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INFLAMMATION, IMMUNOPHARMACOLOGY, AND ASTHMA
Biochemical Pharmacology, Faculty of Biology, University of Konstanz, Konstanz, Germany (M.L., G.K., R.L., H.H., A.W.); Department of Pharmacology, Leo Pharma A/S, Ballerup, Denmark (M.L.); Aurigon Life Science GmbH, Tutzingen, Germany (G.K.); University of Applied Sciences, Krems, Austria (R.L.); and ES Cell International Pte Ltd, Biopolis, Singapore (H.H.)
Received January 15, 2007; accepted March 15, 2007.
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Abstract |
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levels were suppressed, animals still were protected when ATP depletion was initiated after the peak of proinflammatory cytokines upon LPS injection, showing that fructose-induced ATP depletion affects both cytokine release and action. In T cell-dependent necrotic hepatotoxicity elicited by concanavalin A or galactosamine + staphylococcal enterotoxin B, ATP depletion prevented liver injury as well, but here without modulating cytokine release. By attenuating caspase-8 activation, ATP depletion of hepatocytes in vitro impaired TNF receptor signaling by the death-inducing signaling complex, whereas receptor internalization and nuclear factor-
B activation upon TNF stimulation were unaffected. These findings demonstrate that sufficient target cell ATP levels are required for the execution of both apoptotic and necrotic TNF-receptor 1-mediated liver cell death.
; Leist et al., 1997a, 1998). Overactivation of apoptotic hepatocyte death is triggered by an increased release of cytokines (Leist et al., 1997a) such as tumor necrosis factor (TNF) and the CD95 ligand, triggering activation of respective death receptors (Galle et al., 1995; Leist et al., 1998). Controlling hepatocyte death is a desirable goal to handle adverse reactions during therapeutic interventions in liver disease, e.g., during cancer treatment, provided that the necessary specificity is achieved.
The cellular energy status represented by the ATP level is pivotal for controlling apoptosis in different cell types (Leist et al., 1997b). At low concentrations, the glycolytic substrate fructose supplies energy and protects hypoxic hepatocytes from necrosis via ATP generation (Gasbarrini et al., 1992) and inhibits apoptosis during reoxygenation by attenuation of reactive oxygen species formation (Frenzel et al., 2002). However, fructose in high concentrations transiently depletes ATP in hepatocytes (Mäenpää et al., 1968; Raivio et al., 1969) yet leaves sufficient residual ATP (>15% of control), which avoids necrosis induction as it is seen after total ATP loss (Nieminen et al., 1994). Previously, we demonstrated that extensive metabolic ATP depletion by fructose metabolism protected primary murine hepatocytes in vitro and prevented TNF-induced apoptosis in vivo (Latta et al., 2000). Because TNF induces not only apoptosis but also necrosis upon receptor triggering (Lin et al., 2004), we now investigated ATP depletion in three categories of more complex, indirect liver injury models. Curiously and despite the obvious differences in the mode of hepatocyte death, the significance of TNF as a distal mediator of hepatocyte death in all of these models has been shown either by genetic evidence (i.e., resistance of TNF-R1-deficient mice) as well as by passive immunization against TNF prior to the injury. The following models of indirect, inflammatory liver injury were used:
; Leist et al., 1995b; Leist et al., 1998, Mignon et al., 1999, Künstle et al., 1999; Hentze et al., 2000). ; Mignon et al., 1999, Hentze et al., 2000). and TNF release. The distinct periportal destruction is independent of caspase activation and consequently is insensitive to caspase inhibitor treatment (Tiegs et al., 1992; Gantner et al., 1995, 1996; Künstle et al., 1999; Hentze et al., 2000).
We here demonstrate that upon depletion of hepatic ATP, the onset of liver injury was blocked in all TNF-dependent models at the target cell level, i.e., the hepatocyte, regardless whether the nature of hepatocyte death was apoptotic (GalN/LPS or GalN/SEB) or necrotic (LPS or ConA). This protection was associated in the LPS models with a suppression of TNF and IFN- release, which was also seen in mice pretreated with exogenous adenosine. At the molecular level, we show in vitro that inhibition of DISC formation may explain the requirement of an intact ATP status for signaling of TNF-R1.
