Caspase-1 is not involved in experimental hepatitis in mouse

Experimental hepatitis induced by tumor necrosis factor in D-(+)-galactosamine-sensitized mice or by an agonistic anti-Fas antibody in normal mice is accompanied by dramatic apoptosis of hepatocytes. Apoptosis is the final result of activation of a cascade of caspases. We used caspase-1-/- mice, generated by gene targeting, to study the role of this protease in TNF- and anti-Fas-induced lethal hepatitis. We found that mutant mice exhibited the typical caspase-1-/- phenotype, since they resisted to a lethal injection of LPS and released no interleukin-1beta in the circulation, in contrast to wild-type littermates. When caspase-1-/- mice were challenged with different doses of tumor necrosis factor/D-(+)-galactosamine or with anti-Fas, no increased survival was observed compared with control mice. Furthermore, apoptosis in the livers of these mice and serum levels of alanine aminotransferase were not reduced. These data indicate that caspase-1 deficiency does not lead to reduced apoptosis in these models, either because caspase-1 is irrelevant in this model or because of functional redundancy.     

Tumor necrosis factor-induced lethal hepatitis: pharmacological intervention with verapamil, tannic acid, picotamide and K76COOH

Tumor necrosis factor (TNF) induces hepatitis when injected in human beings or in rodents. The molecular mechanism by which TNF induces hepatic distress remains largely unknown, although induction of apoptosis of hepatocytes appears to be an essential step. In order to increase the therapeutic value of TNF, we have studied the protective activity of several molecules and found that four chemically totally different substances confer significant protection in the model of TNF-induced lethal hepatitis in mice sensitized with D-(+)-galactosamine (GalN), but not in mice sensitized with actinomycin-D (ActD) or against anti-Fas-induced lethal hepatitis. Verapamil, a calcium-channel blocker, tannic acid, picotamide, a thromboxane A(2) receptor antagonist, and K76COOH, an inhibitor, amongst others, of complement, protected significantly against induction of lethality, release of the liver-specific enzyme alanine aminotransferase (ALT) and induction of apoptosis in the liver after TNF/GalN, except for K76COOH, which paradoxically increased ALT values after challenge, and which also protected against TNF/GalN in complement-deficient mice. The data suggest that activation of platelets and neutrophils, as well as induction of inflammation occur in the TNF/GalN model, but not in the TNF/ActD or anti-Fas models, in which direct induction of apoptosis of hepatocytes may be more relevant. The protective activity of the drugs may lead to an increase in therapeutic value of TNF.     

Differential response of a(2)-macroglobulin-deficient mice in models of lethal TNF-induced inflammation

Tumor necrosis factor (TNF) is an essential mediator in the pathogenesis of Gram-negative septic shock. Injection of TNF into normal mice leads to systemic, lethal inflammation, which is indistinguishable from lipopolysaccharide (LPS)-induced lethal inflammation. alpha(2)-macroglobulin (A2M) is a major positive acute phase protein with broad-spectrum protease-inhibitory activity. Mouse A2M-deficient (MAM-/-) mice were significantly protected against lethal systemic inflammation induced by TNF. The protection is not due to faster clearance of the injected TNF. The induction of tolerance to TNF-induced lethality by repetitive administration of small doses of human TNF for five consecutive days was equally efficient in both mutant mice compared to wild-type mice. In D-(+)-galactosamine (GalN)-sensitized mice, TNF induces lethal inflammatory hepatitis. MAM(-/-) mice are equally sensitive to the lethal combination of TNF/GalN. Furthermore, interleukin-1-induced desensitization to TNF/GalN was not impaired in MAM(-/-) mice. We conclude that MAM plays a mediating role in TNF-induced lethal shock and that MAM deficiency does not reduce changes in efficiency of tolerance and desensitization to TNF and TNF/GalN-induced lethality, respectively.     

