Mechanisms of the induction of apoptosis in human hepatoma cells by tumour necrosis factor-alpha.

Tumour necrosis factor alpha (TNF-alpha) at 20 ng/ml induced apoptosis in human hepatoma cells in vitro. The effect of TNF-alpha-induced apoptosis was exacerbated by the hypoxanthine-xanthine oxidase (HX/XO) system and cycloheximide (CHX), but alleviated by superoxide dismutase (SOD), suggesting that TNF-alpha-induced apoptosis may be due to oxidative stress, and independent of protein synthesis. TNF-alpha elevated free Ca(2+)concentration, triggered lipid peroxidation and decreased the expression of bcl-2 protein. The findings suggest that TNF-alpha-induced apoptosis may be involved in stimulating Ca(2+)-dependent endonuclease activity and increasing membrane lipid peroxidation. Bcl-2 may play a pivotal role in serving as a Ca(2+)regulator or antioxidant, preventing lipid peroxidation in the process.     

IL-6 is produced by osteoblasts and induces bone resorption

To examine the possible involvement of IL-6 in bone metabolism, a mouse osteoblastic cell line (MC3T3-E1) and primary osteoblast-like cells from fetal mouse calvaria were cultured with several systemic and local bone-resorbing agents and their expression of IL-6 mRNA was determined. Local bone-resorbing agents such as IL-1 alpha, IL-1 beta, TNF-alpha, and LPS greatly induced IL-6 mRNA expression in both MC3T3-E1 cells and primary osteoblast-like cells. Parathyroid hormone slightly increased expression of IL-6 mRNA in primary osteoblast-like cells but not in MC3T3-E1 cells. Neither IL-6 nor 1 alpha,25-dihydroxyvitamin D3 increased expression of IL-6 mRNA in either of the osteoblast-like cells. In agreement with the expression of IL-6 mRNA, biologically active IL-6 was produced in response to the treatment with IL-1 alpha, TNF-alpha, and LPS in MC3T3-E1 cells. Adding IL-6 dose dependently stimulated the release of 45Ca from prelabeled fetal mouse calvaria. Simultaneously adding suboptimal concentrations of IL-6 and IL-1 alpha induced bone resorption cooperatively. In accord with the increase in the release of 45Ca by IL-6, there were three times as many osteoclasts in the bone sections of calvaria cultured with IL-6 for 5 days as in the controls. IL-6 slightly suppressed alkaline phosphatase activity and collagen synthesis in MC3T3-E1 cells. These results indicate that IL-6 is also produced by osteoblasts, preferentially in response to local bone-resorbing agents, and it induces bone resorption both alone and in concert with other bone-resorbing agents.     

Gene, stimulus and cell-type specific regulation of activator protein-1 in mesangial cells by lipopolysaccharide and cytokines

Activator protein-1 (AP-1) plays an important role in the regulation of gene expression in mesangial cells (MC) during the pathogenesis of glomerular inflammatory disease. The precise regulation of the AP-1 family by agents that are known to activate MC is, however, poorly understood. The action of platelet-derived growth factor (PDGF) and, for the first time, lipopolysaccharide (LPS), interleukin-6 (IL-6), interferon-gamma (IFN-gamma) and tumour necrosis factor-alpha (TNF-alpha) on AP-1 gene expression in MC was therefore studied. Whilst the expression of JunD was not affected by any of the mediators, the mRNA levels of c-fos and JunB were induced by LPS, IL-6, IFN-gamma, PDGF and TNF-alpha, and that of c-jun by LPS, IFN-gamma, PDGF and TNF-alpha. Electrophoretic mobility shift assays showed a time-dependent increase in AP-1 DNA binding activity with JunB representing the major mediator-inducible member involved in DNA-protein interactions. However, stimulus-specific changes in the kinetics and magnitude of AP-1 mRNA expression and DNA binding activity were identified and, additionally, the results showed the potential existence of cell-type-specific mechanisms in the regulation of the AP-1 family. These studies provide novel insights into the mediator-specific modulation of AP-1-regulated gene expression and the activation of MC in renal diseases.     

