LPS-induced Tumor Necrosis Factor Research Articles

Activation of adenosine A 3 receptor suppresses lipopolysaccharide-induced TNF-α production through inhibition of PI 3-kinase/Akt and NF-κB activation in murine BV2 microglial cells

Adenosine is an endogenous nucleoside that regulates many processes, including inflammatory responses, through activation of its receptors. Adenosine receptors have been reported to be expressed in microglia, which are major immune cells of brain, yet little is known about the role of adenosine receptors in microglial cytokine production. Thus, we investigated the effect of adenosine and adenosine A 3 receptor ligands on LPS-induced tumor necrosis factor (TNF-α) production and its molecular mechanism in mouse BV2 microglial cells. Adenosine and Cl-IB-MECA, a specific adenosine A 3 receptor agonist, suppressed LPS-induced TNF-α protein and mRNA levels. Moreover, MRS1523, a selective A 3 receptor antagonist, blocked suppressive effects of both adenosine and Cl-IB-MECA on TNF-α. We further examined the effect of adenosine on signaling molecules, such as PI 3-kinase, Akt, p38, ERK1/2, and NF-κB, which are involved in the regulation of inflammatory responses. Adenosine inhibited LPS-induced phosphatidylinositol (PI) 3-kinase activation and Akt phosphorylation, whereas it had no effect on the phosphorylation of p38 and ERK1/2. We also found that adenosine as well as Cl-IB-MECA inhibited LPS-induced NF-κB DNA binding and luciferase reporter activity. Taken together, these results suggest that adenosine A 3 receptor activation suppresses TNF-α production by inhibiting PI 3-kinase/Akt and NF-κB activation in LPS-treated BV2 microglial cells.

Reduced Capacity of Whole Blood Leucocytes to Express Tumour Necrosis Factor-α and Interleukin-10 following Major Orthopaedic Surgery

Background: Severe trauma is a challenge to the immune response and may cause reduced immune capacity. As a marker of decreased cellular activity, studies with ex vivo lipopolysaccharide (LPS) stimulation of whole blood or isolated mononuclear cells from injured patients have revealed reduced production of inflammatory cytokines. To gain further insight into immune alterations in orthopaedic surgery, we studied LPS-induced tumour necrosis factor (TNF)-α and interleukin (IL)-10 in whole blood of patients during peri- and postoperative phases of total hip replacement. Methods: Four females and 3 males undergoing elective total hip replacement were included in the study. Ex vivo LPS-induced TNF-α and IL-10 were measured in a whole blood assay before, during and at 1 and 6 days after operation. In addition, the counts of white blood cells were determined. Results: During the operation, there were significant reductions in the number of monocytes, but at day 1 and 6 after surgery, there were significant increases as compared to the levels before surgery. The capacity of whole blood to express TNF-α and IL-10 did not change significantly during the operation and the following postoperative day. At day 6, however, there were significant reductions in expression of both TNF-α and IL-10 as compared to the levels before the operation. In relation to the values of monocytes, there was a significant reduction in the expression of TNF-α also at day 1 after operation. Conclusion: Our data indicate that in the course of at least 6 days after a major orthopaedic trauma, there is suppression of the whole blood capacity to express the inflammatory cytokine TNF-α and the anti-inflammatory cytokine IL-10 when exposed to LPS. During this time, then, the patient is particular susceptible to septic complications.

Differential expression of function-related antigens on blood monocytes in children with hemolytic uremic syndrome

