Dynamics of cytokine production in human peripheral blood mononuclear cells stimulated by LPS or infected by Borrelia

To specify the role of individual cytokines in the immune response to pyrogens, isolated and cultivated human peripheral blood mononuclear cells (PBMC) were used for the experiments. Different pyrogens (lipopolysaccharide from Escherichia coli - LPS and live Borrelia afzelii) were applied and the time course of changes in concentrations of different cytokines in the medium was followed using the ELISA method. It was found that nonstimulated human PBMC proliferate under in vitro conditions and produce IL-6, TNF-alpha, IL-10 and finally also IL-1beta. Productions of IL-12 and INF-gamma are not changed. Proliferation of PBMC is potentiated after incubation with LPS or live Borrelia. PBMC stimulated by LPS increase the net production (stimulated minus unstimulated) of IL-1beta and TNF-alpha significantly, while production of IL-6 was smaller. A delayed increase in the production of IL-10 was also observed. Productions of IL-12 and INF-gamma were not influenced. In contrast to LPS, stimulation of PBMC with live Borrelia, increases also the production of IL-12 and IFN-gamma, besides IL-1beta, TNF-alpha, IL-6 and IL-10. Productions of IL-1beta, IL-6 and TNFalpha increased immediately after incubation with both LPS and Borrelia, while productions of IL-12 and INF-gamma begin to increase 8 hours and production of IL-10 12 hours after stimulation. Data indicate that stimulation with different pyrogens may activate the cells of the immune cascade in a different way. Stimulation of BPMC by LPS seems to activate the initial steps of the immune response (macrophages and granulocytes) only, while infection with live Borrelia also stimulates the later phase of the immune response, probably due to effect of initially produced cytokines.     

Dynamics of cytokine production in human peripheral blood mononuclear cells stimulated by LPS or infected by Borrelia

To specify the role of individual cytokines in the immune response to pyrogens, isolated and cultivated human peripheral blood mononuclear cells (PBMC) were used for the experiments. Different pyrogens (lipopolysaccharide from Escherichia coli - LPS and live Borrelia afzelii) were applied and the time course of changes in concentrations of different cytokines in the medium was followed using the ELISA method. It was found that nonstimulated human PBMC proliferate under in vitro conditions and produce IL-6, TNF-alpha, IL-10 and finally also IL-1 beta. Productions of IL-12 and INF-gamma are not changed. Proliferation of PBMC is potentiated after incubation with LPS or live Borrelia. PBMC stimulated by LPS increase the net production (stimulated minus unstimulated) of IL-1 beta and TNF-alpha significantly, while production of IL-6 was smaller. A delayed increase in the production of IL-10 was also observed. Productions of IL-12 and INF-gamma were not influenced. In contrast to LPS, stimulation of PBMC with live Borrelia, increases also the production of IL-12 and IFN-gamma, besides IL-1 beta, TNF-alpha, IL-6 and IL-10. Productions of IL-1 beta, IL-6 and TNF alpha increased immediately after incubation with both LPS and Borrelia, while productions of IL-12 and INF-gamma begin to increase 8 hours and production of IL-10 12 hours after stimulation. Data indicate that stimulation with different pyrogens may activate the cells of the immune cascade in a different way. Stimulation of BPMC by LPS seems to activate the initial steps of the immune response (macrophages and granulocytes) only, while infection with live Borrelia also stimulates the later phase of the immune response, probably due to effect of initially produced cytokines.     

Direct stimulation of cytokines (IL-1 beta, TNF-alpha, IL-6, IL-2, IFN-gamma and GM-CSF) in whole blood. I. Comparison with isolated PBMC stimulation.

