Jens Schümann

Importance of Kupffer Cells for T-Cell-Dependent Liver Injury in Mice

T cells seem to be responsible for liver damage in any type of acute hepatitis. Nevertheless, the importance of Kupffer cells (KCs) for T-cell-dependent liver failure is unclear. Here we focus on the role of KCs and tumor necrosis factor (TNF) production after T cell stimulation in mice. T-cell- and TNF-dependent liver injury were induced either by Pseudomonas exotoxin A (PEA), by concanavalin A (Con A), or by the combination of subtoxic doses of PEA and the superantigen Staphylococcus enterotoxin B (SEB). KCs were depleted by clodronate liposomes. Although livers of PEA-treated mice contained foci of confluent necrosis and numerous apoptotic cells, hardly any apoptotic cells were observed in the livers of Con A-treated mice. Instead, large bridging necroses were visible. Elimination of KCs protected mice from PEA-, Con A-, or PEA/SEB-induced liver injury. In the absence of KCs, liver damage was restricted to a few small necrotic areas. KCs were the main source of TNF. Hepatic TNF mRNA and protein production were strongly attenuated because of KC-depletion whereas plasma TNF levels were unaltered. Our results suggest that KCs play an important role in T cell activation-induced liver injury by contributing TNF. Plasma TNF levels are poor diagnostic markers for the severity of TNF-dependent liver inflammation.

Silibinin protects mice from T cell-dependent liver injury.

BACKGROUND/AIMS Silibinin is the major pharmacologically active compound of the Silybum marianum fruit extract silymarin. Its well-known hepatoprotective activities are mostly explained by antioxidative properties, inhibition of phosphatidylcholine synthesis or stimulation of hepatic RNA and protein synthesis. Here, we characterized the hepatoprotective potential of silibinin as an immune-response modifier in T cell-dependent hepatitis in vivo. METHODS Silibinin was tested in the mouse model of concanavalin A (ConA)-induced, T cell-dependent hepatitis. Liver injury was assessed by quantification of plasma transaminase activities and intrahepatic DNA fragmentation. Plasma cytokine concentrations were determined by enzyme-linked immunosorbent assay (ELISA), intrahepatic cytokine and inducible NO synthase (iNOS) mRNA levels by reverse transcriptase polymerase chain reaction, intrahepatic iNOS expression by immunofluorescent staining, and intrahepatic nuclear factor kappa B (NF-kappaB) activation by electrophoretic mobility shift assay. RESULTS Silibinin significantly inhibited ConA-induced liver disease. Silibinin proved to be an immune-response modifier in vivo, inhibiting intrahepatic expression of tumor necrosis factor, interferon-gamma, interleukin (IL)-4, IL-2, and iNOS, and augmenting synthesis of IL-10. In addition, silibinin inhibited intrahepatic activation of NF-kappaB. CONCLUSIONS Silibinin, suppressing T cell-dependent liver injury as an immune-response modifier, might be a valuable drug in therapeutic situations in which intrahepatic immunosuppression is required.

NK Cells, but Not NKT Cells, Are Involved in Pseudomonas aeruginosa Exotoxin A-Induced Hepatotoxicity in Mice

Pseudomonas aeruginosa exotoxin A (PEA) causes T cell- and Kupffer cell (KC)-dependent liver injury in mice. TNF-α as well as IL-18 and perforin are important mediators of liver damage following PEA injection. In this study, we focus on the role of NK and NKT cells in PEA-induced liver toxicity. Depletion of both NK and NKT cells by injection of anti-NK1.1 Ab as well as depletion of NK cells alone by anti-asialo GM1 Ab protected mice from PEA-induced hepatotoxicity, whereas mice lacking only NKT cells were susceptible. Additionally, we observed infiltration of NK cells, T cells, and neutrophils into liver parenchyma after injection of PEA. The number of NKT cells, however, remained unchanged. The increase in intrahepatic NK cells depended on KCs and the TNF-α-dependent up-regulation of the adhesion molecule VCAM-1 in the liver, but not on NKT cells. PEA also augmented the cytotoxicity of hepatic NK cells against typical NK target cells (YAC-1 cells). This effect depended on KCs, but not on TNF-α or NKT cells. Furthermore, only weak expression of MHC class I was detected on hepatocytes, which was further down-regulated in PEA-treated mice. This could explain the susceptibility of hepatocytes to NK cell cytolytic activity in this model. Our results demonstrate that NK cells, activated and recruited independently of NKT cells, contribute to PEA-induced T cell-dependent liver injury in mice.

Pathophysiological mechanisms of TNF during intoxication with natural or man-made toxins.