Monocytes (Mo) mediate central functions in inflammation and immunity. Different subpopulations of Mo with distinct phenotype and functional properties have been described. Here, we investigate the phenotype and function of peripheral Mo from children with hemolytic uremic syndrome (HUS). For this purpose, blood samples from patients in the acute period of HUS (HUS AP) were obtained on admission before dialysis and/or transfusion. The Mo phenotypic characterization was performed on whole blood by flow cytometry, and markers associated to biological functions were selected: CD14 accounting for lipopolysaccharide (LPS) responsiveness, CD11b for adhesion, Fc receptor for immunoglobulin G type I (FcgammaRI)/CD64 for phagocytosis and cytotoxicity, and human leukocyte antigen (HLA)-DR for antigen presentation. Some of these functions were also determined. Moreover, the percentage of CD14+ CD16+ Mo was evaluated. We found that the entire HUS AP Mo population exhibited reduced CD14, CD64, and CD11b expression and decreased LPS-induced tumor necrosis factor production and Fcgamma-dependent cytotoxicity. HUS AP showed an increased percentage of CD14+ CD16+ Mo with higher CD16 and lower CD14 levels compared with the same subset from healthy children. Moreover, the CD14++ CD16- Mo subpopulation of HUS AP had a decreased HLA-DR expression, which correlated with severity. In conclusion, the Mo population from HUS AP patients presents phenotypic and functional alterations. The contribution to the pathogenesis and the possible scenarios that led to these changes are discussed.

Anti-inflammatory potential of Antrodia Camphorata through inhibition of iNOS, COX-2 and cytokines via the NF-κB pathway

Antrodia camphorata ( A. camphorata), well known in Taiwan as a traditional Chinese medicine, has been shown to exhibit antioxidant and anticancer effects. In the present study, therefore, we have examined the effects of the fermented culture broth of A. camphorata (25–100 μg/ml) in terms of lipopolysaccharide (LPS)-induced nitric oxide (NO) and prostaglandin E 2 (PGE 2) production, and inducible nitric oxide synthase (iNOS) and cyclooxygenase-2 (COX-2) protein expression in RAW 264.7 macrophages. Our results indicate concentration-dependent A. camphorata inhibition of LPS-induced NO and PGE 2 production, without appreciable cytotoxicity on the RAW 264.7 cells. A. camphorata also attenuates the production of LPS-induced tumor necrosis factor (TNF-α) and interleukin (IL)-1β. Furthermore, A. camphorata blocks the IκB-α degradation induced by LPS. These results indicate that A. camphorata inhibits LPS induction of cytokine, iNOS and COX-2 expression by blocking NF-κB activation. Therefore, we report the first confirmation of the anti-inflammatory potential of this traditionally employed herbal medicine in vitro.

Src homology 2 domain-containing inositol-5-phosphatase 1 (SHIP1) negatively regulates TLR4-mediated LPS response primarily through a phosphatase activity- and PI-3K-independent mechanism

AbstractSrc homology 2 (SH2) domain-containing inositol-5-phosphatase 1 (SHIP1) plays important roles in negatively regulating the activation of immune cells primarily via the phosphoinositide 3-kinase (PI-3K) pathway by catalyzing the PI-3K product PtdIns-3,4,5P3 (phosphatidylinositol-3,4,5-triphosphate) into PtdIns-3,4P2. However, the role of SHIP1 in Toll-like receptor 4 (TLR4)-mediated lipopolysaccharide (LPS) response remains unclear. Here we demonstrate that SHIP1 negatively regulates LPS-induced inflammatory response via both phosphatase activity-dependent and -independent mechanisms in macrophages. SHIP1 becomes tyrosine phosphorylated and up-regulated upon LPS stimulation in RAW264.7 macrophages. SHIP1-specific RNA-interfering and SHIP1 overexpression experiments demonstrate that SHIP1 inhibits LPS-induced tumor necrosis factor α (TNF-α) and interleukin 6 (IL-6) production by negatively regulating the LPS-induced combination between TLR4 and myeloid differentiation factor 88 (MyD88); activation of Ras (p21ras protein), PI-3K, extracellular signal-regulated kinase 1/2 (ERK1/2), p38, and c-Jun NH2-terminal kinase (JNK); and degradation of IκB-α. SHIP1 also significantly inhibits LPS-induced mitogen-activated protein kinase (MAPK) activation in TLR4-reconstitited COS7 cells. Although SHIP1-mediated inhibition of PI-3K is dependent on its phosphatase activity, phosphatase activity-disrupted mutant SHIP1 remains inhibitory to LPS-induced TNF-α production. Neither disrupting phosphatase activity nor using the PI-3K pathway inhibitor LY294002 or wortmannin could significantly block SHIP1-mediated inhibition of LPS-induced ERK1/2, p38, and JNK activation and TNF-α production, demonstrating that SHIP1 inhibits LPS-induced activation of MAPKs and cytokine production primarily by a phosphatase activity- and PI-3K-independent mechanism. (Blood. 2005;105:4685-4692)

