concanavalin-a and Hepatitis--Animal

Cell vaccination via immunization with attenuated pathogenic cells is an effective preventive method that has been successfully applied in several animal models of inflammatory or autoimmune diseases. Concanavalin A (Con A)-induced hepatitis (CIH) is a commonly used experimental model to study immune-mediated liver injury. Multiple cell types including T lymphocytes, macrophages and neutrophils have been found to be involved in the pathogenesis of CIH. In this study, we used attenuated spleen lymphocytes or peripheral blood lymphocytes as vaccines to investigate whether they could induce protective immune responses to prevent mice from developing CIH. We found that mice receiving such vaccination before CIH induction developed much milder diseases, exhibited a lower level of alanine aminotransferase (ALT) released into their plasma and had less inflammatory lesions in their livers. Such CIH-suppression is dose- and frequency-dependent. The suppressive effect was associated with inhibition of several major inflammatory mediators, pro-inflammatory cytokines and chemokines.

Topics: Alanine Transaminase; Animals; Concanavalin A; Cytokines; Disease Models, Animal; Dose-Response Relationship, Immunologic; Hepatitis, Animal; Liver; Lymphocyte Transfusion; Male; Mice; Mice, Inbred C57BL; T-Lymphocytes; Vaccination; Vaccines

Topics: Animals; Concanavalin A; Disease Models, Animal; Hepatitis, Animal; Hepatocytes; Liver; Mice; Mice, Knockout; Necroptosis; Parenchymal Tissue; Protein Kinases

Acute liver failure is a clinical syndrome of severe hepatic dysfunction. Immune cells play an important role in acute liver failure. In recent years, the immunoregulatory function of extracellular vesicles (EVs) has been reported; therefore, it is inferred that EVs play a role in immune-mediated hepatitis. In this study, we investigated the immunoregulatory function of EVs in concanavalin A (Con A)-induced hepatitis. The mouse model was prepared by a single intravenous administration of 15 mg/kg Con A, in which there was a significant increase in the serum EVs number. In an in vitro study, the number of secreted EVs was also significantly increased in Con A-treated RAW264.7 cells, a mouse macrophage cell line, but not in Hepa1-6 cells, a mouse hepatoma cell line. In an in vitro EVs treatment study, EVs from Con A-treated mouse serum and Con A-treated RAW264.7 cells suppressed inflammatory cytokine production in Con A-stimulated RAW264.7 cells. miRNA sequencing analysis showed that the expression of mmu-miR-122-5p and mmu-miR-148a-3p was commonly increased in these EVs and EVs-treated cells. The pathways enriched in the predicted miRNA target genes included inflammatory response pathways. The mRNA levels of the target genes in these pathways (mitogen-activated protein kinase, phosphoinositide 3-kinase/Akt and Rho/Rho-associated coiled-coil containing protein kinase pathways) were decreased in the EVs-treated cells. In an in vivo RNA interference study, the knockdown of liver RAB27A, an EVs secretion regulator, significantly exacerbated Con A-induced hepatitis. These data suggest that macrophage-derived EVs play an important role in Con A-induced hepatitis through immunoregulation.

Topics: Animals; Cell Line, Tumor; Cell Survival; Concanavalin A; Cytokines; Extracellular Vesicles; Female; Hepatitis, Animal; Liver; Macrophages; Mice; Mice, Inbred BALB C; MicroRNAs; Mitogen-Activated Protein Kinases; Phosphatidylinositol 3-Kinases; RAW 264.7 Cells; rho-Associated Kinases

