concanavalin-a and Necrosis

Signal-transducing adaptor protein-2 (STAP-2) is an adaptor protein involved in inflammatory and immune responses, such as inflammatory bowel disease and allergic responses. In this study, we investigated the role of STAP-2 in the pathogenesis of autoimmune hepatitis. After intravenous injection of concanavalin A (ConA), STAP-2 knock out (KO) mice showed more severe liver necrosis along with substantial lymphocyte infiltration compared to wild type (WT) mice. Serum alanine aminotransferase levels were significantly higher in ConA-injected STAP-2 KO mice than in WT mice. Levels of interferon-γ (IFN-γ), an important factor for liver necrosis, were also significantly increased in sera of STAP-2 KO mice compared to WT mice after ConA injection. Statistically significant upregulation of Fas ligand (FasL) expression was observed in the livers of ConA-injected STAP-2 KO mice compared to WT mice. In accordance with these results, apoptotic signals were facilitated in STAP-2 KO mice compared to WT mice after ConA injection. Correctively, these results suggest that STAP-2 is involved in the pathogenesis of autoimmune hepatitis by regulating the expression of FasL and the production of IFN-γ.

Topics: Adaptor Proteins, Signal Transducing; Animals; Apoptosis; Caspase 3; Concanavalin A; Disease Models, Animal; Fas Ligand Protein; Female; Hepatitis, Autoimmune; Interferon-gamma; Liver; Lymphocytes; Male; Mice, Inbred C57BL; Mice, Knockout; Necrosis; Signal Transduction; Up-Regulation

Topics: Animals; Anti-Inflammatory Agents; Apoptosis; Concanavalin A; Disease Models, Animal; Hepatitis; Hepatocytes; Inflammation; Liver; Male; Mice; Mice, Inbred C57BL; Necrosis; Propane; Sulfides

Simple metabolites released during physical exercise and fasting like lactate (Lac) and β-hydroxybutyrate (BHB) have recently been shown to possess anti-inflammatory properties. However, the effects of these metabolites in immune mediated hepatitis are still unknown. Accordingly, we investigated the role of Lac, BHB and their combination on experimentally induced hepatic inflammation. Adult male mice were administered concanavalin A (Con A, 15 mg/kg, intravenous) for 12 h. In the treatment groups, mice were treated 1 h after Con A-intoxication with Lac (500 mg/kg, intraperitoneal), BHB (300 mg/kg, intraperitoneal) and their combination. The results demonstrated that Lac and BHB, especially when combined together, alleviated Con A-induced hepatocellular injury (ALT, AST and LDH) and necrosis (hematoxylin-eosin and electron microscopy). These beneficial effects correlated with attenuating Con A-induced elevation in hepatic oxidative stress parameters (MDA and NOx). Mechanistically, administration of Lac and BHB led to inhibition of Con A-induced phosphorylation of JNK and AMPK proteins in the liver to the same extent. These effects were concordant with curbing Con A-mediated overexpression of the pro-inflammatory cytokines TNF-α, IL-6 and IL-12 and activation of the transcription factor NF-κB. The marked anti-inflammatory properties of combining Lac and BHB were attributed to their cooperation in repressing immune cells (monocytes and neutrophils) infiltration to the liver. Unlike BHB, Lac administration markedly induced the reparative STAT3 and ERK phosphorylation in the livers of Con A-intoxicated mice at the early time point. In conclusion, the simultaneous use of Lac and BHB might be an auspicious strategy for limiting immune mediated hepatitis.

Topics: 3-Hydroxybutyric Acid; Animals; Anti-Inflammatory Agents; Concanavalin A; Cytokines; Drug Therapy, Combination; Hepatitis, Animal; Inflammation Mediators; Lactic Acid; Liver; Male; Mice; Mice, Inbred BALB C; Necrosis; NF-kappa B; Oxidative Stress; Signal Transduction; STAT3 Transcription Factor

Chlordecone is an organochlorine used in the 1970's as a pesticide in banana plantations. It has a long half-life in the soil and can potentially contaminate humans and animals through food. Chlordecone targets, and mainly accumulates in, the liver, leading to hepatomegaly and neurological signs in mammals. Chlordecone does not cause liver injuries or any inflammation by itself at low doses, but it can potentiate the hepatotoxic effects of other chemicals and drugs. We studied the impact of chlordecone on the progression of acute hepatitis in mouse models of co-exposure to chlordecone with Concanavalin A or murine hepatitis virus type 3. We examined the progression of these two types of hepatitis by measuring hepatic transaminase levels in the serum and inflammatory cells in the liver, liver histological studies. Amplified tremors presented in the MHV3- chlordecone mouse model had led us to study the expression of specific genes in the brain. We show that chlordecone amplifies the auto-immune hepatitis induced by Concanavalin A by increasing the number of liver NKT cells, which are involved in liver damage. Chlordecone also accelerated the death of mice infected by murine hepatitis virus and enhanced the entry of the virus into the cervical spinal cord in infected mice, leading to considerable neurological damage. In conclusion, chlordecone potentiates both the Concanavalin A-induced hepatitis and brain damage caused by an hepatotropic/neurotropic virus.

Topics: Acute Disease; Animals; Brain; Chlordecone; Concanavalin A; Disease Models, Animal; Disease Progression; Hepatitis, Autoimmune; Hepatitis, Viral, Animal; Insecticides; Liver; Male; Mice, Inbred C57BL; Murine hepatitis virus; Necrosis

Necroptosis is a regulated form of cell death involved in several disease models including in particular liver diseases. Receptor-interacting protein kinases, RIPK1 and RIPK3, are the main serine/threonine kinases driving this cell death pathway. We screened a noncommercial, kinase-focused chemical library which allowed us to identify Sibiriline as a new inhibitor of necroptosis induced by tumor necrosis factor (TNF) in Fas-associated protein with death domain (FADD)-deficient Jurkat cells. Moreover, Sib inhibits necroptotic cell death induced by various death ligands in human or mouse cells while not protecting from caspase-dependent apoptosis. By using competition binding assay and recombinant kinase assays, we demonstrated that Sib is a rather specific competitive RIPK1 inhibitor. Molecular docking analysis shows that Sib is trapped closed to human RIPK1 adenosine triphosphate-binding site in a relatively hydrophobic pocket locking RIPK1 in an inactive conformation. In agreement with its RIPK1 inhibitory property, Sib inhibits both TNF-induced RIPK1-dependent necroptosis and RIPK1-dependent apoptosis. Finally, Sib protects mice from concanavalin A-induced hepatitis. These results reveal the small-molecule Sib as a new RIPK1 inhibitor potentially of interest for the treatment of immune-dependent hepatitis.

Topics: Alkaloids; Animals; Apoptosis; Caspase 3; Cell Line, Transformed; Concanavalin A; Cycloheximide; Fibroblasts; Gene Expression Regulation; Hepatitis, Animal; HT29 Cells; Humans; Imidazoles; Immunologic Factors; Indoles; Jurkat Cells; Male; Mice; Molecular Docking Simulation; Necrosis; Protein Kinase Inhibitors; Receptor-Interacting Protein Serine-Threonine Kinases; Signal Transduction; Spiro Compounds; TNF-Related Apoptosis-Inducing Ligand; Tumor Necrosis Factor-alpha

Autoimmune hepatitis (AIH) is a severe necroinflammatory liver disease associated with significant mortality. Although loss of hepatocytes is generally recognised as a key trigger of liver inflammation and liver failure, the regulation of hepatic cell death causing AIH remains poorly understood. The aim of this study was to identify molecular mechanisms that drive hepatocyte cell death in the pathogenesis of acute liver injury.. Acute liver injury was modelled in mice by intravenous administration of concanavalin A (ConA). Liver injury was demonstrated by serum transaminases and histological assessment of liver sections. PGAM5-deficient mice (PGAM5-/-) were used to determine its role in experimental hepatitis. Mdivi-1 was used as an inhibitor of dynamin-related protein 1 (Drp1)-mediated mitochondrial fission. Mitochondrial fission and the expression of PGAM5 were compared between liver biopsies derived from patients with AIH and control patients.. PGAM5 was highly expressed in hepatocytes of patients with AIH and in mice with ConA-induced experimental hepatitis. Deficiency of PGAM5 protected mice from ConA-induced hepatocellular death and liver injury. PGAM5 regulated ConA-induced mitochondrial fission in hepatocytes. Administration of the Drp1-inhibitor Mdivi-1 blocked mitochondrial fission, diminished hepatocyte cell death and attenuated liver tissue damage induced by ConA.. Our data demonstrate for the first time that PGAM5 plays an indispensable role in the pathogenesis of ConA-induced liver injury. Downstream of PGAM5, Drp1-mediated mitochondrial fission is an obligatory step that drives the execution of hepatic necrosis and tissue damage. Our data highlight the PGAM5-Drp1 axis as a potential therapeutic target for acute immune-mediated liver injury.