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Materials and Methods |
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Animal Experiments and Sampling of Material. Specific pathogen-free male in-house BALB/c mice (25 g) received humane care in concordance with legal requirements in Germany as well as with National Advisory Committee for Laboratory Animal Research guidelines. Food was withdrawn overnight before the onset of experiments. Carbohydrates (5 g/kg) were injected i.p. either before the challenge with GalN (700 mg/kg)/LPS (2 µg/kg i.p.), high-dose LPS (10 mg/kg i.p.), ConA (25 mg/kg i.v.), or GalN/SEB (2 mg/kg i.p.) or delayed 2 h after the challenge. 2-Chloroadenosine (50 µg/kg i.v.) was injected 15 min before the challenge in 300 µl of endotoxin-free saline containing 0.1% human serum albumin. Mice were euthanized by i.v. injection of 150 mg/kg pentobarbital plus 0.8 mg/kg heparin. Blood was withdrawn into heparinized syringes, centrifuged (5 min at 14,000g and 4°C) to obtain plasma, and frozen at –80°C. Alanine aminotransferase activity was measured with an EPOS 5060 analyzer as described previously (Bergmeyer, 1983). Livers were perfused for 10 s with cold perfusion buffer (50 mM phosphate, 120 mM NaCl, and 10 mM EDTA, pH 7.4) and subsequently excised. A slice of the large anterior lobe was freeze-clamped with liquid nitrogen-precooled pliers and stored at –80°C for the quantification of ATP and caspase-3-like activity as described previously (Latta et al., 2000).
Isolation and Culture of Mouse Hepatocytes. Hepatocytes from 8-week-old mice were prepared as described previously (Klaunig et al., 1981, Leist et al., 1995b). At 37°C, 5% CO2, 40% O2, and 55% N2, hepatocytes were plated in RPMI 1640 medium containing 10% heat-inactivated fetal calf serum in collagen-coated 24-well plates and allowed to adhere for 4 h before the medium was exchanged for RPMI 1640 without fetal calf serum.
Biochemical Determinations. TNF and IL-10 were determined using enzyme-linked immunosorbent assay kits (OptEIA; PharMingen). The DEVD-afc cleavage assay for caspase-3-like activity was performed as described previously (Latta et al., 2000). For caspase-8-like activity, IETD-afc was used with essentially the same protocol as for DEVD-afc cleavage. For the ATP measurement, an ATP bioluminescence assay kit (HS II; Boehringer Mannheim, Mannheim, Germany) was used. First, a 10% liver homogenate was prepared in ATP Releasing Reagent (Sigma) and centrifuged at 13,000g for 5 min at 4°C, and the supernatants were diluted. Luminescence was measured against standards of ATP in 96-well plates using an automated procedure (Victor2 Multilabel Counter; Wallac Instruments, Turku, Finland). LDH was determined in hepatocyte supernatants (S) and in the remaining cell monolayer (C) after lysis with 0.1% Triton X-100 according to Bergmeyer (1983). The percentage of LDH release was calculated from the ratio of S/(S + C).
Histological Analysis. For microscopy, samples fixed in 4% buffered formalin were embedded in paraffin. Five-micrometer sections (Biocut 2030; Leica Microsystems, Nussloch, Germany) were stained with hematoxylin and eosin and reviewed under a microscope.
Preparation of Nuclear Extracts and Electrophoretic Mobility Shift Assay. Nuclear extracts were prepared from 70-mm2 flasks with a kit from Panomics (Redwood City, CA). Double-stranded NF-B consensus oligonucleotide (sc-2505; Santa Cruz Biotechnology, Santa Cruz, CA) was 5'-end-labeled with [32P]ATP using T4 polynucleotide kinase (Promega, Heidelberg, Germany). Nuclear proteins (10 µg) were incubated at 20°C for 20 min (25 mM HEPES, pH 7.5, 106 cpm of radiolabeled oligonucleotide probe, 2 µg of poly[dIdC], 20% glycerol, 12 mM MgCl2, 100 mM KCl2, 0.1% Nonidet-P40, and 1 mM dithiothreitol). Nucleoprotein-oligonucleotide complexes were resolved by nondenaturing polyacrylamide gel electrophoresis (5%), dried for 2 h at 80°C, and autoradiographed at –80°C for 8 h. The specificity of the DNA-protein complex was confirmed by competition with an excess of unlabeled NF-B sequence versus mutant oligonucleotide (sc-2511; Santa Cruz Biotechnology).