Immunomodulatory Activities of Small Host Defense Peptides

Recent studies have demonstrated that in addition to their antimicrobial activity, cationic host defense peptides, like the human cathelicidin LL-37, perform many activities relating to innate immunity, including the induction or modulation of chemokine and cytokine production, alteration of gene expression in host cells, and inhibition of proinflammatory responses of host cells to bacterial components such as lipopolysaccharide (LPS) in vitro and in vivo. To investigate if these properties are shared by smaller peptides, two cathelicidin peptides derived from bovine neutrophils, the 13-mer indolicidin and Bac2A, a linear 12-amino-acid derivative of bactenecin, were compared to the 37-amino-acid peptide LL-37. Indolicidin, like LL-37, inhibited LPS-induced tumor necrosis factor alpha (TNF-alpha) secretion, even when added up to an hour after the addition of Escherichia coli O111:B4 LPS to the human macrophage/monocyte-like THP-1 cell line. In contrast, Bac2A demonstrated no significant antiendotoxin activity. At low concentrations, indolicidin and LL-37 acted synergistically to suppress LPS-induced production of TNF-alpha. Indolicidin was analogous to LL-37 in its ability to induce the production of the chemokine interleukin-8 (IL-8) in a human bronchial cell line, 16HBE14o(-), but it was unable to induce production of IL-8 in THP-1 cells. In contrast, Bac2A was unable to induce IL-8 in either cell type. Conversely, Bac2A was chemotactic for THP-1 cells at concentrations between 10 and 100 mug/ml, while indolicidin and LL-37 were not chemotactic at these concentrations for THP-1 cells. This indicates that in addition to the potential for direct microbicidal activity, cationic host defense peptides may have diverse and complementary abilities to modulate the innate immune response.

Biological inactivation and impaired detection of IL-10 by suramin

Enzyme-linked immunosorbent assay (ELISA) measures the amount of cytokines secreted by cells but “equivalent” ELISAs from different manufacturers may show different results, dependent on the epitopes used to generate the antibodies employed in the ELISAs. This fact was dramatically illustrated in a series of experiments we conducted to analyze the effect of suramin on the lipopolysaccharide-induced interleukin-10 (LPS-induced IL-10) production in human CD14 + monocytes. Use of the human IL-10 DuoSet® ELISA from R&D Systems showed no influence of suramin on IL-10 levels, whereas the human IL-10 ELISA from Biosource detected only low amounts of IL-10 in the presence of suramin. The results with the Biosource ELISA reflected not only interference of the recognition of IL-10 in the presence of suramin, but also a block in the biological activity of IL-10. Thus, in the presence of suramin the addition of recombinant biological IL-10 protein could not inhibit the LPS-induced tumor necrosis factor alpha (TNF-alpha) or interleukin-12p40 (IL-12p40) production. Furthermore, suramin very efficiently neutralizes endogenous IL-10, resulting in a pronounced increase in IL-12 and TNF-alpha synthesis. Impaired recognition of biologically inactive conformations of IL-10 was not unique to suramin, in that heat-treated or chemically reduced recombinant IL-10 was also no longer recognized by the Biosource ELISA. This suggests that at least one of the antibodies employed in the Biosource ELISA is conformation sensitive, while the antibodies employed in the R&D Systems ELISA are insensitive to a number of conformation changes. All in all, our results show that suramin neutralizes the biological activity of IL-10 produced by monocytes. Furthermore, the amount of IL-10 detected by the Biosource ELISA correlates with the biological activity of IL-10, whereas the amount detected by the R&D Systems ELISA includes active as well as several inactive forms of IL-10.