Hepatitis is an important health problem worldwide. Novel molecular targets are in demand for detection and management of hepatitis. Hepatoma-derived growth factor (HDGF) has been delineated to participate in hepatic fibrosis and liver carcinogenesis. However, the relationship between hepatitis and HDGF remains unclear. This study aimed to elucidate the role of HDGF during hepatitis using concanavalin A (ConA)-induced hepatitis model. In cultured hepatocytes, ConA treatment-elicited HDGF upregulation at transcriptional level and promoted HDGF secretion while reducing intracellular HDGF protein level and cellular viability. Similarly, mice receiving ConA administration exhibited reduced hepatic HDGF expression and elevated circulating HDGF level, which was positively correlated with serum aspartate aminotransferase (AST) and alanine aminotransferase (ALT) levels. By using HDGF knockout (KO) mice, it was found the ConA-evoked cell death was prominently alleviated in KO compared with control. Besides, it was delineated HDGF ablation conferred protection by suppressing the ConA-induced neutrophils recruitment in livers. Above all, the ConA-mediated activation of tumor necrosis factor-α (TNF-α)/interleukin-1β (IL-1β)/interleukin-6 (IL-6)/cyclooxygenase-2 (COX-2) inflammatory signaling was significantly abrogated in KO mice. Treatment with recombinant HDGF (rHDGF) dose-dependently stimulated the expression of TNF-α/IL-1β/IL-6/COX-2 in hepatocytes, further supporting the pro-inflammatory function of HDGF. Finally, application of HDGF antibody not only attenuated the ConA-mediated inflammatory cascade in hepatocytes, but also ameliorated the ConA-induced hepatic necrosis and AST elevation in mice. In summary, HDGF participates in ConA-induced hepatitis via neutrophils recruitment and may constitute a therapeutic target for acute hepatitis.

Topics: Alanine Transaminase; Animals; Aspartate Aminotransferases; Cells, Cultured; Concanavalin A; Hepatitis, Animal; Hepatocytes; Intercellular Signaling Peptides and Proteins; Interleukin-1beta; Interleukin-6; Liver; Liver Cirrhosis; Male; Mice; Mice, Inbred C57BL; Mice, Knockout; Neutrophil Infiltration; Rats; Signal Transduction; Tumor Necrosis Factor-alpha; Up-Regulation

Natural killer group 2D (NKG2D) is a well-characterized activating receptor expressed on many immune cells, including invariant natural killer T (iNKT) cells. These cells were shown to be responsible of liver injury in the model of concanavalin A (Con A)-induced hepatitis, considered to be an experimental model of human autoimmune hepatitis. In this study, we investigated whether NKG2D plays a role in the hepatitis induced by iNKT cell-mediated immune response to Con A. By using killer cell lectin-like receptor subfamily K, member 1 deficient (

Topics: Animals; Concanavalin A; Cytokines; Fas Ligand Protein; Hepatitis, Animal; Hepatitis, Autoimmune; Hepatocytes; Liver; Lymphocyte Activation; Mice; Mice, Knockout; Natural Killer T-Cells; NK Cell Lectin-Like Receptor Subfamily K

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

The elaborate structure of the extracellular matrix (ECM) and the appropriate surface glycoforms upon it are indispensable to CD4+ T cell regulation.. To explore the effects of Glcα1,2Galβ1 glycosylation mediated by GLT25D2 (Colgalt2) for CD4+ T cell regulation, we prepared C57BL/6J Glt25d2-/- mice. In the induction of hepatitis, after concanavalin A (Con A) challenge for 6, 12, and 24 h, more extensive parenchymal injury was noted in Glt25d2-/- mice than in wild-type (WT) mice at 12 h. Immunohistochemistry and laser scanning confocal microscopy were used to detect GLT25D2 expression, and subsets of CD4+T cells was analyzed by flow cytometry. A total of 26 cytokines in serum samples were determined using Luminex technology.. The trend in liver injury score variation was consistent with serum alanine aminotransferase and aspartate aminotransferase levels. The levels of interleukin 4 (IL-4), IL-1β, IL-9, and several chemokines such as macrophage inflammatory protein-2, eotaxin, and growth-related oncogene α were significantly increased in Glt25d2-/- mice compared with WT mice after Con A challenge. A further phenotype analysis of primary Glt25d2-/- CD4+ T cells showed that Glt25d2 knockout increased the frequency of the CD25+CD69- subset but decreased the frequency of the CD25-CD69+ subset after Con A challenge for 6, 12, and 24 h compared with those of WT CD4+ T cells. Activation-induced apoptosis was also significantly increased in Glt25d2-/- CD4+ T cells after Con A challenge compared with WT CD4+ T cells. Lectin microarray hybridization showed that Glt25d2 knockout increased the binding activity of Narcissus pseudonarcissus lectin to CD4+ T cells but Amaranthus caudatus lectin-binding activity was lost during Con A challenge.. The present results suggest that collagen glycosylation mediated by GLT25D2 may regulate a subset of CD4+ T cells and be involved in the pathogenesis of Con A-induced hepatitis.