Measurement of TNF Internalization. According to a method of Pennica et al. (1992), primary mouse hepatocytes (5 x 106) were incubated in phosphate-buffered saline-bovine serum albumin either with 25 nM 125I-TNF (46.2 µCi/µg; GE Healthcare) for 1 h at 4°C or preincubated with fructose (50 mM) for 30 min at 37°C. Cells were washed twice and resuspended in cold phosphate-buffered saline-bovine serum albumin, and aliquots containing 5 x 105 cells were removed for determining bound 125I-TNF. The cells were transferred to 37°C, and samples of 5 x 105 cells were taken and washed twice with cold 0.5 M NaCl and 0.2 M acetic acid to remove surface-bound plus released 125I-TNF. Controls were performed at 4°C. The supernatants were precipitated with 10% trichloroacetic acid (1 h on ice). Acid-washed pellets (internalized counts per minute), trichloroacetic acid pellets (cell surface-associated/released counts per minute), and trichloroacetic acid supernatants (degraded counts per minute) were counted in a Beckman LS6000IC (Beckman Coulter GmbH, Krefeld, Germany). Internalized and cell surface-associated fractions are shown as percentage of total bound 125I-TNF.
Statistics. Data are shown as means ± S.D. or ± S.E.M. as indicated. Statistical differences were determined by one-way analysis of variance followed by Dunnett's multiple comparison test of the control versus other groups. p < 0.05 was considered significant.
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), tagatose represents a more potent ATP-depleting agent than fructose. The depletion of ATP by ketohexose treatment at the doses used here occurred within 30 min and lasted for at least 3 h (Latta et al., 2000).
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). At this late time point, necrotic foci, mild neutrophil infiltration, erythrocyte agglutination, and a massive destruction of the sinusoidal structure of the liver were also observed. All of these hallmarks of TNF-R1 apoptosis were entirely abrogated in animals pretreated with a dose of fructose or tagatose that depletes hepatic ATP by 60 and 80%, respectively, within 1 h (Table 1). No overt histological signs of organ injury were seen after ATP depletion by tagatose (Fig. 2B), and ALT release was dramatically decreased (Table 1), whereas treatment of nondepleting sugars such as mannose or mannitol had no protective effect (Fig. 2C; Table 1). Moreover, mice pretreated with fructose or tagatose completely survived the otherwise lethal GalN/LPS treatment, which shows again that liver injury is the actual cause for lethality in this model (data not shown, summary in Fig. 7B).
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High-dose endotoxin injection elicits hyperinflammatory systemic shock, which affects many organs because of excessive TNF production (Bohlinger et al., 1996; Mignon et al., 1999) and destruction of liver tissue occurs mainly by necrosis of hepatocytes. In mice injected with 10 mg/kg LPS, liver morphology was characterized by a dispersed single-cell necrosis of hepatocytes without any preferential zonation (Fig. 2D). The nuclei of necrotic cells appeared karyolytic, pronounced hepatocyte cell membrane lysis occurred, and infiltration of granulocytes was observed. In mice pretreated with tagatose, essentially no necrotic hepatocytes were seen, and the liver histology appeared normal, except for some optically empty, circular inclusions within hepatocytes (Fig. 2E); this phenomenon was also observed in mice treated with ketohexoses alone but was never associated with any measurable increase of ALT release (Table 1). The ALT release in mice pretreated with fructose or tagatose before LPS injection was entirely abrogated, whereas no protection was seen after pretreatment with the non-ATP-depleting carbohydrates mannose or mannitol (Table 1). In this model, protection from liver injury was not associated with improved survival, because mice become moribund because of systemic shock and multiorgan failure within 24 h in the absence or presence of liver injury (data not shown, summarized in Fig. 7A).
Prevention by ATP-Depleting Carbohydrates of T Cell-Dependent TNF-Mediated Liver Damage Triggered by ConA. Intravenous injection of ConA induces polyclonal T cell activation and release of proinflammatory cytokines (e.g., TNF, IFN-, and granulocyte macrophage–colony-stimulating factor), followed by acute inflammatory liver injury (Tiegs et al., 1992; Gantner et al., 1996). Pretreatment with fructose or tagatose protected mice against liver injury induced by ConA, as demonstrated by a suppression of ALT release by 
90% 8 h after challenge (Table 1). Histologically, ConA injection resulted in sinusoidal destruction, especially in the periportal zone and the appearance of pyknotic nuclei and hepatocyte necrosis, and severe congestions of the microcirculation by agglutinated erythrocytes (Fig. 2G). In contrast, liver specimens from ATP-depleted mice showed only mild erythrocyte agglutination and infiltration of inflammatory cells without necrotic foci (Fig. 2H) and decreased ALT levels (Table 1). Similar to the LPS shock model and despite the absence of liver injury, survival of animals was not prolonged because of massive erythrocyte agglutination and endothelial disruption in this model (data not shown, summary in Fig. 7C). In a further model of T cell-mediated hepatotoxicity based on exposing galactosamine-sensitized mice to the superantigen SEB, fructose pretreatment prevented liver injury as well (Table 1). These findings demonstrate that prevention of hepatotoxicity upon ATP depletion is seen in various in vivo models with different initiation mechanisms and different histopathology, with apoptosis or necrosis as the primary mode of hepatocyte death, but with a common requirement of TNF-R1 activation.