Liposomal methylprednisolone differentially regulates the expression of TNF and IL-10 in human alveolar macrophages

Glucocorticoids (GC) are frequently used for therapy of various inflammatory lung diseases by either systemic or inhalative application. Because the oral application often has various side effects and because the inhalative application is not as potent, new formulations of GCs are required. We evaluated the effect of a liposomal (Lip) formulation of methylprednisolone (MP) on the expression of lipopolysaccharide (LPS)-induced proinflammatory tumor necrosis factor (TNF) and antiinflammatory interleukin-10 (IL-10) in human alveolar macrophages (AM). AM were obtained from bronchoalveolar lavage fluids of patients with various inflammatory lung diseases and precultured 20 h±MP, either liposomal or free, and then stimulated with LPS. Cells were harvested for analysis of mRNA levels by real-time reverse transcriptase polymerase chain reaction (RT-PCR); supernatants were used to measure protein concentrations by ELISA. We confirm the suppression of LPS-induced TNF production by an average of factor 7 at the mRNA level and factor 3 at the protein level. On the other hand, we detected a strong increase of the IL-10 production by MP. At the mRNA level, liposomal MP alone led to an 18-fold increase, and the LPS-induced IL-10 mRNA was enhanced by factor 2. At the protein level, MP alone had no effect, but LPS-induced IL-10 was increased by factor 2.5. Our data show that liposomal MP can consistently induce IL-10 and reduce TNF when macrophages are exposed for a prolonged period of time. In all respects, liposomal MP had similar activities as free MP, but liposomes were selectively taken up by monocytes and macrophages and not by lymphocytes in blood and in the lung. This suggests that liposomal glucocorticoids when applied locally in the lung may act efficiently but with less side effects.

Age and caloric restriction diets are confounding factors that modify the response to lipopolysaccharide by peritoneal macrophages in C57BL/6 mice.

Aging is the result of several detrimental changes that lead to a decrease in homeostasis, an increase in the incidence of degenerative diseases, and death. A caloric-restricted diet (CR), which consists of a significant reduction in calorie intake (40%) without malnutrition, has been shown to delay the onset of age-related diseases and pathologies and to extend life span. The aims of this study were to assess the effects of aging and CR on lipopolysaccharide (LPS)-dependant cytokine production by peritoneal macrophages (PMphis). Resident naïve PMphis were isolated from 2- to 24-month-old male C57BL/6 mice and were stimulated with Escherichia coli LPS (100 ng/mL) for 1 to 5 h in culture conditions. A linear decrease in the production of LPS-induced tumor necrosis factor alpha (TNF-alpha) and interleukin (IL) 10 was observed with age. LPS-induced IL-6 and IL-1beta levels were also reduced with age, but in a nonlinear fashion. Expression of CD14, the major receptor for LPS, on the PMphi surface was also observed to decline with age. Moreover, TNF-alpha production by PMphis was reduced in mice undergoing the two different CR diets of limited daily feeding and intermittent fasting, as compared with ad libitum-fed mice. The results of this study add the new variables age and diet to the paradigm proposing that the response to LPS is modulated by multiple components, including genetic background and sex.

Bacterial endotoxin stimulates macrophages to release HMGB1 partly through CD14- and TNF-dependent mechanisms.

