Effect of cross-tolerance between endotoxin and TNF-alpha or IL-1beta on cellular signaling and mediator production.

Endotoxin [lipopolysaccharide (LPS)] tolerance suppresses macrophage/monocyte proinflammatory-mediator production. This phenomenon also confers cross-tolerance to other stimuli including tumor necrosis factor (TNF) alpha and interleukin (IL)-1beta. Post-receptor convergence of signal transduction pathways might occur after LPS, IL-1beta, and TNF-alpha stimulation. Therefore, it was hypothesized that down-regulation of common signaling molecules induces cross-tolerance among these stimuli. LPS tolerance and cross-tolerance were examined in THP-1 cells. Phosphorylation of MAP kinases and degradation of inhibitor kappaBalpha (IkappaBalpha) DNA binding of nuclear factor-kappaB (NF-kappaB), and mediator production were examined. In naive cells, LPS, TNF-alpha, and IL-1beta induced IkappaBalpha degradation, kinase phosphorylation, and NF-kappaB DNA binding. LPS stimulation induced production of TNF-alpha or TxB2 and degradation of IRAK. However, neither TNF-alpha nor IL-1beta induced IRAK degradation or stimulated TNF-alpha or TxB2 production in naive cells. Pretreatment with each stimulus induced homologous tolerance to restimulation with the same agonist. LPS tolerance also suppressed LPS-induced TxB2 and TNF-alpha production. LPS pretreatment induced cross-tolerance to TNF-alpha or IL-1beta stimulation. Pretreatment with TNF-alpha induced cross-tolerance to LPS-induced signaling events and TxB2 production. Although pretreatment with IL-1beta did not induce cross-tolerance to LPS-induced signaling events, it strongly inhibited LPS TNF-alpha and TxB2 production. These data demonstrate that IL-1beta induces cross-tolerance to LPS-induced mediator production without suppressing LPS-induced signaling to MAP kinases or NF-kappaB activation.     

Effects of Porphyromonas gingivalis and Escherichia coli lipopolysaccharides on mononuclear phagocytes.

The mononuclear phagocyte plays an important role in the regulation of microbe-induced inflammation, in part through its ability to secrete mediators, particularly cytokines, in response to microorganisms and their products. To evaluate the effects of the microbial flora associated with chronic adult periodontitis on cytokine induction, lipopolysaccharide (LPS) from the periodontopathogen Porphyromonas gingivalis was used to stimulate naive and phorbol ester-primed U937 monocytic cells, as well as elutriated human peripheral blood monocytes. We assessed the effect of this LPS, in comparison to that of LPS from Escherichia coli, on cell proliferation, cytokine induction, and surface expression of the LPS receptor CD14. P. gingivalis LPS stimulated proliferation of U937 cells at concentrations of greater than 1 ng/ml, while E. coli LPS inhibited proliferation. Phorbol myristic acid (PMA)-treated U937 cells and elutriated monocytes responded to E. coli LPS activation by producing tumor necrosis factor alpha (TNF-alpha) mRNA and protein; however, P. gingivalis LPS induced greater numbers of TNF-alpha mRNA-positive cells and higher (P < 0.05) levels of protein than did E. coli LPS. Both cell types expressed interleukin-1 beta (IL-1beta) mRNA and protein in response to either LPS treatment. Compared with E. coli LPS, P. gingivalis LPS induced significantly (P < 0.05) higher numbers of IL-1 mRNA-positive U937 cells and elutriated monocytes, as well as production of significantly more (P < 0.05) IL-1 protein by the monocytes. The PMA-treated U937 cells and the monocytes produced high levels of IL-1 receptor antagonist mRNA and protein which were only marginally affected by the LPS preparations. While E. coli LPS induced expression of CD 14 on the surface of PMA-primed U937 cells and monocytes, P. gingivalis LPS exhibited a significantly (P < 0.05) greater ability to enhance receptor levels. Our results indicate that P. gingivalis LPS can activate the mononuclear phagocyte for proliferation, cytokine production, and CD14 expression, providing evidence for the potential of this bacterial component to act as a critical regulatory factor in the chronic inflammatory response associated with periodontitis.     