Topics: Animals; Antigens, CD; Antigens, Differentiation, T-Lymphocyte; Apoptosis; CD4-Positive T-Lymphocytes; Cell Proliferation; Chemokines; Concanavalin A; Cytokines; Galactosyltransferases; Hepatitis, Animal; Interleukin-2 Receptor alpha Subunit; Lectins; Lectins, C-Type; Liver; Male; Mice; Mice, Inbred C57BL; Mice, Knockout; Spleen

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

Chlorogenic acid (CGA), one of the most abundant dietary polyphenolic compounds, has been reported to exhibit anti-inflammatory ability. However, the hepatoprotective effects and molecular mechanisms of CGA on concanavalin A (Con A)-induced hepatitis have not been explored. In the present study, we found that pretreatment with CGA dose-dependently inhibited the elevation of plasma aminotransferases and alleviated hepatic pathological damage as well as hepatocyte apoptosis in Con A-exposed mice. Additionally, CGA markedly suppressed the production of serum tumor necrosis factor (TNF)-α and interferon (IFN)-γ, alleviated the infiltration of hepatic macrophages, neutrophils, and activated CD4

Topics: Animals; Apoptosis; CD4-Positive T-Lymphocytes; Cell Adhesion; Chlorogenic Acid; Concanavalin A; Hepatitis, Animal; Humans; Interferon-gamma; Kupffer Cells; Liver; Male; Mice; Mice, Inbred BALB C; NF-kappa B; Signal Transduction; Toll-Like Receptor 4; Tumor Necrosis Factor-alpha

Death receptor (DR) ligands such as tumor necrosis factor (TNF) have been identified as fundamental mediators of liver damage both in mouse models and in humans. While the essential site of function of DR signaling is conceivably the hepatocyte, a systematic analysis is missing. Using mice with conditional gene ablation, we analyzed the tissue-specific function of DR signaling in T cell-dependent (concanavalin A) and independent (lipopolysaccharide/galactosamine) hepatitis and in models of bacterial infection (Listeria monocytogenes, lipopolysaccharide). We report that lipopolysaccharide/galactosamine-induced liver injury depends on hepatocyte-intrinsic TNF receptor 1 (p55, TNFR1). In contrast, we show that T cell-induced hepatitis was independent of TNFR1 signaling in hepatocytes, T cells, or endothelial cells. Moreover, T cell-induced hepatitis was independent of hepatocyte-intrinsic Fas-associated protein with death domain, TNF-related apoptosis-inducing ligand receptor, or Fas signaling. Instead, concanavalin A-induced hepatitis was completely prevented in mice with myeloid-derived cell (MDC)-specific deletion of TNFR1. Significantly, however, mice lacking TNFR1 in MDCs succumbed to listeria infection, although they displayed similar sensitivity toward endotoxin-induced septic shock when compared to control mice. These results suggest that TNFR1 signaling in MDCs is a critical mediator of both the detrimental and the protective functions of TNF in T cell-induced hepatitis and bacterial infection, respectively.. The critical site of action of DRs is completely dependent on the nature of hepatitis; the data specify MDCs as the essential cell type of TNFR1 function in T cell-mediated hepatitis and in the response to listeria, thereby identifying the opposing role of MDC TNFR1 in autoimmunity and bacterial infection. (Hepatology 2016;64:508-521).

Topics: Animals; Concanavalin A; Endothelial Cells; Hepatitis, Animal; Lipopolysaccharides; Listeria monocytogenes; Liver; Male; Mice, Inbred C57BL; Myeloid-Derived Suppressor Cells; Receptors, Tumor Necrosis Factor, Type I; T-Lymphocytes

Augmenter of liver regeneration (ALR), produced and released by hepatocytes, has cytoprotective and immunoregulatory effects on liver injury, and has been used in many experimental applications. However, little attention has been paid to the effects of ALR on concanavalin A (Con A)-induced hepatitis. The purpose of this paper is to explore the protective effect of ALR on Con A-induced hepatitis and elucidate potential mechanisms. We found that the ALR pretreatment evidently reduced the amount of ALT and AST in serum. In addition, pro-inflammatory cytokines, chemokines and iNOS were suppressed. ALR pretreatment also decreased CD4(+), CD8(+) T cell infiltration in liver. Besides, we observed that ALR pretreatment was capable of suppressing the activation of several signaling pathways in Con A-induced hepatitis. These findings suggest that ALR can obviously weaken Con A-induced hepatitis and ALR has some certain immune regulation function.