Production of interleukin 1 beta (IL-1 beta), interleukin 6 (IL-6), tumor necrosis factor alpha (TNF-alpha), interleukin 2 (IL-2), interferon gamma (IFN-gamma) and granulocyte-macrophage colony-stimulating factor (GM-CSF) after stimulation by lipopolysaccharide (LPS) and phytohemagglutinin (PHA) was studied in 1/10 diluted whole blood (WB) culture and in peripheral blood mononuclear cell (PBMC) culture. Cytokines IL-1 beta, TNF-alpha and IL-6 are preferentially stimulated by LPS whereas IL-2, IFN-gamma and GM-CSF are stimulated by PHA. Combination of 5 micrograms/ml PHA and 25 micrograms/ml LPS gave the most reliable production of the six cytokines studied. IL-1 beta, TNF-alpha and IL-6 represent a homogeneous group of early-produced cytokines positively correlated among themselves and with the number of monocytes in the culture (LeuM3). Furthermore, IL-1 beta was negatively correlated with the number of T8 lymphocytes. IL-2, IFN-gamma and GM-CSF represent a group of late-produced cytokines. Kinetics and production levels of IL-6 and GM-CSF are similar in WB and PBMC cultures. In contrast, production levels of TNF-alpha and IFN-gamma are higher in WB than in PBMC whereas production levels of IL-6 and IL-2 are lower in WB than in PBMC. Individual variation in responses to PHA + LPS was always higher in PBMC cultures than in WB cultures. The capacity of cytokine production in relation to the number of mononuclear cells is higher in WB, or in PBMC having the same mononuclear cell concentration as WB, than in conventional cultures of concentrated PBMC (10(6)/ml). Because it mimics the natural environment, diluted WB culture may be the most appropriate milieu in which to study cytokine production in vitro.     

Modulation of lipopolysaccharide-induced production of tumor necrosis factor, interleukin 1, and interleukin 6 by synthetic precursor Ia of lipid A

Abstract Endotoxin (lipopolysaccharide, LPS) induces the production of mediators of inflammation, which exerts pathophysiological effects such as fever or shock in mammals. In the present study we have investigated the modulation of LPS by the synthetic non-active tetraacylated precursor Ia of lipid A (compound 406) in the induction of tumor necrosis factor (TNF), interleukin 1 (IL-1) and interleukin 6 (IL-6) in human peripheral blood mononuclear cells (PBMC) and in human peripheral blood monocytes (PBMo). PBMC stimulated with LPS released TNF in a concentration dependent manner. Release of biologically active TNF, IL-1 and IL-6 was first detectable 4 h after LPS stimulation. Compound 406 alone in all concentrations tested did not induce TNF, IL-1 or IL-6 release, intracellular TNF or IL-1β, or mRNA for TNF or IL-1. Added to PBMC 1 h before LPS compound 406 enhanced or suppressed TNF release and suppressed IL-1 and IL-6 release depending on the ratio of concentrations between stimulator (LPS) and modulator (compound 406). In contrast to LPS stimulation alone TNF, IL-1 and IL-6 release in presence of compound 406 was delayed and first detectable after 6 to 8 h. Compound 406 was able to suppress LPS-induced intracellular TNF and IL-1β in PBMC. Added to PBMo 1 h before LPS it totally inhibited the production of mRNA for TNF and IL-1. When added to PBMC 1 h after LPS, TNF release was suppressed in a concentration-dependent way and release of biologically active TNF, IL-1 and IL-6 could again be detected for the first time after 4 h. Compound 406 was not able to inhibit phorbol 12-myristate 13-acetate (PMA)-induced TNF and IL-1 release in PBMo which suggests that its modulating effect is LPS-specific. This study provides evidence that the modulating effect of compound 406 on the LPS induction of TNF, IL-, 1 and IL-6 could be due to competitive binding.     

Initial characterization of a lymphokine pathway for the immunologic induction of tumor necrosis factor-alpha release from human peripheral blood mononuclear cells

Under endotoxin-free conditions, unstimulated human PBMC do not release TNF-alpha, as measured in a sensitive assay with 51Cr release in 6 h from actinomycin D-treated WEHI 164 cells. IFN-gamma alone at less than or equal to 10,000 U/ml is insufficient to elicit TNF-alpha release. Similarly, the lymphokine-rich supernatant of PBMC stimulated by allogeneic cells is also insufficient to induce TNF-alpha release in a short term assay. However, when PBMC are first primed with IFN-gamma for 48 h and then exposed to lymphokine supernatant for 6 h, effector cells within the PBMC population are triggered to express TNF-alpha-mediated cytotoxicity. All of the measured cytotoxicity is attributable to TNF-alpha because it could be abolished by a specific anti-TNF-alpha neutralizing mAb. Although IFN-gamma serves to prime PBMC in this assay system, it fails to trigger the release of TNF-alpha. Instead, a second lymphokine (provisionally termed "cytotoxicity triggering factor" (CTF) is required to induce TNF-alpha release from IFN-gamma-primed human PBMC. In kinetic studies, IFN-gamma priming was optimal when PBMC were exposed to IFN-gamma (150 U/ml) for 48 h. In contrast to the prolonged interval for priming, CTF need be present for 6 h or less for maximal induction of TNF-alpha-mediated cytotoxicity. In dose-response studies, IFN-gamma priming (48 h) required at least 4 U/ml and was complete with 20 to 100 U/ml. By using fully primed PBMC, the response to CTF followed a sigmoidal dose-response curve, which allowed the quantitation of CTF in half-maximal units. Activated Th lymphocytes constitute one cellular source for CTF. CTF is produced by cloned allorective T3+T4+T8-M1- Th cells after alloantigen stimulation, and also by nylon wool-purified T cells after stimulation with PMA and A23187 calcium ionophore. Unstimulated T cells do not release CTF. In physicochemical studies, CTF activity elutes from Sephadex G-100 as a major discrete peak of Mr 55 kDa and minor peaks of 14 kDa and greater than 150 kDa. On the basis of multiple criteria, CTF is distinguishable from several other cytokines: IFN-gamma, IL-1, IL-2, GM-CSF, MIF, CSF-1, TNF-alpha, and lymphotoxin (TNF-beta). We conclude that, by acting together, IFN-gamma and CTF provide a lymphokine pathway whereby Ag-responsive human Th cells induce the immunologic release of TNF-alpha from effector cells present in PBMC.     