Blocking NF-kappaB activation may be an effective strategy in the fever therapy

Lipopolysaccharide (LPS) stimulates peripheral mononuclear cells (PBMC) to synthesize or release pyrogenic cytokines, including interleukin-1beta (IL-1beta), IL-6, and tumor necrosis factor-alpha (TNF-alpha). Nuclear factor-kappa B (NF-kappaB) influences inflammatory responses through the regulation of genes encoding cytokines. In the present study, experiments were carried out to determine whether an inhibition of NF-kappaB mechanisms causes an inhibition of pyrogenic cytokine synthesis or release from PBMC and results in antipyresis. Intravenous administration of the supernatant fluids obtained from the human PBMC incubated with LPS caused feverlike hyperthermia in rabbits. The febrile responses were in parallel with the levels of IL-1beta, IL-6, and TNF-alpha in supernatant fluids. Both the fever and the increased levels of these cytokines in supernatant fluids were decreased by incubating LPS-PBMC with NF-kappaB inhibitors, including pyrrolidine dithiocarbamate, sodium pyrithione, N-acetyl-cysteine, and curcumin. Moreover, an intravenous administration of LPS (0.5-2 microg/kg) produced dose-dependent fever in the rabbits. The fevers were in parallel with the levels of IL-1beta, IL-6, and TNF-alpha in rabbit serum. A pretreatment of rabbits with an intravenous injection of pyrrolidine dithiocarbamate, sodium pryithione, N-acetyl-cysteine, or curcumin 1 h before the intravenous administration of LPS significantly attenuated the LPS-induced fever and/or increased levels of these cytokines in the serum of rabbits. Furthermore, pretreatment with an intravenous dose of anti-IL-1beta, anti-IL-6, or anti-TNF-alpha monoclonal antibody significantly attenuated the fever induced by the intravenous injection of LPS in rabbits. The antipyretic effects exerted by anti-L-1beta monoclonal antibody were greater than those exerted by anti-L-6 or anti-NF-alpha monoclonal antibody. The data indicate that NF-kappaB activation correlates with an LPS-induced synthesis or a release of cytokines (in particular, IL-1beta) from PBMC and triggers fever. Blocking NF-kappaB mechanisms in the PBMC with NF-kappaB inhibitors may be an effective strategy in the fever therapy.     

Amiloride blockades lipopolysaccharide-induced proinflammatory cytokine biosynthesis in an IkappaB-alpha/NF-kappaB-dependent mechanism. Evidence for the amplification of an antiinflammatory pathway in the alveolar epithelium.

It has been previously reported that amiloride suppresses inflammatory cytokine biosynthesis. However, the molecular mechanism involved has yet to be ascertained. Therefore, the immunoregulatory potential mediated by amiloride and the underlying signaling transduction pathway was investigated. Exposure of alveolar epithelial cells to amiloride or its analog, 5-(N,N-hexamethylene)-amiloride (HMA), reduced, in a dose-dependent manner, lipopolysaccharide (LPS)-induced secretion of interleukin (IL)-1beta and tumor necrosis factor (TNF)-alpha. This inhibitory effect was associated with the augmentation of a counter antiinflammatory response, mediated by IL-6 and IL-10. Analysis of the mechanism implicated revealed the involvement of an inhibitory kappaB (IkappaB-alpha)/nuclear factor kappaB (NF- kappaB)-sensitive pathway. Amiloride and HMA suppressed the phosphorylation of IkappaB-alpha mediated by LPS, thereby allowing its cytosolic accumulation. Furthermore, both inhibitors interfered with the nuclear translocation of selective NF-kappaB subunits, an effect associated with blockading the DNA-binding activity of NF-kappaB. Recombinant IL-10 blockaded LPS-induced biosynthesis of IL-1beta and TNF-alpha and reduced NF-kappaB activation. Immunoneutralization of endogenous IL-10 reversed the inhibitory effect of amiloride on proinflammatory cytokines and restored the DNA-binding activity of NF-kappaB. These results indicate that amiloride acts as a novel dual immunoregulator in the alveolar epithelium: it downregulates an inflammatory signal and at the same time upregulates an antiinflammatory response. This biphasic effect is IL-10 sensitive and is associated with the selective targeting of the IkappaB-alpha/NF-kappaB signaling transduction pathway.     