Topics: Alanine Transaminase; Animals; Aspartate Aminotransferases; CD4-Positive T-Lymphocytes; CD8-Positive T-Lymphocytes; Cells, Cultured; Concanavalin A; Extracellular Signal-Regulated MAP Kinases; Hepatitis, Animal; Inflammation Mediators; Lymphocyte Activation; Male; Mice; Mice, Inbred BALB C; NF-kappa B; Nitric Oxide Synthase Type II; Oxidoreductases Acting on Sulfur Group Donors; Signal Transduction

Extracellular adenosine regulates inflammatory responses via the A2A adenosine receptor (A2AR). A2AR deficiency results in much exaggerated acute hepatitis, indicating nonredundancy of adenosine-A2AR pathway in inhibiting immune activation. To identify a critical target of immunoregulatory effect of extracellular adenosine, we focused on NKT cells, which play an indispensable role in hepatitis. An A2AR agonist abolished NKT-cell-dependent induction of acute hepatitis by concanavalin A (Con A) or α-galactosylceramide in mice, corresponding to downregulation of activation markers and cytokines in NKT cells and of NK-cell co-activation. These results show that A2AR signaling can downregulate NKT-cell activation and suppress NKT-cell-triggered inflammatory responses. Next, we hypothesized that NKT cells might be under physiological control of the adenosine-A2AR pathway. Indeed, both Con A and α-galactosylceramide induced more severe hepatitis in A2AR-deficient mice than in WT controls. Transfer of A2AR-deficient NKT cells into A2AR-expressing recipients resulted in exaggeration of Con A-induced liver damage, suggesting that NKT-cell activation is controlled by endogenous adenosine via A2AR, and this physiological regulatory mechanism of NKT cells is critical in the control of tissue-damaging inflammation. The current study suggests the possibility to manipulate NKT-cell activity in inflammatory disorders through intervention to the adenosine-A2AR pathway.

Topics: Adenosine; Adenosine A2 Receptor Agonists; Adenosine A2 Receptor Antagonists; Animals; Cells, Cultured; Concanavalin A; Flow Cytometry; Galactosylceramides; Hepatitis, Animal; Interferon-gamma; Liver; Mice; Mice, Inbred C57BL; Mice, Knockout; Natural Killer T-Cells; Phenethylamines; Receptor, Adenosine A2A; Triazines; Triazoles; Tumor Necrosis Factor-alpha

Liver eosinophilia has been associated with incidences of drug-induced liver injury (DILI) for more than 50 years, although its role in this disease has remained largely unknown. In this regard, it was recently shown that eosinophils played a pathogenic role in a mouse model of halothane-induced liver injury (HILI). However, the signaling events that drove hepatic expression of eosinophil-associated chemokines, eotaxins, eosinophil infiltration, and subsequent HILI were unclear. We now provide evidence implicating hepatic epithelial-derived cytokine thymic stromal lymphopoietin (TSLP) and type 2 immunity, in particular, interleukin-4 (IL-4) production, in mediating hepatic eosinophilia and injury during HILI. TSLP was constitutively expressed by mouse hepatocytes and increased during HILI. Moreover, the severity of HILI was reduced in mice deficient in either the TSLP receptor (TSLPR) or IL-4 and was accompanied by decreases in serum levels of eotaxins and hepatic eosinophilia. Similarly, concanavalin A-induced liver injury, where type 2 cytokines and eosinophils play a significant role in its pathogenesis, was also reduced in TSLPR-deficient mice. Studies in vitro revealed that mouse and human hepatocytes produce TSLP and eotaxins in response to treatment with combinations of IL-4 and proinflammatory cytokines IL-1β and tumor necrosis factor alpha.. This report provides the first evidence implicating roles for hepatic TSLP signaling, type 2 immunity, and eosinophilia in mediating liver injury caused by a drug.