Bacterial endotoxin [lipopolysaccharide (LPS)] stimulates macrophages to sequentially release early [tumor necrosis factor (TNF)] and late [high mobility group box 1 (HMGB1)] proinflammatory cytokines. The requirement of CD14 and mitogen-activated protein kinases [MAPK; e.g., p38 and extracellular signal-regulated kinase (ERK)1/2] for endotoxin-induced TNF production has been demonstrated previously, but little is known about their involvement in endotoxin-mediated HMGB1 release. Here, we demonstrated that genetic disruption of CD14 expression abrogated LPS-induced TNF production but only partially attenuated LPS-induced HMGB1 release in cultures of primary murine peritoneal macrophages. Pharmacological suppression of p38 or ERK1/2 MAPK with specific inhibitors (SB203580, SB202190, U0126, or PD98059) significantly attenuated LPS-induced TNF production but failed to inhibit LPS-induced HMGB1 release. Consistently, an endogenous, immunosuppressive molecule, spermine, failed to inhibit LPS-induced activation of p38 MAPK and yet, still significantly attenuated LPS-mediated HMGB1 release. Direct suppression of TNF activity with neutralizing antibodies or genetic disruption of TNF expression partially attenuated HMGB1 release from macrophages induced by LPS at lower concentrations (e.g., 10 ng/ml). Taken together, these data suggest that LPS stimulates macrophages to release HMGB1 partly through CD14- and TNF-dependent mechanisms.

N-linked glycosylation is required for c1 inhibitor-mediated protection from endotoxin shock in mice.

C1 inhibitor (C1INH) prevents endotoxin shock in mice via a direct interaction with lipopolysaccharide (LPS). This interaction requires the heavily glycosylated amino-terminal domain of C1INH. C1INH in which N-linked carbohydrate was removed by using N-glycosidase F was markedly less effective in protecting mice from LPS-induced lethal septic shock. N-deglycosylated C1INH also failed to suppress fluorescein isothiocyanate (FITC)-LPS binding to and LPS-induced tumor necrosis factor alpha mRNA expression by the murine macrophage-like cell line, RAW 264.7, and cells in human whole blood. In an enzyme linked immunosorbent assay, the N-deglycosylated C1INH bound to LPS very poorly. In addition, C1INH was shown to bind to diphosphoryl lipid A (dLPA) but only weakly to monophosphoryl lipid A (mLPA). As with intact LPS, binding of N-deglycosylated C1INH to dLPA and mLPA was diminished in comparison with the native protein. Removal of O-linked carbohydrate had no effect on any of these activities. Neither detoxified LPS, dLPA, nor mLPA had any effect on the rate or extent of C1INH complex formation with C1s or on cleavage of the reactive center loop by trypsin. These data demonstrate that N-linked glycosylation of C1INH is essential to mediate its interaction with the LPA moiety of LPS and to protect mice from endotoxin shock.

The toll-like receptor 4 Asp299Gly variant: no influence on LPS responsiveness or susceptibility to pulmonary tuberculosis in The Gambia

Setting: Tuberculosis (TB) remains a major cause of morbidity and mortality worldwide. Studies in a murine model of pulmonary TB have identified a role for Toll-like receptor 4 (TLR4) in the development of chronic lung infection with Mycobacterium tuberculosis. The Asp299Gly polymorphism in the human TLR4 gene is associated with in vivo hyporesponsiveness to lipopolysaccharide (LPS) in Caucasians. Objective: To determine whether TLR4 Asp299Gly influences LPS responses or susceptibility to pulmonary TB in humans in a Gambian population sample. Design: We compared whole blood monokine responses to LPS in 245 healthy blood donors stratified by TLR4 Asp299Gly genotype to assess whether this polymorphism was functional in this population. A case-control study of 640 subjects was conducted to investigate whether TLR4 Asp299Gly was associated with TB. Results: LPS-induced tumour necrosis factor, interleukin-1 β and interleukin-10 production was not influenced by TLR4 Asp299Gly genotype. There was no association between TLR4 Asp299Gly and TB. Conclusion: Our data suggest that TLR4 Asp299Gly has no influence on monocyte LPS responses or susceptibility to TB in Gambians and could be an ancient neutral polymorphism.