Porphyromonas gingivalis lipopolysaccharide antagonizes Escherichia coli lipopolysaccharide at toll-like receptor 4 in human endothelial cells

E. coli lipopolysaccharide (LPS) induces cytokine and adhesion molecule expression via the toll-like receptor 4 (TLR4) signaling complex in human endothelial cells. In the present study, we investigated the mechanism by which Porphyromonas gingivalis LPS antagonizes E. coli LPS-dependent activation of human endothelial cells. P. gingivalis LPS at 1 micro g/ml inhibited both E. coli LPS (10 ng/ml) and Mycobacterium tuberculosis heat shock protein (HSP) 60.1 (10 micro g/ml) stimulation of E-selectin mRNA expression in human umbilical vein endothelial cells (HUVEC) without inhibiting interleukin-1 beta (IL-1beta) stimulation. P. gingivalis LPS (1 micro g/ml) also blocked both E. coli LPS-dependent and M. tuberculosis HSP60.1-dependent but not IL-1beta-dependent activation of NF-kappaB in human microvascular endothelial (HMEC-1) cells, consistent with antagonism occurring upstream from the TLR/IL-1 receptor adaptor protein, MyD88. Surprisingly, P. gingivalis LPS weakly but significantly activated NF-kappaB in HMEC-1 cells in the absence of E. coli LPS, and the P. gingivalis LPS-dependent agonism was blocked by transient expression of a dominant negative murine TLR4. Pretreatment of HUVECs with P. gingivalis LPS did not influence the ability of E. coli LPS to stimulate E-selectin mRNA expression. Taken together, these data provide the first evidence that P. gingivalis LPS-dependent antagonism of E. coli LPS in human endothelial cells likely involves the ability of P. gingivalis LPS to directly compete with E. coli LPS at the TLR4 signaling complex.     

In vivo vitamin E administration attenuates interleukin-6 and interleukin-1beta responses to an acute inflammatory insult in mouse skeletal and cardiac muscle

Antioxidants are associated with reduced pro-inflammatory cytokine expression in immune cells and isolated tissues; however, no studies have examined whether short-term vitamin E administration is associated with reduced lipopolysaccharide (LPS)-induced cytokine expression in mouse skeletal and cardiac muscle, in vivo. These experiments tested the hypothesis that vitamin E administration attenuates nuclear factor kappaB (NF-kappaB), IL-6, IL-1beta and tumour necrosis factor alpha (TNFalpha) responses in skeletal and cardiac muscle to an inflammatory challenge induced by systemic LPS. We compared IL-6, IL-1beta and TNFalpha mRNA and protein, activated NF-kappaB and total oxidized proteins in skeletal and cardiac muscle 4 or 24 h after saline or LPS injection in mice receiving vitamin E or placebo for 3 days prior to the insult. Skeletal and cardiac IL-6 mRNA and protein were significantly elevated by LPS in both groups, but responses were significantly lower in vitamin E- compared with placebo-treated mice. In skeletal and cardiac muscle, LPS increased IL-1beta mRNA and protein in placebo- but not vitamin E-treated mice. Lipopolysaccharide-induced levels of cardiac IL-1beta mRNA and protein and skeletal IL-1beta mRNA were lower with vitamin E than placebo. Lipopolysaccharide-induced NF-kappaB activation and increases in total oxidized proteins were attenuated with vitamin E compared with placebo in both tissues. Vitamin E decreased LPS-induced increases in plasma IL-1beta but not IL-6 compared with placebo. The major results provide the first in vivo evidence that short-term vitamin E administration reduces IL-6 and IL-1beta responses to LPS in skeletal and cardiac muscle and prevents LPS-induced increases in NF-kappaB activation and total oxidized proteins.     

Bromelain inhibits lipopolysaccharide-induced cytokine production in human THP-1 monocytes via the removal of CD14

Bromelain has been reported to have anti-inflammatory and immunomodulatory effects. However, the anti-inflammatory mechanism of bromelain is unclear. Therefore, we investigated the effect of bromelain on cytokine production from lipopolysaccharide (LPS)-stimulated human peripheral blood mononuclear cells (PBMC) and monocytic leukemia THP-1 cells. The result showed that bromelain (50-100 microg/ml) significantly and reversibly reduced tumor necrosis factor (TNF)-alpha interleukin- (IL)-1beta and IL-6 from LPS-induced PBMC and THP-1 cells. This effect was correlated with reduced LPS-induced TNF-alpha mRNA and NF-kappaB activity in THP-1 cells. In addition, bromelain dose-dependently inhibited LPS-induced prostaglandin E(2), thromboxane B(2) and COX-2 mRNA but not COX-1 mRNA. Importantly, bromelain degraded TNF-alpha and IL-1beta molecules, reduced the expression of surface marker CD14 but not Toll-like receptor 4 from THP-1 cells. Taken together, the results suggest that the suppression of signaling pathways by bromelain's proteolytic activity may contribute to the anti-inflammatory activity of bromelain.     

Opposing effects of tumor necrosis factor alpha and leukemia inhibitory factor in lipopolysaccharide-stimulated myelopoiesis.