High-Dose LPS-Mediated Liver Injury: Cytokine Release Is Modulated by Carbohydrates, but Protection Is Independent from Suppression. Because all models used in this study rely on the early production of proinflammatory cytokines, we investigated whether ATP depletion affected systemic cytokine release. We found that in the ConA model, ATP-depleting carbohydrates did not significantly affect cytokine release (i.e., TNF, IL-4, and IFN-), yet liver injury was prevented under ATP-depleting conditions (Fig. 3, A–C). However in both LPS models, the release of TNF and IFN- was blunted by fructose or tagatose pretreatment (Fig. 4, A and B), whereas IL-6 release was not affected (data not shown). To exclude a direct interaction between ATP-depleting carbohydrates and cytokine-producing macrophages, we determined the release of TNF from primary Kupffer cells upon 6 h of stimulation with LPS (10 µg/ml). We found no differences either in the presence (184 ± 13 pg/ml TNF) or in the absence (191 ± 36 pg/ml TNF) of fructose (50 mM fructose, n = 3 per group). Thus, a direct action of fructose on cytokine-producing Kupffer cells seems to be unlikely.
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). Therefore, we investigated in vivo whether adenosine affects TNF release after GalN/LPS or ConA injection. Exogenous adenosine was capable of significantly suppressing LPS-induced TNF release by 64%, but failed to reduce the systemic release of TNF in the ConA model (Table 2).
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To elucidate whether the suppression of cytokine secretion by fructose or tagatose treatment might be solely responsible for the protection in the LPS models, we varied the time of carbohydrate injection relative to LPS challenge. We found that even when given 2 h after LPS treatment, i.e., after the systemic peak of TNF release, prevention of liver damage by fructose or tagatose was almost complete (Fig. 5). Remarkably, the IL-10 release pattern after LPS was inverse to that of TNF (GalN/LPS, 650 ± 360 pg/ml plasma IL-10; 5 g/kg tagatose 30 min before GalN/LPS, 4630 ± 1560 pg/ml IL-10; tagatose 2 h after GalN/LPS: 400 ± 150 pg/ml IL-10; n = 3/group). It is concluded from these experiments that protection against cell death in the ATP-depleted state results from the resistance of the target cells, i.e., hepatocytes, rather than from differential influences of ATP depletion on cytokine production.
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Abrogation of Initial Signaling Events in TNF-R1 Signaling by Fructose. We then investigated in primary murine hepatocytes whether fructose affects early signaling in TNF-induced apoptosis, where protection is demonstrated by suppression of LDH release after incubation with ATP-depleting but not other carbohydrates (Fig. 6A). Fructose had no influence on activation and subsequent nuclear translocation of NF-B (Fig. 6B). Furthermore, we found that the internalization of the TNF receptor after triggering with its ligand TNF was not inhibited in the presence of fructose (Fig. 6C). These data were confirmed by analyzing the phosphorylation of I-B (Inhibitor of NF-B), which was slightly delayed but not inhibited by fructose (data not shown). In contrast, fructose completely inhibited caspase-8 activity, as assessed by IETD-afc cleavage (Fig. 6D). IETD-afc was only a poor substrate of recombinant caspase-3 (12.7% IETD-afc cleavage compared with DEVD-afc cleavage), excluding the possibility that cleavage of IETD-afc was mainly due to caspase-3 activity.
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Taken together, these findings show that fructose-mediated ATP depletion partially impairs signaling from the DISC of TNF-R1. This may explain the protection against apoptotic as well as necrotic hepatic cell death because it affects an early and common event in TNF-R1 action.
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Discussion |
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).