Muramyl dipeptide enhances osteoclast formation induced by lipopolysaccharide, IL-1 alpha, and TNF-alpha through nucleotide-binding oligomerization domain 2-mediated signaling in osteoblasts

Muramyl dipeptide (MDP) is the minimal essential structural unit responsible for the immunoadjuvant activity of peptidoglycan. As well as bone-resorbing factors such as 1alpha,25-dihydroxyvitamin D3 (1alpha,25(OH)2D3) and PGE2, LPS and IL-1alpha stimulate osteoclast formation in mouse cocultures of primary osteoblasts and hemopoietic cells. MDP alone could not induce osteoclast formation in the coculture, but enhanced osteoclast formation induced by LPS, IL-1alpha, or TNF-alpha but not 1alpha,25(OH)2D3 or PGE2. MDP failed to enhance osteoclast formation from osteoclast progenitors induced by receptor activator of NF-kappaB ligand (RANKL) or TNF-alpha. MDP up-regulated RANKL expression in osteoblasts treated with LPS or TNF-alpha but not 1alpha,25(OH)2D3. Osteoblasts expressed mRNA of nucleotide-binding oligomerization domain 2 (Nod2), an intracellular sensor of MDP, in response to LPS, IL-1alpha, or TNF-alpha but not 1alpha,25(OH)2D3. Induction of Nod2 mRNA expression by LPS but not by TNF-alpha in osteoblasts was dependent on TLR4 and MyD88. MDP also enhanced TNF-alpha-induced osteoclast formation in cocultures prepared from Toll/IL-1R domain-containing adapter protein (TIRAP)-deficient mice through the up-regulation of RANKL mRNA expression in osteoblasts, suggesting that TLR2 is not involved in the MDP-induced osteoclast formation. The depletion of intracellular Nod2 by small interfering RNA blocked MDP-induced up-regulation of RANKL mRNA in osteoblasts. LPS and RANKL stimulated the survival of osteoclasts, and this effect was not enhanced by MDP. These results suggest that MDP synergistically enhances osteoclast formation induced by LPS, IL-1alpha, and TNF-alpha through RANKL expression in osteoblasts, and that Nod2-mediated signals are involved in the MDP-induced RANKL expression in osteoblasts.     

Cytokine-induced apoptosis in epithelial HT-29 cells is independent of nitric oxide formation. Evidence for an interleukin-13-driven phosphatidylinositol 3-kinase-dependent survival mechanism.

A combination of the pro-inflammatory cytokines interleukin (IL)-1alpha, interferon (IFN)-gamma, and tumor necrosis factor (TNF)-alpha induces nitric oxide synthase mRNA expression and nitric oxide (NO) generation in the human colon carcinoma cell line HT-29. This can be inhibited by pretreatment with IL-13 via a phosphatidylinositol (PI) 3-kinase-dependent mechanism (Wright, K., Ward, S. G., Kolios, G., and Westwick, J. (1997) J. Biol. Chem. 272, 12626-12633). Since NO has been implicated in regulating mechanisms leading to cell death, while activation of PI 3-kinase-dependent signaling cascades are thought to be involved with promoting cell survival events, we have investigated the outcome of these cytokine treatments on apoptosis and cell survival of HT-29 cells. Initiation of apoptosis can be achieved by the combinations of IFN-gamma/TNF-alpha, IFN-gamma/CD95, IL-1alpha/IFN-gamma, and IL-1alpha/IFN-gamma/TNF-alpha to varying extents. Induction of apoptotic markers by HT-29 cells in response to cytokine treatment is not dependent on NO production. Pretreatment with IL-13 protects against IL-1alpha/IFN-gamma/TNF-alpha- and IFN-gamma/TNF-alpha- as well as IFN-gamma/CD95-induced (but not IL-1alpha/IFN-gamma-induced) cell death. In addition, IFN-gamma/TNF-alpha and IL-1alpha/IFN-gamma/TNF-alpha stimulate activation of caspase-8 and caspase-3, which IL-13 pretreatment was able to partially inhibit and delay. IL-13 also stimulates activation of the major PI 3-kinase effector, protein kinase B. The PI 3-kinase inhibitors wortmannin and LY294002 inhibit IL-13 stimulation of protein kinase B as well as the cell survival effects of IL-13. These data demonstrate that cytokine-induced apoptosis of HT-29 cells is NO-independent and that the activation of a PI 3-kinase-dependent signaling cascade by IL-13 is a key signal responsible for the inhibition of apoptosis.     