Imidocarb, a potent anti-protozoan drug, up-regulates interleukin-10 production by murine macrophages

Interleukin-10 (IL-10), a potent antiinflammatory and immunosuppressive cytokine, plays an important role in the regulation of immune responses. To discover small molecules that stimulate IL-10 production, a cell-based screening assay was performed using a murine macrophage cell line, RAW264.7. Imidocarb, (3,3 ′-bis-2-imidazolin-2-yl)-carbanilide, which has been used as an anti-protozoan drug for the prevention and treatment of babesiosis in cattle, was thus identified. Imidocarb markedly enhanced LPS-induced IL-10 production not only by RAW264.7 cells but also by murine peritoneal macrophages in a concentration-dependent manner. It also augmented IL-10 production in the presence of zymosan, a yeast cell wall component. In contrast, imidocarb inhibited LPS-induced tumor necrosis factor (TNF)-α production by peritoneal macrophages. In mice, intraperitoneal administration of imidocarb significantly increased serum IL-10 levels and lowered TNF-α levels. Our results suggest that a novel anti-inflammatory property of imidocarb could lead to new therapeutic approaches in inflammatory conditions.

Platelet-activating factor acetylhydrolase inhibits alveolar macrophage activation in vivo.

Platelet-activating factor (PAF) is an important proinflammatory mediator of septic shock. PAF is produced by activated macrophages and acts to perpetuate the response to endotoxin. PAF is metabolized by an endogenous PAF-acetylhydrolase (PAF-AH). Low circulating levels of PAF-AH have been associated with the development of postinjury multiple organ failure. We have previously shown that PAF-AH significantly inhibits macrophage activation by lipopolysaccharide (LPS) in vitro. The purpose of these studies was to determine whether this effect would translate to an in vivo model of remote lung injury. Wistar rats were administered a single intravenous dose of PAF-AH (5 mg/kg) or its carrier solution simultaneous with the induction of zymosan peritonitis. After 24 h, alveolar macrophages were obtained by bronchoalveolar lavage and stimulated in vitro with LPS (1 microg/mL). Supernatants were collected at 18 h for cytokine production and cellular and nuclear protein extractions were performed at 30 and 60 min to assess the activation of p38 and extracellular signal-regulated kinase (ERK) 1/2 kinases and the nuclear translocation of nuclear factor (NF)-kappaB. Administration of PAF-AH significantly inhibited LPS-induced tumor necrosis factor alpha and interleukin-1beta production by alveolar macrophages from zymosan-treated animals. This functional inhibition was associated with inhibition of ERK 1/2 kinase and NF-kappaB activation but not p38 kinase activation. Interleukin 6 production was depressed in the macrophages from zymosan-treated animals but no additional inhibition resulted from PAF-AH treatment. In conclusion, zymosan peritonitis leads to priming of alveolar macrophages such that their subsequent tumor necrosis factor alpha response to LPS is enhanced. In vivo administration of PAF-AH abrogates this response, suggesting that this priming may be PAF dependent. This effect of PAF-AH may be mediated by the inhibition of intracellular signaling via inhibition of ERK kinase and NF-kappaB activation.

Safety, Pharmacokinetics, and Pharmacodynamics of E5564, a Lipid a Antagonist, during an Ascending Single-Dose Clinical Study

E5564, a structural analog of the lipid A portion of lipopolysaccharide (LPS), is a potent antagonist of the biochemical and physiologic effects of LPS in several in vitro and in vivo models and is currently under clinical development as a possible therapeutic for the treatment of sepsis and septic shock. The objectives of this study were to (1) assess the safety and tolerability of E5564 following a 30-minute intravenous (IV) infusion, (2) evaluate the pharmacokinetic profile of E5564, and (3) measure the ability of E5564 to block LPS stimulation ex vivo in blood taken from subjects up to 8 hours after ending the infusion. Healthy male volunteers (n= 7/dose group) were randomly assigned to each of four dose levels (350, 1000, 2000, or 3500 μg). Within each dose group, 5 subjects received drug and 2 received placebo. E5564 or matching placebo was administered by a 30-minute infusion, and blood samples were collected at predetermined time points. All doses of E5564 were demonstrated to be safe and well tolerated. E5564 plasma concentrations were determined using a validated LC/MS/MS method. The Cmax and AUC of E5564 increased in a dose-proportional manner. E5564 pharma- cokinetics were characterized by a slow clearance (0.67–0.95 mL/h/kg), a small volume of distribution (41–54 mL/kg), and a relatively long elimination half-life (42–51 h). As measured in the ex vivo assay, E5564 inhibited LPS-induced tumor necrosis factor–α (TNF-α) in a dose-dependent manner, and at the higher doses (2 and 3.5 mg), antagonistic activity was measurable up to 8 hours postinfusion. E5564 lacked LPS-like agonist activity at doses up to 3.5 mg. Taken together, we believe that E5564 is a safe, potent antagonist of LPS in blood and will likely benefit patients in the treatment of LPS-related diseases.