Topics: Alanine Transaminase; Animals; Aspartate Aminotransferases; Case-Control Studies; Cell Death; Chemical and Drug Induced Liver Injury; Concanavalin A; Dynamins; Gene Expression; Hepatitis, Autoimmune; Hepatocytes; Humans; Liver; Mice; Mice, Knockout; Mitochondrial Dynamics; Mitochondrial Proteins; Necrosis; Phosphoprotein Phosphatases; Quinazolinones

Hepatitis B virus surface antigen (HBsAg) carriers are highly susceptible to liver injury triggered by environmental biochemical stimulation. Previously, we have reported an inverse correlation between γδ T cells and liver damage in patients with hepatitis B virus (HBV). However, whether γδ T cells play a role in regulating the hypersensitivity of HBsAg carriers to biochemical stimulation-induced hepatitis is unknown. In this study, using HBV transgenic (HBs-Tg) and HBs-Tg T-cell receptor-δ-deficient (TCR-δ

Topics: Animals; Cells, Cultured; Concanavalin A; Hepatitis B; Hepatitis B Surface Antigens; Hepatitis B virus; Humans; Interferon-gamma; Interleukin-17; Interleukin-23; Liver; Mice; Mice, Inbred C57BL; Mice, Knockout; Mice, Transgenic; Necrosis; Receptors, Antigen, T-Cell, gamma-delta; T-Lymphocytes

Hepatocyte death, which can be apoptosis or necrosis depending on the context, is a prominent feature of liver disease. The lectin concanavalin A (ConA) activates immune cells, resulting in inflammatory liver injury and hepatocyte necrosis. In this issue of the JCI, Günther et al. demonstrate that the pseudokinase mixed lineage kinase domain-like protein (MLKL) participates in hepatocyte death in ConA injury and that MLKL-mediated death is independent of the receptor-interacting protein kinase RIPK3. RIPK3 was absent in hepatocytes, and MLKL-deficient mice, but not RIPK3-deficient mice, were protected from ConA-induced liver injury. The authors also present evidence that an unidentified kinase activates MLKL, as RIPK1 bound MLKL but did not phosphorylate it. Moreover, ConA rapidly induced MLKL, mediated by the IFN-γ/STAT1 pathway, while activation and translocation to the plasma membrane required TNF. Increased phospho-MLKL staining in liver biopsies from patients with autoimmune hepatitis suggests a role for MLKL in this disease. This study describes a previously unrecognized form of cell death in the liver that should be further explored as a potential therapeutic target in immune-mediated liver injury.

Topics: Animals; Apoptosis; Chemical and Drug Induced Liver Injury; Concanavalin A; Hepatitis, Autoimmune; Hepatocytes; Humans; Interferon-gamma; Liver; Mice; Mice, Mutant Strains; Necrosis; Protein Kinases; Receptor-Interacting Protein Serine-Threonine Kinases; STAT1 Transcription Factor

Necroptosis is a recently described Caspase 8-independent method of cell death that denotes organized cellular necrosis. The roles of RIP1 and RIP3 in mediating hepatocyte death from acute liver injury are incompletely defined. Effects of necroptosis blockade were studied by separately targeting RIP1 and RIP3 in diverse murine models of acute liver injury. Blockade of necroptosis had disparate effects on disease outcome depending on the precise etiology of liver injury and component of the necrosome targeted. In ConA-induced autoimmune hepatitis, RIP3 deletion was protective, whereas RIP1 inhibition exacerbated disease, accelerated animal death, and was associated with increased hepatocyte apoptosis. Conversely, in acetaminophen-mediated liver injury, blockade of either RIP1 or RIP3 was protective and was associated with lower NLRP3 inflammasome activation. Our work highlights the fact that diverse modes of acute liver injury have differing requirements for RIP1 and RIP3; moreover, within a single injury model, RIP1 and RIP3 blockade can have diametrically opposite effects on tissue damage, suggesting that interference with distinct components of the necrosome must be considered separately.

Topics: Acetaminophen; Animals; Apoptosis; Carrier Proteins; Caspase 8; Chemokine CCL2; Concanavalin A; Disease Models, Animal; GTPase-Activating Proteins; Hepatitis, Autoimmune; Hepatocytes; Interleukin-6; Liver; Male; Mice; Mice, Inbred C57BL; Mice, Knockout; Necrosis; NLR Family, Pyrin Domain-Containing 3 Protein; Reactive Oxygen Species; Receptor-Interacting Protein Serine-Threonine Kinases; Tumor Necrosis Factor-alpha

Ghrelin is a 28-amino-acid gut hormone that was first discovered as a potent growth hormone secretagogue. Recently, it has been shown to exert a strong anti-inflammatory effect. The purpose of the study reported here was to explore the effect and mechanism of ghrelin on concanavalin (Con) A-induced acute hepatitis.. Balb/C mice were divided into four groups: normal control (NC) (mice injected with vehicle [saline]); Con A (25 mg/kg); Con A + 10 μg/kg ghrelin; and Con A + 50 μg/kg ghrelin (1 hour before Con A injection). Pro-inflammatory cytokine levels were detected. Protein levels of phosphoinositide 3-kinase (PI3K); phosphorylated Akt (p-Akt); caspase 3, 8, and 9; and microtubule-associated protein 1 light chain 3 (LC3) were also detected. Perifosine (25 mM) (an Akt inhibitor) was used to investigate whether the protective effect of ghrelin was interrupted by an Akt inhibitor. Protein levels of p-AKT; Bcl-2; Bax; and caspase 3, 8, and 9 were also detected.. Aspartate aminotransferase, alanine aminotransferase, and pathological damage were significantly ameliorated by ghrelin pretreatment in Con A-induced hepatitis. Inflammatory cytokines were significantly reduced by ghrelin pretreatment. Bcl-2; Bax; and caspase 3, 8, and 9 expression were also clearly affected by ghrelin pretreatment, compared with the Con A-treated group. However, the Akt kinase inhibitor reversed the decrease of Bax and caspase 3, 8, 9, and reduced the protein level of p-Akt and Bcl-2. Ghrelin activated the PI3K/Akt/Bcl-2 pathway and inhibited activation of autophagy.. Our results demonstrate that ghrelin attenuates Con A-induced acute immune hepatitis by activating the PI3K/Akt pathway and inhibiting the process of autophagy, which might be related to inhibition of inflammatory cytokine release, and prevention of hepatocyte apoptosis. These effects could be interrupted by an Akt kinase inhibitor.

Topics: Animals; Anti-Infective Agents; Apoptosis; Apoptosis Regulatory Proteins; Autophagy; Chemical and Drug Induced Liver Injury; Concanavalin A; Cytokines; Cytoprotection; Disease Models, Animal; Ghrelin; Inflammation Mediators; Liver; Mice, Inbred BALB C; Necrosis; Signal Transduction; Time Factors

Necrostatin-1 (Nec-1) inhibits receptor-interacting protein 1 (RIP1) kinase and programmed necrosis. This study was designed to examine the protective effects and mechanisms of Nec-1 in concanavalin A- (ConA-) induced hepatitis in mice.. C57BL/6 mice were exposed to ConA via tail vein injection and injected intraperitoneally with Nec-1 or vehicle. Levels of serum liver enzymes and histopathology were determined. Levels of inflammatory cytokines with ConA-induced hepatitis were determined with real-time polymerase chain reaction (real-time PCR). The expression of TNF- α , RIP1, and LC3 was detected with immunohistochemical staining. The expression of TNF- α , IFN- γ , IL2, IL6, caspase 3, RIP1, beclin-1, and LC3 protein was assessed by immunofluorescence and western blotting. Autophagosomes were observed with transmission electron microscopy (TEM).. Amelioration in liver functions and histopathological changes and the suppression of inflammatory cytokine production were observed in Nec-1-injected mice. Western blotting analysis showed that the expression of TNF- α , IFN- γ , IL2, IL6, and RIP1 was significantly reduced in the Nec-1-injected mice, which was confirmed by immunofluorescence and immunohistochemistry. Autophagosome formation was significantly reduced by Nec-1 treatment, as the expression of beclin-1 and LC3, determined with immunofluorescence and western blotting.. These results demonstrate that Nec-1 prevents ConA-induced liver injury via RIP1-related and autophagy-related pathways.