Glutathione depletion is involved in the inhibition of procollagen alpha1(I) mRNA levels caused by TNF-alpha on hepatic stellate cells

TNF-alpha has been shown to inhibit procollagen alpha1(I) expression in hepatic stellate cells (HSC), although the molecular mechanisms involved have not been fully established. In the present work, we studied the possible role played by oxidative stress and NFkappaB on the antifibrogenic action of TNF-alpha on a cell line of rat HSC. Treatment of HSC with TNF-alpha did not affect either intracellular levels of reactive oxygen species or lipid peroxidation, but caused a decrease on reduced glutathione (GSH) levels. Restoration of intracellular GSH by incubation with exogenous GSH prevented the inhibition of procollagen alpha1(I) levels caused by TNF-alpha. The effect of GSH was not mimicked by antioxidants like deferoxamine, tempol or trolox. Activation of NFkappaB by TNF-alpha was also abolished by preincubation of HSC with GSH, but not by deferoxamine, tempol or trolox. These results point to GSH depletion as a mediator of TNF-alpha action in HSC.     

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.     

TNF-alpha and H2O2 induce IL-18 and IL-18R beta expression in cardiomyocytes via NF-kappa B activation

Myocardial ischemia/reperfusion is characterized by oxidative stress and induction of proinflammatory cytokines. Interleukin (IL)-18, a member of the IL-1 family, acts as a proinflammatory cytokine, and is induced during various immune and inflammatory disorders. Therefore, in the present study we investigated whether IL-18 expression is regulated by cytokines and oxidative stress in cardiomyocytes. TNF-alpha induced rapid and sustained activation of NF-kappaB whereas H(2)O(2) induced delayed and transient activation. Both TNF-alpha and H(2)O(2) induced IL-18 mRNA and precursor protein in cardiomyocytes, and IL-18 release into culture supernatants. However, only TNF-alpha led to sustained expression. Expression of IL-18Rbeta, but not alpha, was induced by both agonists. TNF-alpha and H(2)O(2) induced delayed expression of IL-18 BP. Pretreatment with PDTC attenuated TNF-alpha and H(2)O(2) induced IL-18 and IL-18Rbeta, but not basal expression of IL-18Ralpha. These results indicate that adult cardiomyocytes express IL-18 and its receptors, and proinflammatory cytokines and oxidative stress regulate their expression via activation of NF-kappaB. Presence of both ligand and receptors suggests IL-18 impacts myocardial biology through an autocrine pathway.     

Taraxacum officinale induces cytotoxicity through TNF-alpha and IL-1alpha secretion in Hep G2 cells

Taraxacum officinale (TO) has been frequently used as a remedy for women's disease (e.g. breast and uterus cancer) and disorders of the liver and gallbladder. Several earlier studies have indicated that TO exhibits anti-tumor properties, but its mechanism remains to be elucidated. In this study, we investigated the effect of TO on the cytotoxicity and production of cytokines in human hepatoma cell line, Hep G2. Our results show that TO decreased the cell viability by 26%, and significantly increased the tumor necrosis factor (TNF)-alpha and interleukin (IL)-1alpha production compared with media control (about 1.6-fold for TNF-alpha, and 2.4-fold for IL-1alpha, P < 0.05). Also, TO strongly induced apoptosis of Hep G2 cells as determined by flow cytometry. Increased amounts of TNF-alpha and IL-1alpha contributed to TO-induced apoptosis. Anti-TNF-alpha and IL-1alpha antibodies almost abolished it. These results suggest that TO induces cytotoxicity through TNF-alpha and IL-1alpha secretion in Hep G2 cells.     