Differential regulation of cytokine release and leukocyte migration by lipopolysaccharide-stimulated primary human lung alveolar type II epithelial cells and macrophages

Bacterial colonization is a secondary feature of many lung disorders associated with elevated cytokine levels and increased leukocyte recruitment. We hypothesized that, alongside macrophages, the epithelium would be an important source of these mediators. We investigated the effect of LPS (0, 10, 100, and 1000 ng/ml LPS, up to 24 h) on primary human lung macrophages and alveolar type II epithelial cells (ATII; isolated from resected lung tissue). Although macrophages produced higher levels of the cytokines TNF-alpha and IL-1beta (p < 0.0001), ATII cells produced higher levels of chemokines MCP-1, IL-8, and growth-related oncogene alpha (p < 0.001), in a time- and concentration-dependent manner. Macrophage (but not ATII cell) responses to LPS required activation of ERK1/2 and p38 MAPK signaling cascades; phosphorylated ERK1/2 was constitutively up-regulated in ATII cells. Blocking Abs to TNF-alpha and IL-1beta during LPS exposure showed that ATII cell (not macrophage) MCP-1 release depended on the autocrine effects of IL-1beta and TNF-alpha (p < 0.003, 24 h). ATII cell release of IL-6 depended on autocrine effects of TNF-alpha (p < 0.006, 24 h). Macrophage IL-6 release was most effectively inhibited when both TNF-alpha and IL-1beta were blocked (p < 0.03, 24 h). Conditioned media from ATII cells stimulated more leukocyte migration in vitro than conditioned media from macrophages (p < 0.0002). These results show differential activation of cytokine and chemokine release by ATII cells and macrophages following LPS exposure. Activated alveolar epithelium is an important source of chemokines that orchestrate leukocyte migration to the peripheral lung; early release of TNF-alpha and IL-1beta by stimulated macrophages may contribute to alveolar epithelial cell activation and chemokine production.     

Gold sodium thiomalate (GSTM) inhibits lipopolysaccharide stimulated tumor necrosis factor-alpha through ceramide pathway

TNF-alpha has emerged as the major pro-inflammatory cytokine involved in the pathogenesis of rheumatoid arthritis (RA). LPS is a potent stimulator of TNF-alpha production by human monocytes. Ceramide, a structural homolog of LPS and a second messenger in the sphingomyelin signal transduction pathway has been shown to stimulate TNF-alpha production from murine macrophages. We have previously shown that GSTM, an anti-rheumatic drug inhibits LPS stimulated TNF-alpha production by normal PBMCs. We studied the ability of ceramide to stimulate TNF-alpha production by human PBMCs and the mechanism of action of GSTM on ceramide and LPS induced TNF-alpha production. LPS induced significant TNF-alpha production in PBMCs and THP-1. However, C(2) ceramide stimulated TNF-alpha production in 5 of 10 PBMCs (ceramide responder); it did not do so in the other 5 PBMCs (ceramide non-responder) or the THP-1 cell line. GSTM inhibited LPS stimulated TNF-alpha productions in PBMCs of all 5 ceramide responders both at protein and mRNA expression level. We also found that GSTM inhibited LPS induced NF-kappaB level only in ceramide responder. Thus, we for the first time report that GSTM inhibits LPS stimulated TNF-alpha production through ceramide pathway and anti-inflammatory activity of GSTM in treatment of RA may depend on its ability to inhibit NF-kappaB activation and TNF-alpha production.     