Topics: Animals; Autophagy; Chemical and Drug Induced Liver Injury; Concanavalin A; Cytokines; Disease Models, Animal; Gene Expression Profiling; Gene Expression Regulation; GTPase-Activating Proteins; Imidazoles; Indoles; Liver; Liver Failure, Acute; Male; Mice; Mice, Inbred C57BL; Microscopy, Electron, Transmission; Microtubule-Associated Proteins; Necrosis; Phagosomes; Real-Time Polymerase Chain Reaction; Transaminases; Tumor Necrosis Factor-alpha

Concanavalin A (Con A) treatment induces severe hepatitis in mice in a manner dependent on T cells, interferon (IFN)-gamma, and tumor necrosis factor (TNF). Treatment with the anticoagulant heparin protects against hepatitis, despite healthy production of IFN-γ and TNF. Here, we investigated molecular and cellular mechanisms for hypercoagulation-mediated hepatitis. After Con A challenge, liver of wild-type (WT) mice showed prompt induction of Ifnγ and Tnf, followed by messenger RNA expression of tissue factor (TF) and plasminogen activator inhibitor-1 (PAI-1), which initiate blood coagulation and inhibit clot lysis, respectively. Mice developed dense intrahepatic fibrin deposition and massive liver necrosis. In contrast, Ifnγ(-/-) mice and Ifnγ(-/-) Tnf(-/-) mice neither induced Pai1 or Tf nor developed hepatitis. In WT mice TF blockade with an anti-TF monoclonal antibody protected against Con A-induced hepatitis, whereas Pai1(-/-) mice were not protected. Both hepatic macrophages and sinusoidal endothelial cells (ECs) expressed Tf after Con A challenge. Macrophage-depleted WT mice reconstituted with hematopoietic cells, including macrophages deficient in signal transducer and activator of transcription-1 (STAT1) essential for IFN-γ signaling, exhibited substantial reduction of hepatic Tf and of liver injuries. This was also true for macrophage-depleted Stat1(-/-) mice reconstituted with WT macrophages. Exogenous IFN-γ and TNF rendered T-cell-null, Con A-resistant mice deficient in recombination-activating gene 2, highly susceptible to Con A-induced liver injury involving TF.. Collectively, these results strongly suggest that proinflammatory signals elicited by IFN-γ, TNF, and Con A in both hepatic macrophages and sinusoidal ECs are necessary and sufficient for the development of hypercoagulation-mediated hepatitis.

Topics: Animals; Concanavalin A; Endothelial Cells; Fibrin; Hepatitis, Animal; Interferon-gamma; Liver; Macrophages; Mice; Mice, Knockout; Mitogens; Necrosis; Plasminogen Activator Inhibitor 1; Signal Transduction; STAT1 Transcription Factor; T-Lymphocytes; Thrombophilia; Thromboplastin; Tumor Necrosis Factor-alpha

We previously reported that whey protein derived from cow milk suppressed inflammation in a variety of animal models. We developed a newly designed enteral formula using peptides prepared from whey protein and fermented milk product and investigated its ability to suppress inflammation in concanavalin A-induced hepatitis in mice.. C57BL/6 mice were fed a standard formula, AIN-93M, or enteral formula for 14 days, and then were intravenously administered concanavalin A. Inflammatory cytokines in plasma, liver, and spleen and markers of hepatic function in plasma were assessed at various time points. Livers were assessed for necrosis and apoptosis.. After concanavalin A treatment, plasma aspartate aminotransaminase, alanine aminotransferase, TNF-α, IL-6, and IFN-γ levels were significantly lower in mice fed enteral formula than in those fed standard formula or AIN-93M. Liver TNF-α and IFN-γ, and spleen IL-6 and IFN-γ levels were lower in enteral formula-fed mice than in standard formula-fed mice 2 h after concanavalin A treatment. Necrosis and apoptosis were suppressed in the livers of enteral formula-fed mice.. The new enteral formula is a potent novel immune-modulating diet that prevents aggravation of local inflammation by modulating systemic cytokine levels.

Topics: Alanine Transaminase; Animal Nutritional Physiological Phenomena; Animals; Apoptosis; Biomarkers; Chemical and Drug Induced Liver Injury; Concanavalin A; Cultured Milk Products; Enteral Nutrition; Inflammation; Interferon-gamma; Interleukin-6; Liver; Male; Mice; Mice, Inbred C57BL; Milk Proteins; Necrosis; Spleen; Tumor Necrosis Factor-alpha; Whey Proteins

Interleukin-1 receptor antagonist (IL-1Ra) is a specific IL-1 inhibitor that possesses anti-inflammatory activities. Several studies in human and mouse suggested a protective role for IL-1Ra in liver inflammation, and we previously demonstrated that hepatocytes produce high levels of IL-1Ra in response to inflammatory challenge in vitro and in vivo. In the present study, we investigated the production and the biological function of hepatocyte-derived IL-1Ra in concanavalin A (ConA)-induced hepatitis in mice. We show that the injured liver produces large amounts of IL-1Ra and that secreted and intracellular IL-1Ra isoforms are produced with different kinetics during the course of hepatitis. By using hepatocyte-specific IL-1Ra-deficient mice (IL-1Ra(ΔH)), we demonstrate that hepatocytes represent the major cellular source of local IL-1Ra. Most interestingly, hepatic necrosis and inflammation were increased in IL-1Ra(ΔH) as compared with wild-type mice during the late phase of the disease, leading to a delayed resolution of hepatitis in IL-1Ra(ΔH) mice. In conclusion, our results show that the local production of IL-1Ra by hepatocytes contributes to the resolution of hepatitis.

Topics: Animals; Concanavalin A; Cytokines; Hepatitis; Hepatocytes; Interleukin 1 Receptor Antagonist Protein; Liver; Male; Mice; Mice, Inbred C57BL; Necrosis

Curcumin, a polyphenol extracted from the plant curcuma longa, exhibits a number of pharmacological properties and has been used for the treatment of inflammatory diseases. However, the potential protective effects of curcumin in inflammatory liver diseases have not been clearly elucidated. Thus, the current study aimed to investigate the beneficial effects of curcumin on hepatic injury induced by concanavalin A (Con A), and its possible molecular mechanisms in mice. Acute live injury model was established successfully by intravenous administration of Con A (15mg/kg) in male C57BL/6 mice. Curcumin was administered to mice 2h prior to Con A injection. It was found that curcumin pretreatment significantly protected animals from T cell-mediated hepatitis as evidenced by decreased elevation of serum ALT, associated with reduced hepatic necrosis, apoptosis and mortality. In addition, curcumin pretreatment markedly reduced hepatic oxidative stress and pro inflammatory cytokines, including TNF-α and IFN-γ. Furthermore, curcumin pretreatment dramatically suppressed the releasing of high-mobility group box-1 (HMGB1) in liver tissues. These results suggest that curcumin pretreatment protects the mice from Con A-induced liver injury via inhibiting hepatocyte oxidative stress, inflammation and releasing of HMGB1.