We showed previously that a sufficient intracellular ATP level is critical for full caspase activation and execution of TNF-mediated apoptosis (Latta et al., 2000). We now addressed the question whether this principle is restricted to apoptotic cell death or whether it may be extended to more complex, inflammatory liver injury situations by comparing three classes of liver injury models that are all strictly dependent on endogenous TNF production but widely differ in hepatocyte death mode as detailed in the Introduction, i.e., caspase-dependent apoptosis models (GalN/LPS and GalN/SEB), macrophage-induced hepatic necrosis (LPS shock), and T cell-triggered caspase-independent necrosis (ConA). The major pathways leading to liver failure and animal death, as well as their interference with ATP levels are depicted in Fig. 7. Clearly, lowering ATP in hepatocytes interferes with TNF release and TNF action in all models, and therefore treatment with ATP-depleting carbohydrates was always hepatoprotective. However, because animal mortality is directly linked to apoptotic liver damage only in the GalN/LPS model but not in the other two models (Fig. 7A), ATP depletion affected animal survival only here as seen before when other hepatoprotective regimens such as caspase inhibitors or glutathione depletion were applied (Leist et al., 1998, Künstle et al., 1999; Hentze et al., 2000).
Our current understanding of the TNF-R1 signaling pathway is that after receptor engagement and internalization of the TNF/TNF-R1 complex, NF-B is activated and the DISC is formed, with the latter activating caspase-8 (Fig. 7B) (Budihardjo et al., 1999). From there on, two alternative pathways emerge: the caspase-3/-7 route or the mitochondrial pathway via Bid cleavage, release of cytochrome c and its binding to Apaf-1 and pro-caspase-9 to form the apoptosome complex with ATP as an integral part (Li et al., 1997). Unlike Jurkat cells, in which complete ATP depletion by a respiratory block prevented activation of caspases downstream of cytochrome c release and switched cell death toward necrosis (Leist et al., 1999), we had found in primary murine hepatocytes that cell death as such was blocked in the ATP-depleted state upstream of apoptosome formation (Latta et al., 2000). Our new findings on the protection against necrotic cell death now add the insight that a direct ATP-dependence of caspase activation or activity is unlikely to be causal. We show here that the two events of TNF-R1 signaling, NF-B activation, and internalization of the TNF receptor are still functional under ATP depletion. We therefore propose that the block of cell death upon metabolic ATP depletion might be due to the incomplete DISC formation and signaling at the TNF-R1. This hypothesis is in concordance with the observation that a dominant negative ***FADD is sufficient to inhibit cell death without affecting NF-B activation and gene induction upon TNF-R1 stimulation (Wajant et al., 1998). TNF-R1 signals via two distinct complexes, one of which, complex I, is at lipid rafts in the membrane consisting of TNF-R1, TRADD, and TRAF-2, whereas a cytosolic complex II consists of FADD and caspase-8 (Harper et al., 2003; Legler et al., 2003; Micheau and Tschopp, 2003). A translocation of the signal from the membrane to a cytosolic complex might be ATP-dependent and could well explain our findings. In fact, we showed that one signal from complex I, NF-B activation, was still operative, whereas the complex II seemed to be inhibited.
Our findings seem to conflict with a previous report by Kim et al. (2003) that ATP depletion by fructose promotes rather than inhibits cell death of hepatocytes. In their studies, cell death was due to complete loss of ATP and damage of mitochondria by ischemia/reperfusion. Fructose increased ATP levels 15-fold and switched cell death from necrosis to apoptosis. With these fundamentally different activation mechanisms, the two studies are hardly comparable.
In our in vivo models, the extent of LPS-induced cytokine release was decreased after ATP depletion. This raises the question of how fructose-induced ATP depletion in hepatocytes can interfere with cytokine production from macrophages, i.e., Kupffer cells, which are the main producers of TNF in the LPS models. The following interpretations seem unlikely to us for the reasons stated: 1) a direct interaction between ketohexoses and LPS, because among the different sugars used (Table 1) only the ATP-depleting ones suppressed cytokine release; 2) a direct cellular effect of fructose, because primary Kupffer cells released similar amounts of TNF upon LPS stimulation in the presence and in the absence of fructose; and 3) a physical interaction between sugars and the lectin-like domain of TNF that has a specificity for trimannoses and N,N'-diacetylchitobiose (Lucas et al., 1994); i.e., an entirely different oligosaccharide from those used here.
