Kazufumi Hiramatsu ◽

Kenji Kishi ◽

Tohru Yamasaki ◽

Tomoku Ichimiya ◽

...

Keyword(s):

Inflammatory Cytokines ◽

Interleukin 1 ◽

Endotoxic Shock ◽

Dependent Manner ◽

Necrosis Factor Alpha ◽

Inflammatory Cytokine Production ◽

E Coli ◽

Factor Alpha ◽

Dose Dependent ◽

Necrosis Factor

ABSTRACT Suppression of endotoxin release and subsequent production of inflammatory cytokines is crucial in the treatment of septic shock. We investigated the effect of clindamycin (CLI) on endotoxic shock induced in mice by Escherichia coli lipopolysaccharide (LPS). Mice were treated with CLI (160 to 600 mg/kg) or saline and then injected with E. coli LPS and d-(+)-galactosamine intraperitoneally 0.5 h after CLI administration. Pretreatment with CLI significantly improved survival in a dose-dependent manner (CLI, at 160, 300, and 440 mg/kg) and significantly lowered the peak concentrations of tumor necrosis factor alpha and interleukin-1β (IL-1β) in serum. However, the peak concentrations of IL-6 in the sera of CLI-treated mice were higher than in control mice. Our findings suggest that CLI alters LPS-induced inflammatory cytokine production and suppresses endotoxin-induced mortality in this murine model.

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Myeloperoxidase and Eosinophil Peroxidase Inhibit the in vitro Endotoxin Activities of Lipopolysaccharide (LPS) and Lipid A and Increase Survival in an in vivo Mouse LPS LD90 model

10.1101/499855 ◽

2018 ◽

Author(s):

Robert C. Allen ◽

Mary L. Henery ◽

John C. Allen ◽

Roger J. Hawks ◽

Jackson T. Stephens

Keyword(s):

Lipid A ◽

Lethal Dose ◽

Dependent Manner ◽

Gram Positive Bacteria ◽

Enzymatic Function ◽

Eosinophil Peroxidase ◽

Kaplan Meier ◽

Endotoxin Activity

Myeloperoxidase (MPO) and eosinophil peroxidase (EPO) are cationic leukocyte haloperoxidases with potent microbicidal and detoxifying activities. MPO selectively binds and kills specific Gram-positive bacteria (GPB) and all Gram-negative bacteria (GNB) tested. Endotoxin, i.e., lipopolysaccharide (LPS) comprising a toxic Lipid A component, is a characteristic of all GNB. The possibility that haloperoxidases bind to and inhibit the endotoxin of GBN was considered and tested by contacting MPO and EPO with LPS and Lipid A and measuring for inhibition of endotoxin activity using either the in vitro gel or chromogenic Limulus amebocyte lysate (LAL) assays. Contacting MPO and EPO with LPS purified from Escherichia coli O55:B5 and with diphosphoryl Lipid A purified from E. coli F583 inhibited their endotoxin activities in proportion to the natural log of the MPO or EPO concentration. Although MPO is less cationic than EPO, MPO consistently demonstrated inhibition of endotoxin activity that is about threefold superior to EPO. Haloperoxidase enzymatic activity was not required for inhibition, and MPO haloperoxidase action did not increase endotoxin inhibition. MPO and EPO inhibition of LPS endotoxin activity was also measured using a 90% lethal dose (LD90) mouse model studied over a five-day period. Based on Kaplan Meier survival analysis, MPO significantly increased mouse survival in a dose-dependent manner. EPO was less effective. In conclusion, contacting MPO and EPO with LPS and Lipid A inhibits in vitro endotoxin activities, but inhibition is independent of haloperoxidase enzymatic function. MPO significantly increases mouse survival against LPS in an in vivo LD90 endotoxin model.

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Pleiotropic functions of TNF-α determine distinct IKKβ-dependent hepatocellular fates in response to LPS

AJP Gastrointestinal and Liver Physiology ◽

10.1152/ajpgi.00043.2006 ◽

2007 ◽

Vol 292 (1) ◽

pp. G242-G252 ◽

Cited By ~ 13

Author(s):

Rana Dajani ◽

Salih Sanlioglu ◽

Yulong Zhang ◽

Qiang Li ◽

Martha M. Monick ◽

...

Keyword(s):

Knockout Mice ◽

Viral Vectors ◽

Endotoxic Shock ◽

Lethal Dose ◽

Dominant Negative ◽

Endotoxin Challenge ◽

Lps Challenge ◽

Differential Outcomes ◽

Tnf Α ◽

Pleiotropic Functions

TNF-α influences morbidity and mortality during the course of endotoxemia. However, the complex pleiotropic functions of TNF-α remain poorly understood. We evaluated how hepatic induction of NF-κB and TNF-α influence survival and hepatocellular death in a lethal murine model of endotoxic shock. Using dominant-negative viral vectors to inhibit the IKK complex, we demonstrate through this study that the liver is a major source of TNF-α during the course of lethal endotoxemia and that IKKβ (but not IKKα) is predominantly responsible for activating NF-κB and TNF-α in the liver after LPS administration. Using TNF-α knockout mice and hepatic-specific inhibition of IKKβ, we demonstrate that the status of TNF-α and NF-κB balances necrotic and apoptotic fates of hepatocytes in the setting of endotoxemia. In the presence of TNF-α, inhibiting hepatic IKKβ resulted in increased survival, reduced serum proinflammatory cytokines, and reduced hepatocyte necrosis in response to a lethal dose of endotoxin. In contrast, inhibiting hepatic IKKβ in TNF-α knockout mice resulted in decreased survival and increased caspase 3-mediated hepatocyte apoptosis after endotoxin challenge, despite a reduced proinflammatory cytokine response. In the presence of TNF-α, NF-κB-dependent hepatocellular necrosis predominated, while in the absence of TNF-α, NF-κB primarily influenced apoptotic fate of hepatocytes. Changes in JNK phosphorylation after LPS challenge were also dynamically affected by both IKKβ and TNF-α; however, this pathway could not solely explain the differential outcomes in hepatocellular fates. In conclusion, our studies demonstrate that induction of NF-κB and TNF-α balances protective (antiapoptotic) and detrimental (proinflammatory) pathways to determine hepatocellular fates during endotoxemia.

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