Three myelopoietically active, lipopolysaccharide (LPS)-stimulated monokines, interleukin-1 alpha (IL-1 alpha), tumor necrosis factor alpha (TNF-alpha), and leukemia inhibitory factor (LIF), were tested for effect in an in vitro model for LPS-induced inflammatory murine monocytopoiesis. Neither cytokine stimulated clonal proliferation of marrow-derived progenitors; however, both IL-1 alpha and TNF-alpha enhanced macrophage colony-stimulating factor (M-CSF)-dependent colony formation. The additional progenitors stimulated by IL-1 alpha and TNF-alpha to form colonies in response to M-CSF were equivalent to the precommitment, transitional progenitors stimulated by M-CSF and bacterial LPS. In addition, the additional colonies elicited by IL-1 alpha and TNF-alpha were not additive in cultures containing both M-CSF and LPS, indicating these colonies arose from the same LPS-responsive, two-signal-dependent transitional progenitors. Leukemia inhibitory factor did not influence M-CSF-stimulated colony formation; however, LIF effected a dose-dependent inhibition of colony formation by transitional progenitors responding to combinations of M-CSF and LPS, IL-1 alpha, TNF-alpha, or an additional transitional cell costimulant, substance P. Neutralizing anti-murine TNF-alpha antibodies abrogated transitional cell colony formation stimulated by combinations of M-CSF and TNF-alpha, IL-1 alpha, LPS, or substance P but had no effect on colony formation stimulated solely by M-CSF. The results indicate that TNF-alpha may be an important positive stimulus for commitment of progenitors to the mononuclear phagocyte lineage and that TNF-alpha may be the endogenous regulator of the costimulatory effects of LPS, IL-1, and substance P. In addition, the results indicate that LIF may play an opposing negative regulatory role acting to inhibit LPS and TNF-alpha stimulation of the transitional progenitors.     

Interaction of surfactant protein A with lipopolysaccharide and regulation of inflammatory cytokines in the THP-1 monocytic cell line

Pulmonary surfactant protein A (SP-A) is involved in innate immunity in the lung. In this study we investigated the interaction of SP-A with different serotypes of lipopolysaccharide (LPS) on the regulation of inflammatory cytokines in vitro. In the human monocytic cell line, THP-1, combining SP-A with lipid A or rough LPS further enhanced lipid A- or rough LPS-stimulated tumor necrosis factor alpha (TNF-alpha) mRNA levels, while SP-A-elicited increases in TNF-alpha mRNA levels were partially neutralized. In contrast, the combination of smooth LPS and SP-A resulted in additive effects on TNF-alpha mRNA levels. We also demonstrated that there was cross-tolerance between SP-A and LPS in THP-1 cells. Pretreatment of THP-1 cells with LPS modestly inhibited the response of these cells to subsequent challenge with SP-A, with regard to the production of TNF-alpha, whereas there was no or little effect on the production of interleukin-1beta (IL-1beta) and IL-8. Conversely, pretreatment of THP-1 cells with SP-A markedly increased the response to subsequent challenge with LPS with regard to the production of IL-1beta and IL-8, although the production of TNF-alpha was modestly decreased. However, a synergistic stimulatory effect was observed when the two agents were added simultaneously to the cells. NF-kappaB formation was downregulated in SP-A- but not in LPS-induced tolerant cells. These results suggested that SP-A exhibits different interactions with distinct serotypes of LPS. In addition, SP-A is different from LPS with regard to the induction of cross-tolerance, and these actions may be mediated, at least in part, through different mechanisms.     

The effects of Porphyromonas gingivalis LPS and Actinobacillus actinomycetemcomitans LPS on human dendritic cells in vitro, and in a mouse model in vivo

Interaction between different bacterial plaque pathogens and dendritic cells may induce different types of T helper (Th) cell response, which is critical in the pathogenesis of periodontitis. In this study we investigated the effects of lipopolysaccharide (LPS) from Porphyromonas gingivalis and Actinobacillus actinomycetemcomitans on human monocyte-derived dendritic cells (Mo-DCs) with respect to co-stimulatory molecule expression, cytokine production and Th cell differentiation. Unlike Escherichia coli and A. actinomycetemcomitans LPS, P. gingivalis LPS induced only low levels of CD40, CD80, HLA-DR and CD83 expression on Mo-DCs. LPS from both bacteria induced considerably lower TNF-alpha and IL-10 than did E. coli LPS. LPS from all three bacteria induced only negligible IL-12 production. In a human mixed-leukocyte reaction, and in an ovalbumin-specific T cell response assay in mice, both types of LPS suppressed IFN-gamma production. In conclusion, stimulation by P. gingivalis LPS and A. actinomycetemcomitans LPS appears to bias Mo-DCs towards Th2 production.     