The development of novel inhibitors of tumor necrosis factor-alpha (TNF-alpha) production based on substituted [5,5]-bicyclic pyrazolones

Novel substituted [5,5]-bicyclic pyrzazolones are presented as inhibitors of tumor necrosis factor-alpha (TNF-alpha) production. Many of these compounds show low nanomolar activity against lipopolysaccaride (LPS)-induced TNF-alpha production in THP-1 cells. This class of molecules was co-crystallized with mutated p38, and several analogs showed good oral bioavailability in the rat. Oral activity of these compounds in the rat iodoacetate model for osteoarthritis is discussed.     

Expression of IL-8 gene in human monocytes and lymphocytes: differential regulation by TNF and IL-1

TNF-alpha and IL-1 were reported to be the most powerful inducers of IL-8 in a multitude of cells, including leukocytes. In this study, we investigated TNF-alpha- and IL-1-mediated regulation of IL-8 gene expression in non-fractionated PBMC, and purified monocyte (MO) and lymphocyte (LY) fractions. Our analysis revealed that purified human MO did not respond to exogenous TNF-alpha with the induction of IL-8 mRNA or protein, nor require endogenous TNF-alpha for IL-8 expression. In contrast, in the presence of exogenous IL-1alpha and IL-1beta a substantial enhancement of IL-8 mRNA and protein expression in MO was observed. Nevertheless, antibodies to IL-1alpha and IL-1beta were unable to downregulate the expression of IL-8 in resting adherent or Staphylococcus aureus Cowan 1 (SAC)-stimulated MO. In contrast with MO, purified LY and non-fractionated PBMC expressed IL-8 in response to exogenous TNF-alpha, similar to exogenous IL-1alpha and IL-1beta. As was seen with MO, antibodies to TNF-alpha, IL-1alpha and IL-1beta did not inhibit the expression of IL-8 in purified LY and non-fractionated PBMC stimulated with SAC and LPS. Taken together, our data demonstrate major differences in responsiveness of MO and LY to exogenous TNF-alpha and IL-1, and suggest relative autonomy of IL-8 gene expression in these cells that does not require accessory cytokines but can be induced directly by exogenous stimuli.     

Surfactant strengthens the inhibitory effect of C-reactive protein on human lung macrophage cytokine release

In this study we investigated the effect of acute-phase levels of C-reactive protein (CRP) on cytokine production by pulmonary macrophages in the presence or absence of pulmonary surfactant. Both human alveolar and interstitial macrophages as well as human surfactant were obtained from multiple organ donor lungs. Precultured macrophages were stimulated with LPS alone or together with IFN-gamma in the presence or absence of CRP, surfactant, and combinations. Releases of TNF-alpha and of IL-1beta to the medium were determined. We found that CRP could modulate lung inflammation in humans by decreasing the production of proinflammatory cytokines by both alveolar and interstitial macrophages stimulated with LPS alone or together with IFN-gamma. The potential interaction between CRP and surfactant phospholipids did not overcome the effect of either CRP or surfactant on TNF-alpha and IL-1beta release by lung macrophages. On the contrary, CRP and pulmonary surfactant together had a greater inhibitory effect than either alone on the release of proinflammatory cytokines by lung macrophages.     

Conjugated linoleic acid can prevent tumor necrosis factor gene expression by inhibiting nuclear factor binding activity in peripheral blood mononuclear cells from weaned pigs challenged with lipopolysaccharide

Twenty-four weanling barrows were fed corn-soybean diets supplemented with 2% conjugated linolenic acid (CLA) or soybean oil. On day 14 and 21, pigs were injected intraperitoneally with lipopolysaccharide (LPS) or sterile saline. Plasma samples were collected 2h after injection. Peripheral blood mononuclear cells (PBMC) were also collected on day 21, 2 h after injection to determine tumor necrosis factor-alpha (TNF-alpha) production and its mRNA expression. The results indicate that dietary CLA inhibited the production of TNF-alpha by pig PBMC both at the protein and mRNA expression level. In a second experiment, PBMC, collected from a healthy pig, were incubated with either c9,t11-CLA or t10,c12-CLA, or without CLA and stimulated with LPS. Both CLA isomers inhibited LPS-stimulated TNF-alpha production and expression, which may be partially due to inhibition of the binding activity of nuclear factor-kappaB. The t10,c12 isomer was more effective than the c9,t11-CLA isomer in reducing TNF-alpha levels and nuclear factor-kappaB activation.     