Topics: Alanine Transaminase; Animals; Anti-Inflammatory Agents; Antioxidants; Apoptosis; Biomarkers; Concanavalin A; Curcumin; Cytoprotection; Disease Models, Animal; Down-Regulation; Hepatitis, Autoimmune; HMGB1 Protein; Interferon-gamma; Liver; Male; Malondialdehyde; Mice; Mice, Inbred C57BL; Necrosis; Oxidative Stress; Time Factors; Tumor Necrosis Factor-alpha

Interferon-gamma (IFN-γ), a potent Th1 cytokine with multiple biological functions, can induce autophagy to enhance the clearance of the invading microorganism or cause cell death. We have reported that Concanavalin A (Con A) can cause autophagic cell death in hepatocytes and induce both T cell-dependent and -independent acute hepatitis in immunocompetent and immunodeficient mice, respectively. Although IFN-γ is known to enhance liver injury in Con A-induced hepatitis, its role in autophagy-related hepatocyte death is not clear. In this study we report that IFN-γ can enhance Con A-induced autophagic flux and cell death in hepatoma cell lines. A necrotic cell death with increased lysosomal membrane permeabilization (LMP) is observed in Con A-treated hepatoma cells in the presence of IFN-γ. Cathepsin B and L were released from lysosomes to cause cell death. Furthermore, IFN-γ induces immunity related GTPase family M member 1(IRGM1) translocation to lysosomes and prolongs its activity in Con A-treated hepatoma cells. Knockdown of IRGM1 inhibits the IFN-γ/Con A-induced LMP change and cell death. Furthermore, IFN-γ(-/-) mice are resistant to Con A-induced autophagy-associated necrotic hepatocyte death. We conclude that IFN-γ enhances Con A-induced autophagic flux and causes an IRGM1-dependent lysosome-mediated necrotic cell death in hepatocytes.

Topics: Animals; Autophagy; Carcinoma, Hepatocellular; Cathepsin B; Cathepsin L; Cell Line, Tumor; Cell Membrane; Concanavalin A; Endocytosis; GTP-Binding Proteins; Hep G2 Cells; Hepatocytes; Humans; Interferon-gamma; Liver Neoplasms; Lysosomes; Mice; Mice, Inbred BALB C; Necrosis; Permeability

Previous studies demonstrate that both membrane B7-H4 and B7-H4-Ig fusion protein could inhibit T-cell responses. In the present study, we explored the potential effect of B7-H4-Ig on liver injury in a hepatitis mouse model induced by concanavalin A (ConA). A B7-H4-Ig construct was introduced into animals by the hydrodynamic gene delivery approach. It was found that ectopic expression of B7-H4-Ig could inhibit ConA-induced elevation of serum levels of ALT and AST, suppress liver necrosis and even mortality of mice. Furthermore, we observed that pretreatment of B7-H4-Ig dramatically decreased serum levels and the expression of mRNA for IL-2, IFN-gamma and IL-4, but increased IL-10 in ConA-treated mice. Our results suggest that B7-H4-Ig may protect animals from liver injury induced by ConA, which could be associated with reduced serum levels for IL-2, IFN-gamma and IL-4 as well as enhanced IL-10 production.

Topics: Alanine Transaminase; Animals; Aspartate Aminotransferases; B7-1 Antigen; Chemical and Drug Induced Liver Injury; Concanavalin A; Gene Expression; Gene Transfer Techniques; Genetic Therapy; Humans; Immunoglobulin Fc Fragments; Immunoglobulin G; Interferon-gamma; Interleukin-10; Interleukin-2; Interleukin-4; Leukocytes, Mononuclear; Liver; Mice; Mice, Inbred BALB C; Necrosis; Recombinant Fusion Proteins; Survival Analysis; V-Set Domain-Containing T-Cell Activation Inhibitor 1

The aim of this experiment is to investigate the antioxidative and antiapoptotic roles of ellagic (EA) acid in in vitro and in in vivo experiment. We measured protective properties of EA against oxidative stress-induced hepatocyte damage in vitro and Concanavalin (ConA)-induced liver damage in vivo. EA, a potent antioxidant, exhibited protective properties against oxidative stress-induced hepatocyte damage by preventing vitamin k3 (VK3)-induced reactive oxygen species (ROS) productions, apoptotic and necrotic cellular damage and mitochondrial depolarization, which is a main cause of ROS production. EA also protects against cell death and elevation of glutathione (GSH), alanine transaminase (ALT) and asparatate transaminase (AST) in Con A-induced fulminant liver damage in mice. These results show that antioxidant and cytoprotective properties of EA prevent liver damage induced by various type of oxidative stress.

Topics: Alanine Transaminase; Animals; Antioxidants; Apoptosis; Aspartate Aminotransferases; Cell Line; Concanavalin A; Cytoprotection; Ellagic Acid; Glutathione; Hepatocytes; Humans; Male; Membrane Potential, Mitochondrial; Mice; Mice, Inbred BALB C; Mitochondria, Liver; Necrosis; Oxidative Stress; Reactive Oxygen Species; Vitamin K 3

Tumor necrosis factor-alpha-mediated liver injury can be induced by several different means; however, the signaling events and mechanisms of cell death are likely different. We investigated the mechanism of both apoptotic and necrotic hepatocyte cell death as well as the role of c-Jun NH2-terminal kinase (JNK) in the ConA and ConA/D-galactosamine (GalN) models of murine liver injury. ConA alone induced primarily necrotic cell death with no caspase activation, whereas ConA/GalN induced apoptosis in addition to necrotic cell death. The bi-modal death pattern in the ConA/GalN model was confirmed by the use of transgenic mice expressing a dominant-negative form of Fas-associated death domain in which the mice were resistant to apoptotic but not necrotic cell death. JNK1 and, more significantly, JNK2 participated in the induction of hepatocyte apoptosis in response to ConA/GalN. Deletion of JNK led to the stabilization of FLIP L, reduced caspase-8 activation, decreased Bid cleavage, and inhibition of the mitochondrial apoptosis pathway. In contrast, JNK did not participate in necrotic death induced by ConA either alone or in combination with GalN. As such, JNK-deficient mice remained susceptible to necrotic liver injury in both model systems. Thus, ConA and ConA/GalN mouse models induce liver injury with different mechanisms of cell death, and JNK contributes to apoptotic but not necrotic cell death. These findings further elucidate the specific pathways involved in tumor necrosis factor-alpha-mediated liver injury.

Topics: Animals; Apoptosis; BH3 Interacting Domain Death Agonist Protein; Caspase 8; Chemical and Drug Induced Liver Injury; Concanavalin A; Disease Models, Animal; Enzyme Activation; Fas-Associated Death Domain Protein; Galactosamine; Gene Deletion; JNK Mitogen-Activated Protein Kinases; Liver Diseases; Mice; Mice, Inbred C57BL; Mitochondria, Liver; Mutant Proteins; Necrosis; Phosphorylation

Both nuclear factor kappa B (NF-kappaB) activation and inducible nitric oxide synthase (iNOS) expression increase in the liver injury, and there are NF-kappaB binding sites in the iNOS promoter. The aim of this study was to investigate the correlation between iNOS expression and NF-kappaB activation in hepatitis induced by concanavalin A (con A).. Eighty-eight male BALB/c mice were randomly divided into three groups: vehicle control group, con A group and pyrrolidine dithiocarbamate (PDTC) plus con A group. In the vehicle control group, the mice were treated with saline (0.3 mL, i.v.). In the con A group, the mice were treated with con A (20 mg/kg, i.v.). In the PDTC + con A group, the mice were pretreated with PDTC (120 mg/kg, i.p.) 30 min before administration of con A (20 mg/kg, i.v.). Blood samples were taken from the retro-orbital venous plexus at 0.5, 1, 4, 8 and 16 h after con A injection and the mice were killed immediately. The plasma alanine aminotransferase (ALT) levels were measured by the standard photometric method. Nitric oxide (NO) levels in the liver homogenate were assayed by spectroscopy. Liver tissues were sectioned and stained with hematoxylin-eosin for histological examination. Activation of NF-kappaB, degradation of inhibitor of kappa B alpha (IkappaBalpha), and expression of iNOS were measured by western blot.. In the con A group, the plasma ALT activity and NO levels in the liver increased significantly at 1 h (P < 0.05, n = 8) and reached a peak at 4 h after con A injection. The liver injury in this group was characterized by liver necrosis, cell swelling and fatty degeneration. Cytosolic IkappaBalpha decreased slightly at 30 min after con A challenge, was undetectable at 1 h and reappeared at 4 h. Correspondingly, the NF-kappaB level in the nucleus was highest at 1 h. The iNOS expression increased at 30 min after con A injection and reached a maximum at 4 h. Pretreatment with PDTC prevented these changes and attenuated the liver injury.. Con A-induced iNOS expression in the liver is dependent on the activation of NF-kappaB.