Selective sensitization to tumor necrosis factor-alpha-induced apoptosis by blockade of NF-kappaB in primary glomerular mesangial cells.

Recent data have implicated nuclear factor kappaB (NF-kappaB) in the prevention of apoptosis in transformed cell lines exposed to tumor necrosis factor alpha (TNF-alpha). However, it is obscure whether NF-kappaB plays an anti-apoptotic role in nontransformed cells, and it is not clear whether NF-kappaB inhibits apoptosis triggered by other mediators. We investigated the effect of specific inhibition of NF-kappaB on cytokine-induced apoptosis of glomerular mesangial cells, which is important in determining the outcome of glomerulonephritis. Cultured rat mesangial cells were stably transfected with the dominant negative mutant inhibitor of NF-kappaB (IkappaBalphaM). IkappaBalphaM was resistant to stimulus-dependent degradation and suppressed NF-kappaB activation induced by TNF-alpha (10 ng/ml) or IL-1beta (10 ng/ml). IkappaBalphaM significantly sensitized mesangial cells to TNF-alpha-induced apoptosis in a dose- and time-dependent manner but had no significant effects on the level of apoptosis in the presence of proinflammatory or apoptosis-inducing stimuli including Fas ligand, IL-1alpha, IL-1beta, hydrogen peroxide, lipopolysaccharide, cycloheximide, or serum deprivation. Moreover, IkappaBalphaM-mediated sensitization to TNF-alpha overcame the protective effect of mesangial cell survival factors present in serum, which usually inhibit killing of mesangial cells by the proapoptotic stimuli used. These data show that inhibition of NF-kappaB selectively sensitizes primary adult glomerular mesangial cells to TNF-induced apoptosis but not to other mediators of cell death including the Fas ligand.     

Weak functional coupling of the melanocortin-1 receptor expressed in human adipocytes

The melanocortin (MC) receptor type-1 (MC1-R) is the only one of the five MC receptor subtypes expressed in human adipose tissue explants, human mesenchymal stem cells (MSCs), and MSC-derived adipocytes. Following our recent expression studies (Obesity 2007, 15, 40-49), we now investigated the functional role of MC1-R in these tissues and cells to deduce the coupling state of MC1-R to intracellular output signals in human fat cells and tissue. Expression of MC1-R by undifferentiated and differentiated MSCs was quantified by real-time TaqMan PCR. Intracellular output signals (cAMP, lipolysis, secretion of IL-6, IL-10, and TNF-alpha), as well as effects on the metabolic rate and proliferation of human MSCs were analyzed by standard assays, exposing undifferentiated and differentiated MSCs and, in part, human adipose tissue explants to the potent MC1-R agonist, [Nle(4), D-Phe(7)]-alpha-MSH (NDP-MSH). This agonist induced a weak cAMP signal in MSC-derived adipocytes. However, it did not affect lipolysis in these cells or in adipose tissue explants, nor did it modulate cytokine release and mRNA expression of IL-6, IL-8, and TNF-alpha upon LPS stimulation. In undifferentiated MSCs, NDP-MSH did not alter the metabolic rate, but it showed a significant antiproliferative effect. Therefore, it appears that MC1-R-effector coupling in (differentiated) human adipocytes is too weak to induce a regulatory effect on lipolysis or inflammation; by contrast, MC1-R stimulation in undifferentiated MSCs induces an inhibitory signal on cell proliferation.     

Differential biological activities of human interleukin-1 alpha and interleukin-1 beta