The existence of a metabolic link between the lack of ATP in hepatocytes and the cytokine release from Kupffer cells is likely, despite the above reservations. As to the kinetics, only seconds after ATP depletion by fructose, the content of inorganic phosphate in hepatocytes decreases, resulting in a sudden decline of the total amount of adenosine nucleotides (Mäenpää et al., 1968), whereas the energy charge is maintained, as ATP, ADP, and AMP levels are affected equally (Hultman et al., 1975). Biochemically, a rapid loss of adenosine nucleotides results in the metabolization of AMP to adenosine or IMP via adenosine deaminase and nucleotidase, respectively, leading to a slow increase of inosine, uric acid, and allantoin in the plasma, which is in fact measured (van den Berghe et al., 1977). The consequence is an early local release of adenosine and/or inosine from hepatocytes (Bode et al., 1973; van den Berghe et al., 1977; Liaudet et al., 2001), which results in suppression of TNF production in Kupffer cells via adenosine receptor A2 and cAMP up-regulation (Reinstein et al., 1994; Liaudet et al., 2001).
Indeed, we found that in the GalN/LPS model, exogenous adenosine attenuated TNF release, whereas adenosine had no such effect in the ConA model. This, together with the interpretation outlined above, might explain why the Kupffer cell-derived TNF was suppressed by ATP depletion in the LPS models, but not in the T cell models. In the ConA model, lymphocytes are the primary target cells of the T cell activator, whereas macrophages depend on cross-talk with T cells and are activated by these via release of (a) soluble factor(s) different from IFN- and granulocyte macrophage–colony-stimulating factor (Gantner et al., 1996). The experiments with the T cell models clearly demonstrate that the attenuation of cytokine release alone in the ATP-depleted state is not the only factor preventing cell death, but the action of TNF on the hepatocyte itself, which requires ATP, is also a factor.
To ultimately demonstrate a causal relationship between ATP depletion and protection in the in vivo models used in this study, we aimed to replete intrahepatic ATP levels by means of phosphate supplementation. Several attempts to approach this repletion pharmacologically failed, and to our knowledge these levels are experimentally inaccessible in vivo. For instance, systemic supplementation of phosphate would be counter-regulated by renal phosphate excretion and lower bone resorption. However, the fact that non-ATP-depleting sugars (mannose and mannitol) failed to prevent liver injury (Table 1; Fig. 2) and our previous finding that repletion of ATP via phosphate supplementation in vitro restored sensitivity to apoptosis in primary hepatocytes (Fig. 3 in Latta et al., 2000) suggest a causal relationship between ATP depletion and prevention of cell death. Moreover, as was clearly shown by us previously (Fig. 8 in Latta et al., 2000), the degree and time course of ATP depletion correlated well with refractoriness to TNF-induced cell death, i.e., when TNF was injected 4 h after treatment with fructose, which means after ATP levels recovered to control values again, no protection was observed.
For a long time, necrosis has been viewed as a passive, unregulated process, in which ATP has no apparent active role. However, there is more and more evidence that necrosis is not just lysis of organelles and plasma membrane but rather is controlled by a postulated specific "necrotic program." Chiarugi (2005) recently published an excellent review on the energy requirement of various steps in both apoptosis and necrosis, and here we provide further evidence that a reduction in cellular ATP can halt necrosis in vivo. More research needs to be done to understand better the necrotic program and the underlying regulatory mechanisms.
In total, this study emphasizes the crucial role of sufficient intrahepatic ATP levels for TNF-mediated macrophage-dependent as well as T cell-dependent liver injury models at the target cell level, i.e., the hepatocyte. This requirement holds true for both apoptotic and necrotic cell death.
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Acknowledgements |
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Footnotes |
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Article, publication date, and citation information can be found at http://jpet.aspetjournals.org.
ABBREVIATIONS: TNF, tumor necrosis factor; TNF-R1, tumor necrosis factor receptor 1; GalN, D-galactosamine; LPS, lipopolysaccharide; SEB, staphylococcal enterotoxin B; ConA, concanavalin A; IFN, interferon; DISC, death-inducing signaling complex; DEVD-afc, N-acetyl-Asp-Glu-Val-Asp-7-amino-4-trifluoromethylcoumarin; IETD-afc, N-acetyl-Ile-Glu-Thr-Asp-7-amino-4-trifluoromethylcoumarin; IL, interleukin; LDH, lactate dehydrogenase; NF, nuclear factor; ALT, alanine aminotransaminase.
Address correspondence to: Dr. Hannes Hentze, ES Cell International, 11 Biopolis Way, Helios Building #05-06, 138667 Singapore. E-mail: hhentze{at}escellinternational.com
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) was determined at +90 min and that of IFN-
) at +8h.