Topics: Active Transport, Cell Nucleus; Alanine Transaminase; Animals; Concanavalin A; Disease Models, Animal; Enzyme Induction; Hepatitis; I-kappa B Proteins; Liver; Male; Mice; Mice, Inbred BALB C; Necrosis; NF-KappaB Inhibitor alpha; Nitric Oxide; Nitric Oxide Synthase Type II; Pyrrolidines; Severity of Illness Index; Thiocarbamates; Time Factors; Transcription Factor RelA; Up-Regulation

Rutin is a flavonoid obtained from Dimorphandra mollis (Benth.), a medicinal Brazilian plant used as antioxidative, antihemorrhagic, and blood vessel protector. The present study has examined its effects on the viability and function of immune system cells in vitro. Rat spleen and thymus cells were cultured with 10 nM, 1 microM, and 10 microM of the drug in the presence or absence of PWM, LPS, or ConA mitogens. Cellular proliferation was analyzed by H(3)-thymidin uptake and IFN-gamma and IL-10 were measured by ELISA after 48 and 72 hr. Viability was measured by flow cytometry using Annexin V and PI after 24 and 48 hr. The flavonoid rutin inhibited splenocytes and thymocytes proliferation under ConA stimulation observed by an increase on apoptosis levels of thymocytes stimulated with PWM in 24 hr and on splenocytes stimulated with PWM in 48 hr. Function studies showed a decrease on IFN-gamma production by splenocytes and thymocytes stimulated with PWM or ConA. Spleen cells cultured with LPS and rutin showed a decrease on apoptosis after 24 hr and an increase on the IL-10 levels after 48 hr. There was no significant variation on the necrosis rate, viability, and function of cells treated with rutin in the absence of mitogenic stimulus.

Topics: Animals; Annexin A5; Apoptosis; Cell Proliferation; Cell Survival; Concanavalin A; Cytokines; Interferon-gamma; Interleukin-10; Lipopolysaccharides; Male; Mitogens; Necrosis; Pokeweed Mitogens; Rats; Rutin; Spleen; Stimulation, Chemical; T-Lymphocytes

The outcome of immunological assays is markedly influenced by the method of isolation of lymphocytes. It is, therefore, important to comparatively assess various techniques of isolation of lymphocytes, an aspect thus far not thoroughly addressed. In particular, the potential of isolation techniques to influence cell recovery, viability, and function has not yet been evaluated. These studies were designed to determine the effect of different mechanical tissue dissociation methods on the viability and function of lymphocytes. Following spleen and thymus removal, the lymphoid organs were dissociated by one of four different tissue dissociation techniques: metallic screen, sheer force slide, commercial stomacher, or plunger-screen. Cells were then enumerated and a trypan blue exclusion technique and 7-amino-actinomycin D (7-AAD) were both employed to assess viability. Mitogen-induced lymphocyte proliferation was measured using the Alamar Blue assay. Cell viability and lymphocyte surface antigen expression were assessed using flow cytometry. No significant differences in lymphocyte viability, morphology, or surface antigen expression were observed among the different techniques. Likewise, cellular apoptosis and necrosis were comparable across all the techniques. However, mitogen induced splenic T-cell proliferation was higher in cells collected using the metallic screen and plunger-screen isolation methods as compared to the sheer force slide or commercial stomacher procedures. These data suggest that cell recovery, morphology, and viability are not affected by isolation techniques. However, lymphocyte function, as assessed by mitogen induced proliferation, was negatively affected by the sheer force slide or commercial stomacher isolation techniques.

Topics: Animals; Antigens, CD; Apoptosis; Cell Count; Cell Proliferation; Cell Separation; Cell Survival; Concanavalin A; Female; Lymphocytes; Mice; Mice, Inbred BALB C; Necrosis; Spleen; Thymus Gland

Macrophage migration inhibitory factor (MIF) is involved in inflammatory and immune-mediated diseases but the role of MIF in liver injury has not yet been elucidated.. We investigated biochemically, histologically and immunologically the character of MIF in concanavalin A (Con A)-induced T-cell-mediated liver injury using MIF knockout (KO) mice and wild-type (WT) mice.. MIF KO mice showed significantly decreased serum alanine aminotransferase values and suppressed histological change with massive necrosis of the hepatic parenchymal cells and infiltration of inflammatory cells compared with their WT counterparts. This protection was not mediated by either tumor necrosis factor-alpha or interferon-gamma, which are critical mediators of Con A-induced liver injury, as their serum concentrations were shown to be similar in MIF KO and WT mice. On the other hand, a flow cytometric analysis demonstrated that the number of activated hepatic leukocytes decreased more in the MIF KO mice than in the WT mice.. A lack of MIF protected the mice from Con A-induced liver injury. Controlling the MIF activity may be a useful therapeutic strategy for treating such T-cell activation-associated liver diseases as autoimmune hepatitis and viral hepatitis.

Topics: Alanine Transaminase; Animals; Antigens, CD; Antigens, Differentiation, T-Lymphocyte; Biomarkers; CD3 Complex; Concanavalin A; Disease Models, Animal; Female; Inflammation Mediators; Interferon-gamma; Lectins, C-Type; Leukocytes; Liver; Lymphocyte Activation; Macrophage Migration-Inhibitory Factors; Mice; Mice, Inbred BALB C; Mice, Knockout; Mitogens; Necrosis; RNA, Messenger; T-Lymphocytes; Tumor Necrosis Factor-alpha

Major physical traumas provoke a systemic inflammatory response and immune dysfunction. In a model of thermal injury in rats, we previously showed that an overproduction of nitric oxide (NO) was responsible for the collapse of lymphoproliferative responses. In the present work, we performed a time-course analysis of cell proliferation and cell death parameters in order to establish the sequence of events triggered by the high NO output in Wistar/Han rat splenocytes activated with Con A, 10 days after burn injury. We demonstrate that activated T cells from burned rats never divided whereas normal T cells underwent four division cycles. However, T cells from both burned and normal rat entered the G1 phase as shown by increase of cell size, mitochondria hyperpolarization, and expression of cyclin D1. Burned rat T cells progressed to the late G1 phase as shown by expression of the nuclear Ki-67 antigen, but they never entered the S phase. They underwent apoptosis as shown by morphological parameters, disruption of transmembrane mitochondrial potential, and DNA fragmentation. Persistent accumulation of the p53 protein accompanied these phenomena. NO synthase inhibitors antagonize alterations of cell proliferation and cell death parameters in burned rat T cells and accelerated p53 turnover.

Topics: Animals; Apoptosis; Burns; Cell Division; Cell Proliferation; Concanavalin A; Enzyme Inhibitors; Flow Cytometry; G1 Phase; Interleukin-2; Isothiuronium; Lymphocyte Activation; Male; Models, Biological; Necrosis; Nitric Oxide; Rats; Rats, Wistar; Spleen; T-Lymphocytes; Thiazines; Tumor Suppressor Protein p53

Massive liver necrosis can develop as a consequence of imbalance between T-helper (Th)1 and Th2 immune reactions in the liver. Osteopontin is a glycoprotein secreted for the initiation of the Th1 immune reaction, as well as for extracellular matrix formation and calcium deposition in the bone and kidney. Osteopontin is overexpressed in Kupffer cells, macrophages, and stellate cells activated in injured livers. We established transgenic mice expressing osteopontin exclusively in hepatocytes, using a vector containing human serum amyloid P component promoter. The relation of Th1/Th2 immune imbalance to massive liver necrosis was studied using these transgenic mice.. Transgenic mice and C27BL/6 mice, wild-type controls of the transgenic mice, were given an intravenous injection of concanavalin-A, and the histological extent of liver injuries and plasma cytokine levels were evaluated.. When the transgenic mice received concanavalin-A, massive necrosis and mononuclear cell infiltration developed in the liver, the extent of which was greater in the female mice than in the male mice. This treatment produced minimal liver injury and focal liver necrosis in male and female C57BL/6 mice. In these transgenic and control mice, plasma concentrations of interleukin (IL)-10 and interferon (IFN)-gamma were increased after concanavalin-A treatment. However, the upregulation of plasma IL-10 concentration was smaller in the male and female transgenic mice than in the control mice, and the upregulation of the IFN-gamma concentration was greater in the female transgenic mice than in the female control mice.. Th1 and Th2 immune reactions were deranged after concanavalin-A treatment, with Th1 immunity predominating in transgenic mice expressing osteopontin in hepatocytes; this immunological imbalance may contribute to massive liver necrosis.