Long term production of acute-phase proteins by adult rat hepatocytes co-cultured with another liver cell type in serum-free medium

Three acute-phase proteins, haptoglobin, alpha 2-macroglobulin and hemopexin, as well as albumin, have been measured daily in the hydrocortisone-supplemented serum-free medium of pure and mixed cultures of adult rat hepatocytes for 5 and 20 days respectively. Whereas plasma protein production rapidly declined in pure culture, it remained relatively stable when hepatocytes were co-cultured with rat liver epithelial cells. In the latter cultures, an early stimulation of albumin and alpha 2-macroglobulin secretion was observed. In addition, four other plasma proteins, fibrinogen, alpha 1-acute-phase protein, alpha 1-acid glycoprotein and alpha 1-antitrypsin were shown by immunodiffusion to still be produced by day 20 of co-culture. These results suggest that hepatocyte co-cultures represent a suitable model for studying the mechanism which controls synthesis of plasma proteins, including acute-phase proteins by liver cells.     

Induction of several acute-phase protein genes by heavy metals: a new class of metal-responsive genes.

Acute-phase reactants, metallothioneins, and heat-shock proteins are the products of three families of genes that respond to glucocorticoids and cytokines. Metallothioneins and heat-shock proteins, however, are also stimulated by heavy metals, whereas very little is known about the effect of heavy metals on acute-phase-reactant genes. We have studied the effect of heavy metals (Hg, Cd, Pb, Cu, Ni, and Zn) and Mg on the acute-phase reactants alpha 1-acid glycoprotein, C-reactive protein, alpha 1-antitrypsin and alpha 1-antichymotrypsin. alpha 1-Acid glycoprotein and C-reactive protein mRNA levels were increased severalfold in livers of heavy-metal-treated Balb/c mice. The strongest induction was mediated by Hg, followed in order of response by Cd greater than Pb greater than Cu greater than Ni greater than Zn greater than Mg. None of the metals affected the mRNA levels of albumin, alpha 1-antitrypsin, and alpha 1-antichymotrypsin. Furthermore, failure to repress albumin, a negative acute-phase reactant, indicated that the induction of these genes was not due to a metal-mediated inflammatory response. The metals also induced alpha 1-acid glycoprotein and C-reactive protein in adrenalectomized animals, indicating that induction by the heavy metals is not mediated by the glucocorticoid induction pathway. Sequence analysis has revealed a region of homology to metal-responsive elements in the alpha 1-acid glycoprotein and C-reactive protein promoters. Additionally, an alpha 1-acid glycoprotein expression vector, pAGP(-595)CAT, responded to Hg and Cd when transfected into human HepG2 cells.(ABSTRACT TRUNCATED AT 250 WORDS)     

In vivo evidence that caspase-3 is required for Fas-mediated apoptosis of hepatocytes.

Caspase-3 is essential for Fas-mediated apoptosis in vitro. We investigated the role of caspase-3 in Fas-mediated cell death in vivo by injecting caspase-3-deficient mice with agonistic anti-Fas Ab. Wild-type controls died rapidly of fulminant hepatitis, whereas the survival of caspase-3-/- mice was increased due to a delay in hepatocyte cell death. Bcl-2 expression in the liver was dramatically decreased in wild-type mice following anti-Fas injection, but was unchanged in caspase-3-/- mice. Hepatocytes from anti-Fas-injected wild-type, but not caspase-3-/-, mice released cytochrome c into the cytoplasm. Western blotting confirmed the lack of caspase-3-mediated cleavage of Bcl-2. Presumably the presence of intact Bcl-2 in caspase-3-/- hepatocytes prevents the release of cytochrome c from the mitochondria, a required step for the mitochondrial death pathway. We also show by Western blot that Bcl-xL, caspase-9, caspase-8, and Bid are processed by caspase-3 in injected wild-type mice but that this processing does not occur in caspase-3-/- mice. This study thus provides novel in vivo evidence that caspase-3, conventionally known for its downstream effector function in apoptosis, also modifies Bcl-2 and other upstream proteins involved in the regulation of Fas-mediated apoptosis.     