Taurine supplementation induces multidrug resistance protein 2 and bile salt export pump expression in rats and prevents endotoxin-induced cholestasis

The effect of oral taurine supplementation on endotoxin-induced cholestasis was investigated in rat liver. At 12 h following lipopolysaccharide (LPS) injection (4 mg/kg body weight i.p.) bile flow and bromosulfophthalein (BSP) and taurocholate (TC) excretion were determined in the perfused liver and the expression of the canalicular transporters multidrug resistance protein 2 (Mrp2) and bile salt export pump (Bsep) was analyzed. Injection of LPS induced a significant decrease of bile flow ( 2.2±0.2 μl/ g liver wet weight/min vs 3.3±0.1 μl/ g liver wet weight in controls), biliary BSP excretion (10.8±2.2 nmol/g/min vs 21.0±3.8 nmol/g/min), and biliary TC excretion (114±23 nmol/g/min vs 228±8 nmol/g/min). These effects were due to transporter retrieval from the canalicular membrane and downregulation of Mrp2 and Bsep expression. In taurine-supplemented rats bile flow was 30% higher than that in untreated rats and the expression of Mrp2 and Bsep protein was increased two- to threefold. In taurine-supplemented rats there was no significant reduction of bile flow or of BSP and TC excretion at 12 h following LPS injection. This protective effect of taurine was due to higher Mrp2 and Bsep protein levels compared to nonsupplemented LPS-treated rats, whereas relative Mrp2 retrieval from the canalicular membrane induced by LPS was not significantly different. LPS-induced tumor necrosis factor α and interleukin-1β release were lower in taurine-fed rats; however, downregulation of Mrp2 and Bsep expression by LPS was delayed but not prevented. The data show that oral supplementation of taurine induces Mrp2 and Bsep expression and may prevent LPS-induced cholestasis.

Interferon-gamma enhances the pulmonary CXC chemokine response to intratracheal lipopolysaccharide challenge.

CXC chemokines are major chemoattractants for pulmonary polymorphonuclear leukocyte (PMNL) recruitment. To study the effects of interferon (IFN)-gamma on the pulmonary chemokine response to lipopolysaccharide (LPS) challenge, rats were treated with intratracheal IFN-gamma (1x10(5) U/rat) 24 h before an intratracheal LPS (100 microg/rat) challenge. Intratracheal LPS caused significant increases in both cytokine-induced neutrophil chemoattractant (CINC) and macrophage inflammatory protein-2 in bronchoalveolar lavage (BAL) fluid and pulmonary PMNL recruitment. IFN-gamma enhanced these responses. IFN-gamma also increased LPS-induced tumor necrosis factor (TNF)-alpha in BAL fluid. LPS-induced TNF-alpha and CINC mRNA expression in alveolar macrophages was increased by IFN-gamma. CD11b/c and CD18 expression on circulating PMNLs was not affected by IFN-gamma, nor was the chemotaxis of these cells. IFN-gamma increases the pulmonary CXC chemokine response, which may serve as one mechanism underlying enhanced PMNL delivery into the lung.