Topics: Animals; Concanavalin A; Cytokines; Disease Models, Animal; Female; Immunity, Cellular; Liver; Male; Mice; Mice, Inbred C57BL; Mice, Transgenic; Necrosis; Osteopontin; Sialoglycoproteins; Th1 Cells; Th2 Cells; Up-Regulation

RNA interference (RNAi) is a powerful tool to silence gene expression post-transcriptionally. However, its potential to treat or prevent disease remains unproven. Fas-mediated apoptosis is implicated in a broad spectrum of liver diseases, where inhibiting hepatocyte death is life-saving. We investigated the in vivo silencing effect of small interfering RNA (siRNA) duplexes targeting the gene Fas (also known as Tnfrsf6), encoding the Fas receptor, to protect mice from liver failure and fibrosis in two models of autoimmune hepatitis. Intravenous injection of Fas siRNA specifically reduced Fas mRNA levels and expression of Fas protein in mouse hepatocytes, and the effects persisted without diminution for 10 days. Hepatocytes isolated from mice treated with Fas siRNA were resistant to apoptosis when exposed to Fas-specific antibody or co-cultured with concanavalin A (ConA)-stimulated hepatic mononuclear cells. Treatment with Fas siRNA 2 days before ConA challenge abrogated hepatocyte necrosis and inflammatory infiltration and markedly reduced serum concentrations of transaminases. Administering Fas siRNA beginning one week after initiating weekly ConA injections protected mice from liver fibrosis. In a more fulminant hepatitis induced by injecting agonistic Fas-specific antibody, 82% of mice treated with siRNA that effectively silenced Fas survived for 10 days of observation, whereas all control mice died within 3 days. Silencing Fas expression with RNAi holds therapeutic promise to prevent liver injury by protecting hepatocytes from cytotoxicity.

Topics: Animals; Apoptosis; Cells, Cultured; Concanavalin A; Disease Models, Animal; fas Receptor; Gene Silencing; Hepatitis, Autoimmune; Hepatocytes; In Situ Nick-End Labeling; Liver; Male; Mice; Necrosis; RNA Interference; RNA, Small Interfering

P-selectin (CD62P) is an adhesion molecule that mediates the initial attachment of leukocytes to activated platelets and endothelial cells in damaged tissues. We evaluated the role of P-selectin in concanavalin A (Con A)-induced hepatitis, a model characterized by CD4(+) T cell activation and infiltration of the liver. Con A injection induced transient P-selectin expression on hepatic venules and platelets. Mice lacking P-selectin showed impaired lymphocyte adhesion to liver venules and sinusoids, a striking reduction in intrasinusoidal occlusion, and decreased lymphocyte infiltration of liver parenchyma. The reduction in transaminase levels and the almost complete abolition of necrotic injury demonstrated that liver damage was lower in P-selectin-deficient mice. In wild-type mice, pretreatment with the P-selectin-blocking monoclonal antibody attenuated the sinusoidal occlusion and reduced the rise in transaminases after Con A treatment. These results implicate P-selectin in the development of Con A-induced liver injury and reveal the protective effect of blocking P-selectin in this hepatitis.

Topics: Alanine Transaminase; Animals; Antibodies, Monoclonal; Aspartate Aminotransferases; Autoimmune Diseases; Blood Platelets; CD4-Positive T-Lymphocytes; Cell Adhesion; Chemical and Drug Induced Liver Injury; Chemotaxis, Leukocyte; Concanavalin A; Disease Models, Animal; Endothelium, Vascular; Gene Expression Regulation; Hemostasis; Liver; Mice; Mice, Inbred C57BL; Mice, Knockout; Necrosis; P-Selectin; Specific Pathogen-Free Organisms

CD7 and CD28 are Ig superfamily molecules expressed on thymocytes and mature T cells that share common signaling 0mechanisms and are co-mitogens for T cell activation. CD7-deficient mice are resistant to lipopolysaccharide (LPS)-induced shock syndrome, and have diminished in vivo LPS-triggered IFN-gamma and tumor necrosis factor (TNF)-alpha production. CD28-deficient mice have decreased serum Ig levels, defective IgG isotype switching, decreased T cell IL-2 production and are resistant to Staphylococcus aureus enterotoxin-induced shock. To determine synergistic roles CD7 and CD28 might play in thymocyte development and function, we have generated and characterized CD7/CD28 double-deficient mice. CD7/CD28-deficient mice were healthy, reproduced normally, had normal numbers of thymocyte subsets and had normal thymus histology. Anti-CD3 mAb induced similar levels of apoptosis in CD7-deficient, CD28-deficient and CD7/CD28 double-deficient thymocytes as in control C57BL/6 mice (P = NS). Similarly, thymocyte viability, apoptosis and necrosis following ionomycin or dexamethasone treatment were the same in control, CD7-deficient, CD28-deficient and CD7/CD28-deficient mice. CD28-deficient and CD7/CD28-deficient thymocytes had decreased [3H]thymidine incorporation responses to concanavalin A (Con A) stimulation compared to control mice (P < or = 0.01 and P < or = 0.05 respectively). CD7/CD28 double-deficient mice had significantly reduced numbers of B7-1/B7-2 double-positive cells compared to freshly isolated wild-type, CD7-deficient and CD28-deficient thymocytes. Con A-stimulated CD4/CD8 double-negative (DN) thymocytes from CD7/CD28 double-deficient mice expressed significantly lower levels of CD25 when compared to CD4/CD8 DN thymocytes from wild-type, CD7-deficient and CD28-deficient mice (P < 0.05). Anti-CD3-triggered CD7/CD28-deficient thymocytes also had decreased IFN-gamma and TNF-alpha production compared to C57BL/6 control, CD7-deficient and CD28-deficient mice (P < or = 0.05). Thus, CD7 and CD28 deficiencies combined to produce abnormalities in the absolute number of B7-1/B7-2-expressing cells in the thymus, thymocyte IL-2 receptor expression and CD3-triggered cytokine production.

Topics: Animals; Antigens, CD; Antigens, CD7; Apoptosis; B7-1 Antigen; B7-2 Antigen; CD28 Antigens; Cell Differentiation; Cell Division; Cell Survival; Concanavalin A; Cytokines; Lymphocyte Activation; Membrane Glycoproteins; Mice; Mice, Inbred C57BL; Mice, Knockout; Muromonab-CD3; Necrosis; Receptors, Interleukin-2; T-Lymphocyte Subsets; Thymus Gland

The activity principle of the mistletoe (Viscum album L.) phytotherapeutics could be considered as combined cytotoxic and "biological response modifying" activities (increasing host defense against cancer) that result from the activities of the plant lectins and the other biologically relevant substances. We found before that the aqueous extract Isorel, produced by Novipharm GmbH (Pörtschach, Austria) from the entire plant (planta tota) of fresh mistletoe under standardized conditions with bioassay validated batch consistency, can be valuable in experimental adjuvant cancer therapy increasing efficiency of cyclophosphamide chemotherapy. In current study we found that Isorel increases the reactivity of the tumor-bearing mice lymphocytes to the mitogens (ConA and LPS) in vitro, thus indicating its immune stimulating effects for the cancer-immunosuppressed lymphocytes. Moreover, Isorel inhibited the incorporation of 3H-labelled amino acids (protein synthesis) in various malignant cell lines. For the growth inhibition mostly higher MW components were responsible, although even less than 500 Da components were also active. We further analyzed the effects of drug application in vicinity of tumor (murine mammary carcinoma) and compared it with systemic effects. The animals carried mammary carcinoma in both hind limbs and were also injected with tumor cells i.v. to develop artificial lung metastases. Isorel was applied only at the right side (in the limb distal from the tumor) and caused persistent and almost complete inhibition of the tumor growth for 2/7 animals. Anticancer effects were less pronounced on the contralateral side tumors, although tumor growth rate was transiently reduced for some mice. Histology revealed that Isorel treatment, both at the side of tumor and systemically, increased the incidence of apoptosis and necrosis in the tumors, while reduction of mitosis was noticed only for the tumors in vicinity of the tumor exposed to Isorel. Finally, animals treated with Isorel had, on the average, three times less lung metastases than the controls. Thus, we conclude that both local and systemic effects of the application of Isorel could be of benefit for the tumor-bearing organism resulting in immunomodulation combined with tumor growth inhibition and reduction of metastases. According to the in vitro results, antitumorous effects could be the result not only of the mistletoe lectins and the other high MW factors, but also of the very low MW (< 500 Da)