The mitochondrial permeability transition augments Fas-induced apoptosis in mouse hepatocytes

Tumor necrosis factor-alpha receptor 1 and Fas recruit overlapping signaling pathways. To clarify the differences between tumor necrosis factor alpha (TNFalpha) and Fas pathways in hepatocyte apoptosis, primary mouse hepatocytes were treated with TNFalpha or an agonist anti-Fas antibody after infection with an adenovirus expressing an IkappaB superrepressor (Ad5IkappaB). Treatment with TNFalpha induced apoptosis in Ad5IkappaB-infected mouse hepatocytes, as we previously reported for rat hepatocytes. Ad5IkappaB plus anti-Fas antibody or actinomycin D plus anti-Fas antibody rapidly induced apoptosis, whereas anti-Fas antibody alone produced little cytotoxicity. The proteasome inhibitor (MG-132) and a dominant-negative mutant of nuclear factor-kappaB-inducing kinase also promoted TNFalpha- and Fas-mediated apoptosis. Expression of either crmA or a dominant-negative mutant of the Fas-associated death domain protein prevented TNFalpha- and Fas-mediated apoptosis. In addition, the caspase inhibitors, DEVD-cho and IETD-fmk, inhibited TNFalpha- and Fas-mediated apoptosis. In Ad5IkappaB-infected hepatocytes, caspases-3 and -8 were activated within 2 h after treatment with anti-Fas antibody or within 6 h after TNFalpha treatment. Confocal microscopy demonstrated onset of the mitochondrial permeability transition (MPT) and mitochondrial depolarization by 2-3 h after anti-Fas antibody treatment and 8-10 h after TNFalpha treatment, followed by cytochrome c release. The combination of the MPT inhibitors, cyclosporin A, and trifluoperazine, protected Ad5IkappaB-infected hepatocytes from TNFalpha-mediated apoptosis. After anti-Fas antibody, cyclosporin A and trifluoperazine decreased cytochrome c release but did not prevent caspase-3 activation and cell-death. In conclusion, nuclear factor-kappaB activation protects mouse hepatocytes against both TNFalpha- and Fas-mediated apoptosis. TNFalpha and Fas recruit similar but nonidentical, pathways signaling apoptosis. The MPT is obligatory for TNFalpha-induced apoptosis. In Fas-mediated apoptosis, the MPT accelerates the apoptogenic events but is not obligatory for them.     

Recombinant human interleukin-6 (IL-6/BSF-2/HSF) regulates the synthesis of acute phase proteins in human hepatocytes

Recombinant human IL-6 (rhIL-6) is a potent inducer of the synthesis of acute phase proteins in adult human hepatocytes. A wide spectrum of acute phase proteins is regulated by this mediator. After labeling of rhIL-6 stimulated human hepatocytes with [35S]methionine acute phase protein synthesis was measured by immunoprecipitation. Serum amyloid A, C-reactive protein, haptoglobin, alpha 1-antichymotrypsin and fibrinogen were strongly induced (26-, 23-, 8.6-, 4.6- and 3.8-fold increases, respectively). Moderate increases were found for alpha 1-antitrypsin (2.7-fold) and alpha 1-acid glycoprotein (2.7-fold). RhIL-6 had no effect on alpha 2-macroglobulin, whereas fibronectin, albumin and transferrin decreased to 64, 56 and 55% of controls. In the cases of serum amyloid A, haptoglobin, alpha 1-antichymotrypsin, alpha 1-antitrypsin and alpha 1-acid glycoprotein, dexamethasone enhanced the action of rhIL-6. We conclude that rhIL-6 controls the acute phase response in human liver cells.     

A Bcl-2 transgene expressed in hepatocytes does not protect mice from fulminant liver destruction induced by Fas ligand

We compared the biological mechanism of cell death during hepatotoxicity induced by ligation of the Fas receptor in wild-type and liver-specific bcl-2 transgenic mice. Transgenic overexpression of Bcl-2 in mouse hepatocytes can prevent lethal hepatitis induced by agonistic anti-Fas antibodies. In contrast, Fas ligand (FasL)-induced death cannot be overcome in bcl-2 transgenic mice, indicating that anti-Fas antibodies do not reliably mimic the more physiological ligand. Different apoptotic parameters, viz. caspase activation, cytochrome c release and nuclear DNA degradation were analysed. No differences, however, could be observed between wild-type and bcl-2 transgenic mice after injection with a lethal dose of soluble FasL, indicating that apoptosis by FasL-dependent ligation is not modulated by Bcl-2 in vivo. These results demonstrate that the stimulus determines the outcome between type I mitochondria-independent apoptosis, in the case of FasL, or type II mitochondria-dependent and Bcl-2-inhibitable apoptosis, in the case of anti-Fas antibodies.     