Pavlovian conditioning of endotoxin-tolerance in rats

The most fascinating example of the bi-directional interaction between the central nervous system (CNS) and immune system is the behavioral conditioning of immune functions. We therefore investigated the behavioral conditioning of lipopolysaccharide (LPS)-induced endotoxin tolerance using the taste aversion paradigm. The conditioned stimulus (CS) saccharin was paired with the unconditioned stimulus (UCS) LPS over a five (CONDl) or four (COND2) days learning trial. Controls received drinking water with (SHAM) or without (UNT) LPS. Endotoxin tolerance was tested by determination of LPS-induced tumor necrosis factor (TNF)-α release. After the avoidance of the induced endotoxin-tolerance the CS saccharin was re-presented in all experimental groups. A the end of the re-exposure period a complete endotoxin tolerance was noticed in the CONDl- and COND2-group. In contrast, no effect of saccharin administration was observed in the SHAM- or UNT-group. Our data demonstrate for the first time the behavioral conditioning of endotoxin tolerance. Furthermore, these results contribute new aspects to the mechanisms underlying the development and modulation of endotoxin tolerance.

Preclinical and clinical assessment of the safety and potential efficacy of thalidomide in heart failure

Background: Inflammatory mediators, especially tumor necrosis factor (TNF), have been implicated in heart failure (HF). Thalidomide has anti-inflammatory properties and selectively inhibits TNF. Thus far, thalidomide or thalidomide analogues have not been evaluated in patients with heart failure. Methods: Thalidomide was assessed in preclinical and clinical studies. First, isolated cardiac myocytes were pretreated with thalidomide or thalidomide analogues, and TNF production was assessed after lipopolysaccharide (LPS) provocation. Second, to determine the safety and potential efficacy of thalidomide, an open-label dose escalation safety study was conducted in seven patients with advanced heart failure. Results: Thalidomide and thalidomide analogues inhibited LPS-induced TNF biosynthesis in cardiac myocytes in a dose-dependent manner. Thalidomide analogues had a greater inhibitory effect on TNF production than did thalidomide. In patients with advanced HF, thalidomide was safe and potentially effective when used at lower doses. However, dose-limiting toxicity was observed in two patients. There was a significant increase in the 6-minute walk distance and a trend toward improvement in left ventricular ejection fraction and quality of life after 12 weeks of maintenance therapy with thalidomide. Conclusions: Taken together these results suggest that thalidomide or its derivatives may be useful in selected patients with HF. This potential needs to be studied in larger clinical trials.

S-Adenosylmethionine, cytokines, and alcoholic liver disease

Hepatic deficiency of S-adenosylmethionine (AdoMet) is a critical acquired metabolic abnormality in alcoholic liver disease (ALD) and in many experimental models of hepatotoxicity. Subnormal AdoMet, elevated serum tumor necrosis factor (TNF), and endotoxemia (LPS) are hallmarks of ALD and experimental liver injury. AdoMet deficiency is attributed to its subnormal synthesis, but mechanisms for increased TNF are not known. AdoMet deficiency may affect the critical balance of proinflammatory (e.g., TNF) and antiinflammatory [e.g., interleukin (IL)-10] cytokines. Rats maintained on a choline-deficient diet with limited amounts of methionine (MCD diet) developed AdoMet deficiency. When challenged with LPS, rats fed MCD diet had significantly increased serum TNF levels and worse liver injury compared with findings for controls. Exogenous AdoMet attenuated liver injury and serum TNF levels. Results of in vitro studies with the use of RAW 264.7 cells demonstrated that exogenous AdoMet supplementation lowered LPS-induced TNF formation in a dose-dependent manner, and AdoMet deficiency enhanced TNF secretion and TNF gene expression. AdoMet also dose-dependently decreased LPS-stimulated TNF production from monocytes obtained from patients with alcoholic hepatitis. Finally, AdoMet supplementation stimulated production of the antiinflammatory cytokine IL-10. Interleukin-10 plays a critical role in the modulation of TNF production, and IL-10 may inhibit hepatic fibrosis. This article will review (1) the role of AdoMet in ALD/liver injury, (2) the role of TNF/proinflammatory cytokines in ALD, (3) potential roles of AdoMet in TNF/proinflammatory cytokine regulation in ALD, and (4) conclusions and future directions.