In vivo exposure to Porphyromonas gingivalis up-regulates nitric oxide but suppresses tumour necrosis factor-alpha production by cultured macrophages.

The present study was designed to test whether the functional response of mouse macrophages elicited by chronic exposure to bacteria will be different from that of cells elicited by a non-bacterial irritant. Macrophage elicitation was conducted by Porphyromonas gingivalis, a major periodontal pathogen, in comparison to a standard elicitation by thioglycollate (TG). We measured lipopolysaccharide (LPS)-induced nitric oxide (NO) and tumour necrosis factor-alpha (TNF-alpha) secretion by the elicited macrophages, and the expression of inflammatory cytokines in the whole elicited cell population. In addition, we tested the response of TG-elicited macrophages to pretreatment with P. gingivalis LPS in vitro. Mouse peritoneal macrophages were harvested 4 days after intraperitoneal injection of TG or heat-killed P. gingivalis. TG-elicited macrophages produced undetectable levels of TNF-alpha and approximately 0.5 microM of NO. The stimulation of the macrophages with LPS resulted in the secretion of NO and TNF-alpha in a dose-dependent manner. The P. gingivalis-elicited macrophages produced basal levels of approximately 5 microM NO, but TNF-alpha was not detectable. LPS stimulation of these cells further increased the secretion of NO eightfold while TNF-alpha remained undetectable. The NO secretion by P. gingivalis-elicited cells was significantly higher than that by TG-elicited cells. Examination of cytokine expression in the whole elicited cell population revealed that both P. gingivalis-elicited cells and TG-elicited cells expressed messenger RNA for interleukin-2 (IL-2), TNF-alpha and interferon-gamma (IFN-gamma), but not for IL-4. IL-6 was expressed in P. gingivalis-elicited cells only. Pretreatment of TG-elicited macrophages with P. gingivalis LPS for 24 hr prior to a second LPS challenge resulted in down-regulation of TNF-alpha secretion and up-regulation of NO secretion, a response similar to that seen in P. gingivalis-elicited peritoneal macrophages. The results suggest that the in vivo exposure of resident macrophages to P. gingivalis induces functional changes in peritoneal macrophages. These changes might be due to the effect of P. gingivalis LPS.     

Hyperthermia differentially regulates TLR4 and TLR2-mediated innate immune response

Fever influences multiple parameters of the immune response. However, the mechanisms by which fever manipulates immune response remain undefined. Here we present the evidences that fever range hyperthermia differentially regulates immune response to lipopolysaccharide (LPS) and lipoteichoic acids (LTA) through modulating Toll-like receptor (TLR) signaling. Pretreatment with 39.5 degrees C temperature enhanced LPS, but not LTA, induced NF-kappaB activation and TNF-alpha, IL-6 production in human macrophages. Consistently, expression of TLR4, but not TLR2, was up-regulated by 39.5 degrees C treatment. The increase in LPS-induced cytokine production was inhibited by TLR4-blocking antibody, indicating the enhancement of LPS-induced cytokine production by 39.5 degrees C pretreatment was TLR4-dependent. Pretreatment of mice with 39.5 degrees C temperature also enhanced LPS, but not LTA, induced TNF-alpha and IL-6 production in vivo. These results support the concept that fever range hyperthermia might activate innate immune response by promoting TLR4 expression and signaling, providing a possible mechanistic explanation for the function of fever in regulating innate immune responses.     

Corticotropin-releasing hormone augments proinflammatory cytokine production from macrophages in vitro and in lipopolysaccharide-induced endotoxin shock in mice