The role of complement activation in tumour necrosis factor-induced lethal hepatitis.

Injection of tumour necrosis factor (TNF) in animals causes severe liver cell toxicity, especially when D-(+)-galactosamine (GalN) is co-administered. After challenge with TNF/GalN, serum complement activity (CH50 and APCH50) decreased dramatically, suggesting strong activation of both the classical and the alternative pathways. TNF or GalN alone had no such effect. A cleavage product of complement protein C3 [C3(b)] was deposited on the surface of hepatocytes of TNF/GalN-treated mice. Intravenous administration of cobra venom factor (CVF), which depletes complement, inhibited the development of hepatitis. However, CVF pretreatment also protected C3-deficient mice. Pretreatment of mice with a C1q-depleting antibody did not prevent TNF/GalN lethality, although the anti-C1q antibody had depleted plasma C1q. Factor B-deficient and C3-deficient mice, generated by gene targeting, proved to be as sensitive to TNF/GalN as control mice. Furthermore, induction of lethal shock by platelet-activating factor, an important mediator in TNF-induced hepatic failure, was not reduced in C3-deficient mice. These data indicate that complement, although activated, plays no major role in the generation of acute lethal hepatic failure in this model and that CVF-induced protection is independent of complement depletion.     

Hepatocyte specific long lasting inhibition of protein N-glycosylation by D-galactosamine

The effect of D-galactosamine on protein N-glycosylation was studied in rat hepatocyte primary cultures for alpha 1-antitrypsin (three complex type oligosaccharide chains) and alpha 1-acid glycoprotein (six complex type oligosaccharide chains). D-Galactosamine at a concentration of 4 mM inhibited partially de novo N-glycosylation leading to the formation of alpha 1-antitrypsin lacking one to two and of alpha 1-acid glycoprotein lacking one to five of its carbohydrate side chains. In addition D-galactosamine interfered with oligosaccharide processing, leading to the formation of some carbohydrate side chains remaining in an endoglucosaminidase H sensitive, i.e., not completely processed, form. D-Galactosamine impaired the secretion of alpha 1-antitrypsin and of alpha 1-acid glycoprotein but did not inhibit the secretion of the unglycosylated albumin. The inhibitory effect of D-galactosamine on de novo glycosylation as well as on oligosaccharide processing lasted for at least 24 h after it had been removed from the cells. D-Galactosamine impaired the glycosylation of alpha 1-antitrypsin only in hepatocytes, but not in human monocytes. Furthermore, D-galactosamine did not impair the N- and O-glycosylation of interleukin-6 in human monocytes and in MRC 5 fibroblasts. The results indicate that the effect of D-galactosamine on protein glycosylation is restricted to D-galactosamine metabolizing hepatocytes and is not exerted by the drug itself but by its metabolites.     

Sensitivity of metallothionein-null mice to LPS/D-galactosamine-induced lethality

Mice treated with lipopolysaccharide (LPS)/D-galactosamine (GalN) selectively develop hepatic failure. The acute-phase protein alpha(1)-acid glycoprotein (AGP) has been demonstrated to protect mice from LPS/GalN-induced lethality. Metallothionein (MT), which is a low-molecular weight, cysteine-rich, metal-binding protein, is also induced in the acute-phase reaction. However, the specific function of MT in acute-phase response remain to be elucidated. We showed that MT-null mice were more sensitive to LPS/GalN-induced lethality than wild-type mice. The increase in vital mediator levels, TNF-alpha and NO were of similar levels in wild-type and MT-null mice. A remarkable increase in plasma platelet-activating factor levels was not observed in our experimental conditions. On the other hands, the mRNA level of AGP in the response to LPS/GalN was decreased in MT-null mice compared to wild-type mice. These results indicated that MT may have the potential to prevent LPS/GalN-induced lethality, at least through the attenuation of AGP induction.     

Insulin modulation of acute-phase protein production in a human hepatoma cell line.