We examined the biological effects induced by both human recombinant interleukin-1 alpha (IL-1 alpha) and beta (IL-1 beta) in five different cell types of human, rat and mouse origin. IL-1 alpha and beta preparations were standardized in terms of biological activity in the EL-4/CTLL bioassay and, in parallel, employed to stimulate PGE2 secretion in human fibroblasts, mesangial cells (MC), C57B1/6 mouse MC, DBA/2 mouse macrophages and Sprague Dawley rat MC. In addition, the co-mitogenic effects of IL-1 alpha and beta were determined in freshly prepared Sprague Dawley rat thymocytes. No significant differences in IL-1 alpha and beta concentration dependent PGE2 production were detectable in the different cell types (MC, fibroblasts and macrophages) of human or mouse origin. Incubation of Sprague Dawley rat MC with both IL-1 alpha and beta resulted in a concentration dependent production of PGE2. However, in contrast to mouse or human MC the potency of IL-1 beta to induce PGE2 in Sprague Dawley rat MC was 26-fold higher compared to IL-1 alpha. In addition, the potency of IL-1 beta to enhance co-stimulated proliferation of Sprague Dawley thymocytes was 200-fold higher than that of equal biological activities of IL-1 alpha. When we tested the additive effects on Sprague Dawley cells, increasing IL-1 beta concentrations added to a fixed IL-1 alpha concentration resulted in a cumulative rise in both, PGE2 secretion by MC and thymocyte proliferation.(ABSTRACT TRUNCATED AT 250 WORDS)     

Interleukin-1 enhances the response of osteoblasts to platelet-derived growth factor through the alpha receptor-specific up-regulation.

Both platelet-derived growth factor (PDGF) and interleukin-1 (IL-1) are produced by activated macrophages and are thought to contribute to bone remodeling, but their precise roles remain to be clarified. The interaction between PDGF and IL-1 was, therefore, studied in normal osteoblast-like cells (MC3T3-E1). The expression of alpha- and beta-PDGF receptors on MC3T3-E1 cells was detected by RNA blot analysis and confirmed by immunoblot analysis. PDGF-induced chemotactic as well as mitogenic activities were synergistically enhanced by either IL-1 alpha or IL-1 beta (40 units/ml) pretreatment in serum-free medium, although IL-1 alone did not show any detectable chemotactic activities. This biological enhancement by IL-1 was accompanied by a selective increase of alpha-PDGF receptor expression, following the augmentation of alpha receptor autophosphorylation and inositol phosphate hydrolysis induced by PDGF-AA. These findings suggest that PDGF and IL-1 are jointly involved in the bone-remodeling microenvironment as local coupling factors.     

The bcl, NFkappaB and p53/p21WAF1 systems are involved in spontaneous apoptosis and in the anti-apoptotic effect of TGF-beta or TNF-alpha on activated hepatic stellate cells

Activated hepatic stellate cells (HSC) are thought to play a pivotal role in development of liver fibrosis which takes place in chronic liver diseases. Previous studies have shown that "activated" rat HSC undergo spontaneous apoptosis probably through the CD95/CD95L pathway. TGF-beta as well as TNF-alpha reduced spontaneous apoptosis and CD95L expression. The aim of this study was to investigate the possible mechanisms responsible for the spontaneous apoptosis and for the anti-apoptotic effect of TGF-beta and TNF-alpha on activated HSC. While bcl-2, bax, NFkappaB and p53 gene expression were spontaneously upregulated, bcl-xL and p21WAF1 gene expression decreased and IkappaB remained unchanged during the activation process in vitro. TGF-beta as well as TNF-alpha induced activation of NFKB and upregulated bcl-xL. The latter was inhibited by overexpression of IkappaB. By suppressing spontaneous apoptosis TGF-beta as well as TNF-alpha inhibited p53 gene expression while that of the p21WAF1 gene was increased. We conclude that TGF-beta as well as TNF-alpha may act as surviving factors for activated rat HSC not only through reduction of CD95L gene expression but also by upregulating the anti-apoptotic factors NFKB, bcl-xL and p21WAF1 and by downregulating the proapoptotic factor p53. The interaction with these factors may lead to the generation of new antifibrotic drugs.     