Porphyromonas gingivalis lipopolysaccharide induces shedding of syndecan-1 expressed by gingival epithelial cells

Syndecans are constitutively shed from growing epithelial cells as the part of normal cell surface turnover. However, increased serum levels of the soluble syndecan ectodomain have been reported to occur during bacterial infections. The aim of this study was to evaluate the potential of lipopolysaccharide (LPS) from the periodontopathogen Porphyromonas gingivalis to induce the shedding of syndecan-1 expressed by human gingival epithelial cells. We showed that the syndecan-1 ectodomain is constitutively shed from the cell surface of human gingival epithelial cells. This constitutive shedding corresponding to the basal level of soluble syndecan-1 ectodomain was significantly increased when cells were stimulated with P. gingivalis LPS and reached a level comparable to that caused by phorbol myristic acid (PMA), an activator of protein kinase C (PKC) which is well known as a shedding agonist. The syndecan-1 shedding was paralleled by pro-inflammatory cytokine interleukin-1 beta (IL-1beta), IL-6, IL-8, and tumor necrosis factor alpha (TNF-alpha) release. Indeed, secretion of IL-1beta and TNF-alpha increased following stimulation by P. gingivalis LPS and PMA, respectively. When recombinant forms of these proteins were added to the cell culture, they induced a concentration-dependent increase in syndecan-1 ectodomain shedding. A treatment with IL-1beta converting enzyme (ICE) specific inhibitor prevented IL-1beta secretion by epithelial cells stimulated by P. gingivalis LPS and decreased the levels of shed syndecan-1 ectodomain. We also observed that PMA and TNF-alpha stimulated matrix metalloproteinase-9 secretion, whereas IL-1beta and P. gingivalis LPS did not. Our results demonstrated that P. gingivalis LPS stimulated syndecan-1 shedding, a phenomenon that may be mediated in part by IL-1beta, leading to an activation of intracellular signaling pathways different from those involved in PMA stimulation.     

Relationship between immune system and gram-negative bacteria: monocyte chemotaxis induced by supernatants from human peripheral blood OKT8+ lymphocytes stimulated with smooth and rough Salmonella strains

It has been recently reported that smooth (S) Salmonella typhimurium LT-2 and rough (R) mutants, S. minnesota R345 (Rb) and R595 (Re), spontaneously adhere to human peripheral blood mononuclear cells (PBMC). The binding is confined to T cells and is mediated by the lipopolysaccharide (LPS) moiety of the bacteria outer membrane. In this study, we have investigated the monocyte chemotactic responsiveness induced by supernatants recovered from human PBMC stimulated with either S or R Salmonella strains. All supernatants were able to trigger monocyte chemotaxis, even if to a different degree according to the bacterial strain used. These data were further supported by experiments which showed that stimulation of PBMC by purified homologous LPS led to the release of a chemotactic lymphokine (CLK) for human monocytes. Moreover, this CLK seems to be released by T cells, and in particular OKT8+ cells, since OKT8- PBMC failed to produce CLK.     

Splenectomy differentially influences immune responses in various tissue compartments of the body

Patients without spleens have an increased risk of infection. Previous studies have shown that splenectomy (Spx) influences Kupffer cells (KC), peritoneal macrophages (pMphi) and alveolar macrophages (aMphi) phagocytosis and bactericidal activity. This study examined the effect of Spx on the cytokine production by peripheral blood monocular cells (PBMC), KC, aMphi, pMphi and cells from mesenteric lymph nodes (MLN). We also determined if Spx influences survival following sepsis induced by cecal ligation and puncture (CLP). C57BL/6J male mice (8-10 weeks old) underwent sham operation or Spx. Two weeks after sham or Spx, KC, pMphi, aMphi, PBMC and cells from MLN were isolated and stimulated with LPS. Cell-free supernatants were analyzed for TNF-alpha, IL-6, and IL-10. A significant increase in KC, pMphi and aMphi TNF-alpha and IL-6 release was observed following Spx. The production of IL-10 was also significantly higher in KC under those conditions. In contrast, the release of TNF-alpha, IL-6 and IL-10 was significantly decreased in PBMC after Spx. Similarly, TNF-alpha and IL-6 was also decreased in MLN after Spx. Overall survival after CLP was not different in mice subjected to either sham or Spx. However, the mean time to death was significantly decreased in mice subjected to Spx compared to sham injured mice. These findings suggest that Spx modulates immune cell functions in various tissue compartments of the body and results in early deaths following sepsis.     