Corticotropin-releasing hormone (CRH) exerts an anti-inflammatory effect indirectly, via cortisole production, and a proinflammatory effect directly on immune cells. The aim of the present work was to examine the effect of CRH on macrophage-derived cytokines both in vitro and in vivo. For the in vitro experiments we used two types of macrophages: (i) the RAW264.7 monocyte/macrophage cell line and (ii) thioglycolate-elicited peritoneal macrophages from BALB/c mice. We have found that CRH enhanced lipopolysaccharide (LPS)-induced tumor necrosis factor alpha (TNF-alpha), interleukin-1beta (IL-1beta), and IL-6 production. For the in vivo experiments we have used the LPS-induced endotoxin shock model in BALB/c mice, an established model for systemic inflammation in which macrophages are the major source of the proinflammatory cytokines responsible for the development of the shock. Administration of antalarmin, a synthetic CRH receptor 1 (CRHR1) antagonist, prior to LPS prolonged survival in a statistically significant manner. The effect was more evident at the early stages of endotoxin shock. CRHR1 blockade suppressed LPS-induced elevation of the macrophage-derived cytokines TNF-alpha, IL-1beta, and IL-6, confirming the role of CRH signals in cytokine expression. In conclusion, our data suggest that CRH signals play an early and crucial role in augmenting LPS-induced proinflammatory cytokine production by macrophages. Our data suggest that the diffuse neuroendocrine system via CRH directly affects the immune system at the level of macrophage activation and cytokine production.     

Enhanced interleukin-1beta, interleukin-6 and tumor necrosis factor-alpha production by LPS stimulated human monocytes isolated from HIV+ patients

Periodontal disease and tooth loss is a common finding among advanced HIV+ patients. In addition to local oral lipopolysaccharide (LPS) stimulation, systemic up-regulation of monocyte pro-inflammatory cytokine secretion may also be involved in the pathogenesis of HIV disease. A study was undertaken to investigate IL-1beta, IL-6 and TNF-alpha production by resting and LPS stimulated monocytes isolated from HIV+ patients and also to investigate the relationship of the patient's HIV viral load status to the cytokine production. Whole blood samples in EDTA were collected from 39 HIV-1 infected patients and 20 age and sex matched uninfected controls. Plasma was separated by centrifugation. Viral load was determined using a quantitative RT-PCR. Monocytes were isolated by Ficoll-hypaque gradient separation followed by overnight plastic adherence. Cultured monocytes (1x10(6)/ml) were stimulated with LPS (1 microg/ml) of either P. gingivalis or F. nucleatum for 2, 8, 24 and 48 h and supernatant fluids were collected. IL-1beta, IL-6, and TNF-alpha levels in supernatant fluids were estimated by ELISA. Increased overall production of IL-1beta, IL-6 and TNF-alpha by LPS stimulated monocytes isolated from HIV-1 infected patients was observed when compared to HIV-1 uninfected controls. LPS stimulated monocytes from HIV-1 infected patients with high viral load (HVL) produced significant (p<0.05) elevations in these pro-inflammatory cytokines when compared to HIV-1 uninfected controls. Both LPS of P. gingivalis and F. nucleatum produced a comparable cytokine production by monocytes after 8 h of stimulation. These data suggest that enhanced IL-1beta, IL-6 and TNF-alpha is produced by monocytes/macrophages isolated from HVL HIV+ patients and may be involved in the overall pathogenesis of HIV-1 infection.     

Bikunin inhibits lipopolysaccharide-induced tumor necrosis factor alpha induction in macrophages

Bikunin, a Kunitz-type protease inhibitor, exhibits anti-inflammatory activity in protection against cancer and inflammation. To investigate the molecular mechanism of this inhibition, we analyzed the effect of bikunin on tumor necrosis factor alpha (TNF-alpha) production in human peripheral mononuclear cells stimulated by lipopolysaccharide (LPS), an inflammatory inducer. Here, we show the following results. (i) LPS induced TNF-alpha expression in time- and dose-dependent manners through phosphorylation of extracellular signal-regulated kinases 1 and 2 (ERK1/2), c-Jun N-terminal kinase (JNK), and p38 mitogen-activated protein kinase pathways. (ii) Bikunin inhibits LPS-induced up-regulation of TNF-alpha protein expression in a dose-dependent manner, reaching 60% inhibition at the highest doses of bikunin tested (5.0 microM). (iii) Inhibition by bikunin of TNF-alpha induction correlates with the suppressive capacity of ERK1/2, JNK, and p38 signaling pathways, implicating repressions of at least three different signals in the inhibition. (iv) Bikunin blocks the induction of TNF-alpha target molecules interleukin-1beta (IL-1beta) and IL-6 proteins. (v) Bikunin is functional in vivo, and this glycoprotein blocks systemic TNF-alpha release in mice challenged with LPS. (vi) Finally, bikunin can prevent LPS-induced lethality. In conclusion, bikunin significantly inhibits LPS-induced TNF-alpha production, suggesting a mechanism of anti-inflammation by bikunin through control of cytokine induction during inflammation. Bikunin might be a candidate for the treatment of inflammation, including septic shock.