Insulin is widely used as a growth factor in hepatocyte culture but its effect on the production of acute-phase proteins has not been studied. By measuring four positive (fibrinogen, alpha 1-antitrypsin, alpha 1-acid glycoprotein, and alpha 1-antichymotrypsin) and four negative (albumin, prealbumin, transferrin, and retinol binding protein) acute-phase proteins produced by the Hep G2 hepatoma cell line, we have shown that insulin is an important modulator of acute-phase protein production. Our data show that insulin is able to inhibit the synthesis of prealbumin, transferrin, and fibrinogen. The results also show a complex interaction between insulin, interleukin 6, and glucocorticoids because insulin is able to inhibit the dexamethasone induction of alpha 1-antichymotrypsin, and in the presence of interleukin 6, dexamethasone is able to regulate the production of fibrinogen and prealbumin. The regulatory role of insulin in fibrinogen production was confirmed by pulse chase labeling followed by immunoprecipitation and fluorography.     

Serine proteases of the fibrinolysis pathway are not involved in lethal hepatitis and fibrinogen breakdown induced by tumor necrosis factor

Tumor necrosis factor (TNF) plays a key role in several types of fulminant and acute hepatitis, and induces massive apoptosis and necrosis of hepatocytes. Our previous studies described the central role played by several matrix metalloproteinases (MMPs) and one or more unknown serine proteases. The aim of this study was to investigate the involvement of serine proteases of the fibrinolytic pathway, known to be activators of several MMPs, in TNF-induced hepatitis and fibrinogen (FG) breakdown. Experiments were performed in a model of TNF-induced hepatitis, consisting of administration of TNF in combination with D-(+)-galactosamine (GalN) to mice deficient in urokinase-type plasminogen (PG) activator (u-PA), tissue-type PG activator (t-PA) or PG. Lethality, transaminase release, increased plasma clotting time and FG levels were measured. In PA- and PG-deficient mice, TNF/GalN still induced hepatitis, as well as increased clotting time and FG breakdown. MMP-9 activation still occurred in the liver despite the lack of plasmin. The data suggest that the serine proteases involved in TNF-induced lethal hepatitis are no constituents of the fibrinolytic cascade.     

Biosynthesis of abnormally glycosylated hepatoma secretory proteins in cell cultures

We studied, by electrophoretic techniques, the physiochemical properties of 4 glycoproteins, alpha 1-antitrypsin, alpha 1-antichymotrypsin, alpha 1-acid glycoprotein and transferrin synthesized by three different human hepatoma cell lines. A common feature was the export of glycoproteins with retarded electrophoretic mobility, indicating incomplete sialylation, and a predominance of atypical, highly branched carbohydrate chains. The abnormal glycosylation pattern may be specific for malignant transformation of hepatocytes and possibly related to the intracellular accumulation of some of these proteins in malignant cells.     

Distinct sets of acute phase plasma proteins are stimulated by separate human hepatocyte-stimulating factors and monokines in rat hepatoma cells

A subline of the rat hepatoma (H-35) cells has been identified which responds to hepatocyte-stimulating factors (HSFs) of human squamous carcinoma cells by increased synthesis of all major rat acute phase plasma proteins. The regulation occurs at the level of mRNA. Two HSFs (HSF-I and HSF-II) have been purified from conditioned medium of the squamous carcinoma cells. HSF-I is a protein with an Mr = 18,000 and pI 5.5, and HSF-II is a glycoprotein with an Mr = 34,000 and a broad, neutral to basic charge. In H-35 cells, HSF-I predominantly stimulates the synthesis of complement C3 and haptoglobin and acts synergistically with dexamethasone to stimulate alpha 1-acid glycoprotein. HSF-II stimulates cysteine protease inhibitor, alpha 1-antichymotrypsin, alpha 1-antitrypsin, fibrinogen, and hemopexin, and acts synergistically with dexamethasone to stimulate alpha 2-macroglobulin. Each HSF is between 10 and 100 times less effective in regulating proteins of the other set. Human tumor necrosis factor and interleukin-1 increase complement C3, haptoglobin, and alpha 1-acid glycoprotein, as does HSF-I, but are unable to modulate any of the other acute phase proteins. The monokines differ from HSF-I is their low activity in HepG2 cells and rat hepatocytes.     