Topics: Adjuvants, Immunologic; Animals; Antineoplastic Agents, Phytogenic; Apoptosis; Concanavalin A; Drug Synergism; Female; Lipopolysaccharides; Lung Neoplasms; Lymphocyte Activation; Lymphocytes; Mammary Neoplasms, Experimental; Melanoma, Experimental; Mice; Mice, Inbred C57BL; Necrosis; Phytotherapy; Plant Extracts; Plant Lectins; Plants, Medicinal; Tumor Cells, Cultured

Glucocorticoids (GCs) have been considered to regulate immune cell systems through induction of apoptosis in thymocytes and mature peripheral-blood lymphocytes. Here we report that apoptosis induced by cortisol in mitogen-activated peripheral-blood mononuclear cells (PBMC) is suppressed by cortisone, an oxidized metabolite of cortisol. Apoptosis in PBMCs is quantified by a cell death ELISA procedure, which can specifically detect fragmented DNA. Cortisol induced PBMC-apoptosis at concentrations more than 10 ng/ml (28 nM) in concanavalin A-stimulated PBMCs and cortisone suppressed this apoptosis at a concentration range of 1-10,000 ng/ml (2.8-28,000 nM) dose-dependently. Prednisone, a synthetic oxidized-GC, also suppressed the apoptosis-inducing effect of cortisol in a dose-dependent manner. Suppression of cortisol-induced apoptosis by cortisone was consistently observed in PBMCs derived from 16 healthy subjects. Examination for inhibitory activities of the steroids against [3H]dexamethasone binding to PBMCs suggested that cortisone can bind cellular GC-receptors (GC-Rs), but the affinity of cortisone to GCRs is 1/30 or less than that of cortisol. The results raised a possible role of cortisone in cortisol-mediated regulation of apoptosis in activated human PBMCs. The counteracting action of cortisone against cortisol-induced apoptosis may take place partially through intervention of GC-receptors (GC-Rs), but may also be due to unknown pathway(s) different from those mediated by cellular GC-Rs.

Topics: Adult; Apoptosis; Cells, Cultured; Concanavalin A; Cortisone; Dexamethasone; DNA Fragmentation; Dose-Response Relationship, Drug; Female; Humans; Hydrocortisone; Leukocytes, Mononuclear; Lymphocyte Activation; Male; Necrosis; Prednisone; Receptors, Glucocorticoid

Weekly injections of Concanavalin A (Con A) were performed in BALB/c mice to evaluate the pattern of cytokine production and liver injury. High serum levels of tumor necrosis factor alpha (TNF-alpha), interleukin 2 (IL-2), IL-4, and interferon gamma (IFN-gamma) were found in the serum after the first 2 injections of Con A but rapidly decreased from the third injection. Conversely, IL-10 serum levels after repeated Con A challenge increased by 7 times from week 1 to 20. In vivo depletion studies indicated that CD4(+) T cells are essential in IL-10 production. Hepatocyte necrosis was only observed after the first injections of Con A whereas centrilobular inflammatory infiltrates persisted up to 20 weeks. Perisinusoidal liver fibrosis was also increasingly detected in BALB/c mice, whereas no fibrous change was observed in nude mice after 6 weeks of Con A challenge. The number of stellate cells, detected by immunostaining, increased after 20 weeks of Con A injections. Liver cytokine messenger RNA (mRNA) expression after 20 weeks showed expression of transforming growth factor beta1 (TGF-beta1), IL-10, and IL-4 whereas IL-2 was no more expressed. The present study shows that mice repeatedly injected with Con A develop liver fibrosis. The cytokine-release pattern observed after 1 injection of Con A is rapidly shifted towards an immunomodulatory phenotype characterized by the systemic production of large amounts of IL-10.

Topics: Animals; CD4-Positive T-Lymphocytes; Chemical and Drug Induced Liver Injury; Concanavalin A; Drug Administration Schedule; Female; Interleukin-10; Liver; Liver Cirrhosis, Experimental; Mice; Mice, Inbred BALB C; Necrosis; Phenotype; Time Factors

The activation of the death receptors, tumor necrosis factor-receptor-1 (TNF-R1) or CD95, is a hallmark of inflammatory or viral liver disease. In different murine in vivo models, we found that livers depleted of gamma-glutamyl-cysteinyl-glycine (GSH) by endogenous enzymatic conjugation after phorone treatment were resistant against death receptor-elicited injury as assessed by transaminase release and histopathology. In apoptotic models initiated by engagement of CD95, or by injection of TNF or lipopolysaccharide into galactosamine-sensitized mice, hepatic caspase-3-like proteases were not activated in the GSH-depleted state. Under GSH depletion, also caspase-independent, TNF-R1-mediated injury (high-dose actinomycin D or alpha-amanitin), as well as necrotic hepatotoxicity (high-dose lipopolysaccharide) were entirely blocked. In the T-cell-dependent model of concanavalin A-induced hepatotoxicity, GSH depletion resulted in a suppression of interferon-gamma release, delay of systemic TNF release, hepatic nuclear factor-kappaB activation, and an abrogation of sinusoidal endothelial cell detachment as assessed by electron microscopy. When GSH depletion was initiated 3 hours after concanavalin A injection, ie, after the peak of early pro-inflammatory cytokines, livers were still protected. We conclude that sufficient hepatic GSH levels are a prerequisite for the execution of death receptor-mediated hepatocyte demise.

Topics: Amanitins; Animals; Antigens, CD; Apoptosis; Caspases; Cell Death; Concanavalin A; Cytokines; Dactinomycin; Dose-Response Relationship, Drug; Glutathione; Liver; Male; Mice; Mice, Inbred BALB C; Necrosis; Nucleic Acid Synthesis Inhibitors; Receptors, Tumor Necrosis Factor; Receptors, Tumor Necrosis Factor, Type I; Shock, Septic

Wild-type mammalian adenoviruses are known to inhibit programmed cells death in infected cells. This study demonstrated for the first time that an avian type II adenovirus, the hemorrhagic enteritis virus (HEV) of turkeys, induced apoptosis in turkey spleen cells at 3 and 4 days post infection. The increased apoptosis rate in spleens of HEV-infected turkeys was associated with increased virus replication. Increased apoptosis preceded extensive virus-induced cellular necrosis. At 3 days post infection, spleen cells from HEV-infected turkeys released tumor necrosis like factor and nitric oxide inducing factors after ex vivo stimulation with concanavalin A. Spleen cells from HEV-exposed turkeys also secreted an interleukin 6-like factor when cultured in vitro. These cytokines may have contributed to HEV-pathogenesis and HEV-induced apoptosis and necrosis in the spleen. Induction of apoptosis by an avian adenovirus but not by wild-type mammalian adenoviruses indicates that evolutionarily distant adenoviruses may have different pathogenic mechanisms.

Topics: Adenoviridae Infections; Animals; Apoptosis; Aviadenovirus; Bird Diseases; Cell Count; Cells, Cultured; Concanavalin A; Immunohistochemistry; In Situ Hybridization; In Situ Nick-End Labeling; Interleukin-6; Necrosis; Nitric Oxide; Spleen; Tumor Necrosis Factor-alpha; Turkeys

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.

Topics: ADP Ribose Transferases; Animals; Antigens, Bacterial; Bacterial Toxins; Chemical and Drug Induced Liver Injury; Concanavalin A; Drug Combinations; Enterotoxins; Exotoxins; Kupffer Cells; Liver; Liver Diseases; Male; Mice; Mice, Inbred BALB C; Necrosis; Pseudomonas aeruginosa Exotoxin A; T-Lymphocytes; Tumor Necrosis Factor-alpha; Virulence Factors

Recombinant human interleukin-11 (rhIL-11) is a multifunctional cytokine that can reduce inflammation through the downregulation of multiple pro-inflammatory mediators from activated macrophages. rhIL-11 also inhibits production of several immunostimulatory cytokines such as IL-12 and interferon gamma (IFN-gamma) and has shown biological activity in multiple animal models of inflammatory disease consistent with immunomodulatory effects on macrophages and T cells. To further elucidate the anti-inflammatory activity of rhIL-11 in vivo, the effect of rhIL-11 in a model of Concanavalin A (Con-A)-induced T-cell-mediated hepatotoxicity was examined. Administration of a single dose of rhIL-11 before Con-A administration reduced centrilobular liver necrosis and enhanced survival. A dose-dependent reduction in serum levels of liver enzymes, tumor necrosis factor alpha (TNF-alpha), and IFN-gamma corresponded with this amelioration of liver damage. No significant change in infiltrating lymphocyte populations in the liver was observed following rhIL-11 treatment. Taken together, these results indicate that rhIL-11 ameliorates T-cell-mediated hepatic injury and suggests its therapeutic potential to treat inflammatory liver disease.