Interleukin-22 Protects against Acute Alcohol-Induced Hepatotoxicity in Mice

The protective effects of interleukin-22 (IL-22) on acute alcohol-induced liver injury were investigated. Mice were gavaged with 7 doses of alcohol (56% wt/vol, 15.2 mL/kg of body weight for each dose) over the 24 h, and IL-22 (0.5 mg/kg BW) was given to the mice by injection into the tail vein 1 h after alcohol administration. The results indicated that acute alcohol administration caused prominent hepatic microvesicular steatosis and an elevation of serum transaminase activities, induced a significant decrease in hepatic glutathione in conjunction with enhanced lipid peroxidation, and increased hepatocyte apoptosis as well as hepatic TNF-alpha production. IL-22 treatment attenuated these adverse changes induced by acute alcohol administration. The protective effects of IL-22 on alcohol-induced hepatotoxicity were due mainly to its anti-inflammatory, anti-oxidant, and anti-apoptotic features.     

Interleukin-22 protects against acute alcohol-induced hepatotoxicity in mice

The protective effects of interleukin-22 (IL-22) on acute alcohol-induced liver injury were investigated. Mice were gavaged with 7 doses of alcohol (56% wt/vol, 15.2 mL/kg of body weight for each dose) over the 24 h, and IL-22 (0.5 mg/kg BW) was given to the mice by injection into the tail vein 1 h after alcohol administration. The results indicated that acute alcohol administration caused prominent hepatic microvesicular steatosis and an elevation of serum transaminase activities, induced a significant decrease in hepatic glutathione in conjunction with enhanced lipid peroxidation, and increased hepatocyte apoptosis as well as hepatic TNF-alpha production. IL-22 treatment attenuated these adverse changes induced by acute alcohol administration. The protective effects of IL-22 on alcohol-induced hepatotoxicity were due mainly to its anti-inflammatory, anti-oxidant, and anti-apoptotic features.     

Interleukin-1 beta (IL-1beta) induces tumor necrosis factor alpha (TNF-alpha) expression on mouse myeloid multipotent cell line 32D cl3 and inhibits their proliferation

Interleukin-1 alpha (IL-1alpha) and beta (IL-1beta) are well known factors that stimulate hematopoiesis, nevertheless there are reports that show that they can also inhibit this activity. While both IL-1alpha and IL-1beta induce the expression of hematopoietic cytokines, such as growth factors and their receptors on myeloid cells, helping thus to regulate hematopoiesis, it is not known if their inhibitory activity is also mediated through the induction of other specific cytokines. In this work we show that recombinant human IL-1beta (rhIL-1beta) inhibits the proliferation of a mouse IL-3-dependent myeloid multipotent cell line (32D cl3), without inducing its differentiation. We show that rhIL-1beta induces in 32D cl3 cells the expression of the tumor necrosis factor alpha (TNF-alpha) gene, a well known growth inhibitor, and that the rhIL-1beta growth inhibition property on 32D cl3 cells is partially due to this secreted TNF-alpha, hinting thus that the inhibition of hematopoiesis by IL-1 is mediated through other induced cytokines.     

F2-isoprostane, inflammation, cardiac function and oxygenation in the endotoxaemic pig

Prostaglandins are profoundly involved in endotoxaemic shock. Twenty pigs were given endotoxin at various doses (0.063-16 microg kg(-1) h(-1)). Three non-endotoxaemic pigs served as controls. Two eicosanoids were measured in plasma (8-iso-PGF(2alpha), a free radical-mediated lipid peroxidation product, and 15-keto-dihydro-PGF(2alpha) a major metabolite of COX activity) and evaluated against the pathophysiological responses that occur during endotoxaemic shock. Endotoxin mediates an increase in both 8-iso-PGF(2alpha) and 15-keto-dihydro-PGF(2alpha). An increase in the endotoxin dose induced significant log-linear responses in 8-iso-PGF(2alpha) and 15-keto-dihydro-PGF(2alpha). Oxidative injury correlated to the TNF-alpha, IL-6, reductions in cardiac performance and to oxygen delivery and utilisation. COX-mediated inflammatory responses correlated to TNF-alpha, IL-6 and to reductions in arterial oxygen tension. Thus, oxidative injury and COX-mediated inflammation play a central role in the manifestation of endotoxaemic shock. Furthermore, formation of these eicosanoids on endotoxin-mediated alterations in pulmonary hypertension, oxygen delivery and oxygen utilisation seems to be independent of the administered endotoxin dose.