Reverse signaling through transmembrane TNF confers resistance to lipopolysaccharide in human monocytes and macrophages

We have previously reported that the CD14+ monocytic subpopulation of human PBMC induces programmed cell death (apoptosis) in cocultured endothelial cells (EC) when stimulated by bacterial endotoxin (LPS). Apoptosis is mediated by two routes, first via transmembrane TNF-alpha (mTNF) expressed on PBMC and, in addition, by TNF-independent soluble factors that trigger apoptosis in EC. Neutralizing anti-TNF mAb completely blocked coculture-mediated apoptosis, despite the fact that there should have been additional soluble cell death factors. This led to the hypothesis that a reverse signal is transmitted from the TNF receptor on EC to monocytes (MO) via mTNF that prevents the production of soluble apoptotic factors. Here we have tested this hypothesis. The results support the idea of a bidirectional cross-talk between MO and EC. Peripheral blood MO, MO-derived macrophages (MPhi), or the monocytic cell line Mono Mac 6 were preincubated with human microvascular EC that constitutively express TNF receptor type I (TNF-R1) and subsequently stimulated with LPS. Cell-free supernatants of these preparations no longer induced EC apoptosis. The preincubation of MO/MPhi with TNF-reactive agents, such as mAb and soluble receptors, also blocked the production of death factors, providing further evidence for reverse signaling via mTNF. Finally, we show that reverse signaling through mTNF mediated LPS resistance in MO/MPhi as indicated by the down-regulation of LPS-induced soluble TNF and IL-6 as well as IL-1 and IL-10.     

The stimulating action of lipopolysaccharide and muramyl dipeptide on the activation of mononuclear cells in human peripheral blood by recombinant interleukin-2 in vitro

Muramyl dipeptide (MDP) and lipopolysaccharide (LPS) were effective in augmentation of killer cells generation from human peripheral blood mononuclear cells (PBMC) in response to recombinant human interleukin-2 (IL-2). Pretreatment of PBMC with combination of LPS and MDP resulted in most significant their proliferation stimulated by IL-2. Thus our results show the enhancement of PBMC sensitivity to IL-2 by action of LPS, MDP and most of all by their combination in vitro.     

Regulated expression of galectin-1 during T-cell activation involves Lck and Fyn kinases and signaling through MEK1/ERK,p38 MAP kinase and p70S6 kinase

It has been shown that staphylococcal enterotoxin A (SEA) acts through human peripheral blood mononuclear cells (PBMC) to stimulate synthesis or release of pyrogenic cytokines. Nuclear factor-kappa B (NF-kappaB) is thought to play an important role in inflammatory responses through the regulation of genes encoding pro-inflammatory cytokines. The purpose of the present study was to determine whether the NF-kappaB mechanisms in human PBMC are involved in SEA-induced fever. Western blot evaluation revealed SEA was able to induce nuclear translocation of NF-kappaB from cytosol to nucleus in PBMC, which could be abolished by a NF-kappaB inhibitor such as pyrrolidine dithiocarbamate (PDTC), sodium pyrithione (Pyri), N-acetyl-L-cysteine (NAC), or curcumin (Cur). Electrophoretic mobility shift assay also showed that the NF-kappaB DNA-binding activity was increased in the SEA-treated PBMC. Again, the SEA-induced increased NF-kappaB binding activity was significantly attenuated by either PDTC, Pyri, NAC or Cur. The pyrogenic responses to supernatant fluids obtained from human PBMC stimulated with SEA were associated with increased levels of interleukin 1-beta (IL-1beta), IL-6, and tumor necrosis factor-alpha (TNF-alpha) in the supernatant fluids. Both the fever and the increased levels of IL-1beta, IL-6, and TNF-alpha in supernatant fluids obtained from the SEA-stimulated PBMC were decreased by incubating SEA-PBMC with either PDTC, Pyri, NAC, or Cur. Furthermore, the fever induced by systemic or central administration of SEA in rabbits were attenuated by pre-treatment with an systemic or central dose of either PDTC, Pyri, NAC, or Cur. The data indicate that inhibition of NF-kappaB prevents SEA-induced fever.