Topics: Alanine Transaminase; Animals; Aspartate Aminotransferases; Chemical and Drug Induced Liver Injury; Concanavalin A; Dose-Response Relationship, Drug; Enzyme Induction; Female; Humans; Interferon-gamma; Interleukin-11; Interleukin-2; Liver; Mice; Mice, Inbred BALB C; Necrosis; Recombinant Proteins; T-Lymphocytes; Tumor Necrosis Factor-alpha

Experimental T-cell-mediated hepatitis induced by concanavalin A (Con A) involves the production of proinflammatory cytokines. Because interleukin (IL)-10 is a potent anti-inflammatory cytokine derived from macrophages and T cells and is produced within the liver, we investigated the role of IL-10 in modulating the hepatotoxicity and the secretion of cytokines following in vivo injection of Con A. IL-10 is produced early in the serum after Con A challenge. Neutralization of endogenous IL-10 by monoclonal antibodies (mAbs) increases the secretion of tumor necrosis factor alpha (TNF-alpha) (+111%), interferon gamma (IFN-gamma) (+92%), and IL-12 (+730%) 8 hours after Con A injection, and increases the hepatotoxicity, assessed by serum alanine transaminase (ALT) (+174%) measurement and by histology, 24 hours after induction of hepatitis. Conversely, preadministration of recombinant IL-10 reduces the production of these proinflammatory cytokines (-47%, -80%, and -47% for TNF-alpha, IL-12, and IFN-gamma, respectively), and decreases neutrophil infiltration and ALT serum concentration (-74%) 8 hours after Con A challenge. We conclude that IL-10, either endogenously produced or exogenously added, has a hepatoprotective role in Con A-induced hepatitis, through its suppressive property on proinflammatory cytokine production, and that it might be of therapeutic relevance in human liver diseases involving activated T cells.

Topics: Animals; Chemical and Drug Induced Liver Injury; Concanavalin A; Interferon-gamma; Interleukin-10; Interleukin-12; Liver; Mice; Mice, Inbred C3H; Necrosis; Recombinant Proteins; Tumor Necrosis Factor-alpha

Concanavalin A (Con A) can induce an immune-mediated hepatitis. Since direct evidence of immune mechanism for this hepatitis is lacking, we employed adoptive transfer to study the mechanism of Con A-induced hepatitis. Intravenous administration of Con A (20 mg/kg) to Balb/c mice was accompanied by elevations of serum alanine aminotransferase (ALT) levels and midzonal necrosis with lymphocyte infiltration in the liver. None of the Balb/c nu/nu mice showed biochemical or pathologic hepatic abnormalities with the same dose of Con A. In the area of midzonal necrosis, CD4-positive T lymphocytes appeared at 24 hr after injection, and then both CD4-positive and CD8-positive T lymphocytes were found at the margin of zonal necrosis at 48 hr. Pretreatment with carrageenan, a potent inhibitor of macrophages, prevented these biochemical and pathologic changes. Mononuclear cells infiltrating in the liver of Balb/c mice 24 hr after priming with Con A were harvested and injected into Balb/c nu/nu mice injected with Con A 24 hr previously. Serum ALT levels elevated and the same pathologic changes observed in Con A-treated Balb/c mice were observed. These changes were not observed when the splenic cells from Con A-treated Balb/c mice were transferred to Con A-treated nude mice. These results suggest that Con A-induced hepatic injury is mediated by macrophages and T lymphocytes sensitized by Con A or its metabolites.

Topics: Adoptive Transfer; Alanine Transaminase; Animals; Carrageenan; Concanavalin A; Hepatitis, Animal; Inflammation; Liver; Lymphocyte Transfusion; Male; Mice; Mice, Inbred BALB C; Mice, Nude; Necrosis; Spleen

The structural proteins of infectious pancreatic necrosis virus (IPNV) have been analyzed. Two-dimensional gel electrophoresis showed that IPNV proteins are slightly acidic with apparent pIs ranging from 5.8 to 6.6. To identify the IPNV surface-located proteins, purified virus was labelled either with fluorescein isothiocyanate (FITC) or with Na 125I. After analysis by SDS-PAGE, only the major viral protein, VP2, was labelled by either procedure. The accessibility of VP2 to these reagents suggests that this protein is externally located. In addition, using Concanavalin A conjugated with FITC and IPNV labelling with 3H-mannose, evidence is present that VP2 contains carbohydrate residues.

Topics: Animals; Capsid; Capsid Proteins; Concanavalin A; Fluorescein-5-isothiocyanate; Fluoresceins; Glycosylation; Isoelectric Point; Necrosis; Pancreatic Diseases; Salmonidae; Viral Structural Proteins; Viruses

Changes in the cytoplasm of skeletal muscle fibres during necrosis, regeneration, and neurogenic atrophy have been studied in a wide range of human neuromuscular diseases with a panel of eleven biotinylated lectins and by immunohistochemical staining for the cytoskeletal protein desmin. Increased binding of several lectins was observed in both necrotic and regenerating fibres, with Concanavalin A the most consistently positive lectin. Staining for desmin was strong in the cytoplasm of regenerating and partially damaged fibres and was lost in necrotic fibres, although there were differences in the staining reactions of the two antidesmin antibodies used. In fibres which had undergone neurogenic atrophy, cytoplasmic lectin binding was seen only with Griffonia simplicifolia 1 lectin, and desmin was expressed more strongly than in normal fibres. Lectin binding and immunohistochemical staining from desmin can supplement the information obtained from muscle biopsies by conventional histochemical methods and lead to a better understanding of the mechanisms of muscle damage.

Topics: Adolescent; Adult; Aged; Aged, 80 and over; Child; Child, Preschool; Concanavalin A; Cytoplasm; Desmin; Humans; Infant; Lectins; Middle Aged; Muscles; Muscular Atrophy; Necrosis; Protein Binding; Regeneration

Sequential study of permeability and cellular responses following intradermal concanavalin A in the chicken skin, using the colloidal carbon technique, revealed an increase in vascular permeability which was mostly confined to venules. A noteworthy feature of the reaction was marked accumulation of basophils, even in the later stages, and the early appearance of perivascular lymphoid aggregations. The occurrence of well formed giant cells, hypertrophy and hyperplasia of vascular endothelium and marked acanthosis of the epidermis were the other prominent changes. The findings suggest that Con A, in the chicken, appears to have a more general effect on the different types of cells and that it may act as a mitogen not only for T lymphocytes but also for endothelial and epithelial cells.

Topics: Animals; Basophils; Capillary Permeability; Chickens; Concanavalin A; Endothelium; Female; Hyperplasia; Injections, Intradermal; Leukocytes; Male; Necrosis; Poultry Diseases; Skin; Time Factors; Venules

Topics: Animals; Carbohydrates; Cell Membrane; Cell Survival; Chick Embryo; Concanavalin A; Extremities; Glycoproteins; Microscopy, Electron; Morphogenesis; Necrosis; Polysaccharides; Ruthenium Red; Staining and Labeling

Topics: Agglutinins; Animals; Antigens, Surface; Arachis; Cell Differentiation; Cell Separation; Concanavalin A; Female; Male; Mice; Necrosis; Neoplasms, Experimental; Plant Lectins; T-Lymphocytes

Topics: Animals; Azo Compounds; Biphenyl Compounds; Concanavalin A; Cyanosis; Edema; Forelimb; gamma-Globulins; Humans; Immunotherapy; Lectins; Mice; Mice, Inbred Strains; Multiple Myeloma; Necrosis; Paresis; Skin Diseases; Time Factors; Vaccines