Intoxication with different natural toxins or man-made toxicants has been associated with the induction of tumor necrosis factor alpha (TNF). These include endotoxin, superantigens, Pseudomonas aeruginosa exotoxin A, bacterial DNA, T cell stimulatory agents such as agonistic anti-CD3 mAbs or concanavalin A, alpha-amanitin, paracetamol, ethanol, carbon tetrachloride, dioxin, and dimethylnitrosamine. In this paper we compile and discuss the current knowledge on the pathophysiological role of TNF during intoxication with all mentioned toxins and toxicants. A possible role of gut-derived endotoxin in several TNF-dependent toxic events has been considered. The development of pharmaceuticals that selectively interfere with the detrimental pathways induced by TNF during intoxication with bacteria, viruses, drugs, or other chemicals requires detailed knowledge of the signaling pathways originating from the two TNF receptors (TNFR1 and TNFR2). Major characteristics of these signaling pathways are described and put together.

Synergism of Pseudomonas aeruginosa exotoxin A with endotoxin, superantigen, or TNF results in TNFR1- and TNFR2-dependent liver toxicity in mice.

Pseudomonas aeruginosa is a potentially dangerous Gram-negative nosocomial pathogen, causing bacteremia in debilitated patients, and a prominent cause of bacterial cholangitis. Opportunistic infections with other nosocomial pathogens, e.g. Staphylococcus aureus, are common. Hence, multi-intoxication with P. aeruginosa exotoxin A (PEA) and other bacterial toxins, including endotoxin (LPS) and the superantigen S. aureus enterotoxin B (SEB), is very likely. Here we show that PEA synergistically interacted with LPS, SEB, or recombinant murine tumor necrosis factor alpha (rmuTNF) in mice, resulting in severe liver injury. Enhanced and prolonged circulation of cytokines, including TNF, which depended on the presence of T cells, was a remarkable feature of synergistic PEA/LPS- or PEA/SEB-induced hepatotoxicity. PEA/LPS-, PEA/SEB- or PEA/rmuTNF-induced liver injury was mediated by both TNF receptors (TNFRs), i.e. TNFR1 and TNFR2. In view of the fact that TNFR1, but not TNFR2, signaling is unequivocally required for host defense, our results suggest that anti-TNFR2 strategies might be beneficial to protect the liver from inflammatory damage caused by synergistic interactions of PEA with other TNF-inducing bacterial toxins.

Parenchymal, But Not Leukocyte, TNF Receptor 2 Mediates T Cell-Dependent Hepatitis in Mice

TNF-α is a central mediator of T cell activation-induced hepatitis in mice, e.g., induced by Pseudomonas exotoxin A (PEA). In this in vivo mouse model of T cell-dependent hepatitis, liver injury depends on both TNFRs. Whereas TNFR1 can directly mediate hepatocyte death, the in vivo functions of TNFR2 in pathophysiology remained unclear. TNFR2 has been implicated in deleterious leukocyte activation in a transgenic mouse model and in enhancement of TNFR1-mediated cell death in cell lines. In this study, we clarify the role of hepatocyte- vs leukocyte-expressed TNFR2 in T cell-dependent liver injury in vivo, using the PEA-induced hepatitis model. Several types of TNFR2-expressing leukocytes, especially neutrophils and NK cells, accumulated within the liver throughout the pathogenic process. Surprisingly, only parenchymal TNFR2 expression, but not the TNFR2 expression on leukocytes, contributed to PEA-induced hepatitis, as shown by analysis of wild-type → tnfr2° and the reciprocal mouse bone marrow chimeras. Furthermore, PEA induced NF-κB activation and cytokine production in the livers of both wild-type and tnfr2° mice, whereas only primary mouse hepatocytes from wild-type, but not from tnfr2°, mice were susceptible to cell death induced by a combination of agonistic anti-TNFR1 and anti-TNFR2 Abs. Our results suggest that parenchymal, but not leukocyte, TNFR2 mediates T cell-dependent hepatitis in vivo. The activation of leukocytes does not appear to be disturbed by the absence of TNFR2.

Ultrastructural Alterations of Mitochondria in Pre-apoptotic and Apoptotic Hepatocytes of TNFα-treated Galactosamine-sensitized Micea

The electron microscopical studies presented here show that characteristic morphological alterations in mitochondria are a very early hallmark of the hepatocellular apoptotic program. Before chromatin condensation occurs, the outer mitochondrial membrane is focally disrupted and the inner membrane protrudes through this gap forming a hernia. The demonstration of cytochrome oxidase in mitochondria revealed a very strong activity in pre‐apoptotic and apoptotic cells as well as in apoptotic bodies.