Caspases are the main executioners of Fas-mediated apoptosis, irrespective of the ceramide signalling pathway

Tumor necrosis factor alpha (TNF) or cytotoxic anti-Fas antibodies lead to the activation of apoptotic proteases (caspases) and to sphingomyelinase-mediated ceramide generation. Caspases and ceramide are both known to induce apoptosis on its own, but their relative contribution to Fas- and TNF-induced cell death is not well established. We report here that rapid apoptosis induced by TNF in U937 cells or anti-Fas in Jurkat cells, in the presence of cycloheximide, induced only a very low increase (<20%) in the cell ceramide content. Neither treatment with inhibitors of sphingomyelinases nor incubation of cells with fumonisin B1, which inhibits de novo ceramide synthesis, prevented TNF and Fas-mediated apoptosis. Increasing or depleting the cell ceramide content by prolonged culture in the presence of monensin or fumonisin B1, respectively, did not prevent TNF and Fas-mediated apoptosis. Treatment of cells with sphingomyelinase inhibitors did not affect to the activation of CPP32 (caspase-3) induced by TNF or anti-Fas antibodies. Chromatin condensation and fragmentation in cells treated with anti-Fas or TNF was abrogated by peptide inhibitors of caspases, which also inhibited Fas-, but not TNF-induced cell death. These results indicate that while ceramide does not seem to act as a critical mediator of TNF and Fas-induced apoptosis, it is generated as a consequence of CPP32 activation and could contribute to the spread of the intracellular death signal.     

Ontogenetic peculiarities of regulation of apoptosis of hypothalamic neurosecretory cells in TNF-knockout mice

Tumor necrosis factor (TNF) participates in regulation of many processes, including carcinogenesis and apoptosis. However, at present, there are practically no studies on peculiarities of apoptosis control in tnf-knockout (tnf-/-) mice. These mice develop without morphologic abnormalities, but they seem to have impairment of many biological processes, such as inflammation, programmed cell death, etc. Therefore, the goal of our work was to study possible pathways of regulation of apoptosis in the absence of TNF in neurosecretory cells (NSC) of young and old mice. For this purpose, we determined immunohistochemically expression of apoptosis markers caspase-8, caspase-9, Bax, Bcl-2, Mcl-1, neuropeptide vasopressin and the apoptosis level in hypothalamus of tnf-knockout mice of different ages as compared with mice with unchanged level of TNF synthesis. It was shown that the apoptosis activation observed during aging did not depend on the tnf gene and that apoptosis at aging was caspase-dependent. It was revealed that at aging in mouse NSC the external cell death pathway with participation of caspase-8 is activated. The pathways mediating cell death in different neurosecretory centers at aging are different. Thus, in supraoptic nucleus (SON), in all studied animal groups, an important cause of the NSC apoptosis is Bax. In paraventricular nucleus (PVN), of the greater importance is a decrease of the anti-apoptotic protection. Hence, misbalance of synthesis of proteins of the Bcl-2 family plays an important role in development of senescent apoptosis.     

High-level Constitutive Expression of α1-acid Glycoprotein and Lack of Protection Against Tumor Necrosis Factor-induced Lethal Shock in Transgenic Mice

1-Acid glycoprotein (AGP) is an acute phase protein produced by hepatocytes. Although its exact biological function remains controversial, it was shown to protect galactosamine-sensitized or normal mice against hepatitis and lethal shock induced by tumor necrosis factor (TNF). Rat-AGP- transgenic mice, constitutively producing several mg AGP per ml serum were tested for their response to a combined challenge with TNF and D-(+)-galactosamine. A previously characterized, single transgenic line (9.5–5) was used. In contrast to our expectations both heterozygous or homozygous transgenic mice were not protected by the endogenously overproduced AGP. However, both transgenic and non-transgenic mice were protected by pretreatment with interleukin-1, an effect which we believe is mediated by the induction of acute phase proteins like AGP. Furthermore, both types of mice were protected by exogenous bovine AGP, suggesting that the lack of protection by endogenous AGP is not because of a repressed response to AGP. Finally, we demonstrate that purified AGP from the serum of transgenic mice is as protective as the AGP from non-transgenic mice or rats. The results suggest that AGP is protective only when its concentration is rapidly induced, perhaps because the endogenous steady state synthesis of AGP, in non-transgenic as well as transgenic mice, is coupled to the production of an AGP-binding factor. This study provides an interesting example of differences in outcome to a lethal challenge between an acute administered and a chronically produced protective protein.