Topics: Anesthetics, Inhalation; Animals; Chemical and Drug Induced Liver Injury; Concanavalin A; Cytokines; Female; Halothane; Hepatitis, Animal; Hepatocytes; Humans; Interleukin-4; Mice, Inbred BALB C; Thymic Stromal Lymphopoietin

Previous investigations demonstrated that protein tyrosine phosphatase, receptor type, O (PTPRO) acts as a tumor suppressor in liver cancer; however, little is known about its role in liver inflammation. Thus, we investigated the role of PTPRO in fulminant hepatitis (FH) using a Con A-induced mouse model. Significantly more severe liver damage, but attenuated inflammation, was detected in PTPRO-knockout (KO) mice, and PTPRO deficiency could confer this phenotype to wild-type mice in bone marrow transplantation. Moreover, hepatocytes with PTPRO depletion were more sensitive to TNF-α-induced apoptosis, and secretion of cytokines was significantly decreased in both T and NK/NKT cells and led to marked impairment of NF-κB activation. Intriguingly, wild-type and PTPRO-KO cells responded equally to TNF-α in activation of IKK, but NF-κB activation was clearly decreased in PTPRO-KO cells. PTPRO associated with ErbB2, and loss of PTPRO potentiated activation of the ErbB2/Akt/GSK-3β/β-catenin cascade. Increased β-catenin formed a complex with NF-κB and attenuated its nuclear translocation and activation. Importantly, in humans, PTPRO was much decreased in FH, and this was associated with enhanced β-catenin accumulation but reduced IFN-γ secretion. Taken together, our study identified a novel PTPRO/ErbB2/Akt/GSK-3β/β-catenin/NF-κB axis in FH, which suggests that PTPRO may have therapeutic potential in this liver disease.

Topics: Acute Disease; Animals; beta Catenin; Concanavalin A; Gene Expression Regulation; Glycogen Synthase Kinase 3; Glycogen Synthase Kinase 3 beta; Hepatitis, Animal; Hepatocytes; Humans; Inflammation; Interferon-gamma; Killer Cells, Natural; Liver; Male; Mice; Mice, Knockout; NF-kappa B; Proto-Oncogene Proteins c-akt; Receptor-Like Protein Tyrosine Phosphatases, Class 3; Receptor, ErbB-2; Severity of Illness Index; Signal Transduction; Survival Analysis; T-Lymphocytes

Although ursodeoxycholic acid (UDCA) has long been used for patients with chronic cholestatic liver diseases, particularly primary biliary cirrhosis, it may modulate the host immune response. This study investigated the effect of UDCA feeding on experimental hepatitis, endotoxin shock, and bacterial infection in mice. C57BL/6 mice were fed a diet supplemented with or without 0.3% (wt/vol) UDCA for 4 wk. UDCA improved hepatocyte injury and survival in concanavalin-A (Con-A)-induced hepatitis by suppressing IFN-γ production by liver mononuclear cells (MNC), especially NK and NKT cells. UDCA also increased survival after lipopolysaccharide (LPS)-challenge; however, it increased mortality of mice following Escherichia coli infection due to the worsening of infection. UDCA-fed mice showed suppressed serum IL-18 levels and production of IL-18 from liver Kupffer cells, which together with IL-12 potently induce IFN-γ production. However, unlike normal mice, exogenous IL-18 pretreatment did not increase the serum IFN-γ levels after E. coli, LPS, or Con-A challenge in the UDCA-fed mice. Interestingly, however, glucocorticoid receptor (GR) expression was significantly upregulated in the liver MNC of the UDCA-fed mice but not in their whole liver tissue homogenates. Silencing GR in the liver MNC abrogated the suppressive effect of UDCA on LPS- or Con-A-induced IFN-γ production. Furthermore, RU486, a GR antagonist, restored the serum IFN-γ level in UDCA-fed mice after E. coli, LPS, or Con-A challenge. Taken together, these results suggest that IFN-γ-reducing immunomodulatory property of UDCA is mediated by elevated GR in the liver lymphocytes in an IL-12/18-independent manner.

Topics: Animals; Cells, Cultured; Concanavalin A; Escherichia coli; Escherichia coli Infections; Hepatitis, Animal; Hepatocytes; Immunologic Factors; Interleukin-18; Kupffer Cells; Lipopolysaccharides; Liver; Lymphocytes; Mice; Mice, Inbred C57BL; Receptors, Glucocorticoid; Shock, Septic; Transcription, Genetic; Ursodeoxycholic Acid

Mesenchymal stromal stem cells (MSCs) are an attractive therapeutic model for regenerative medicine due to their pluripotency. MSCs are used as a treatment for several inflammatory diseases, including hepatitis. However, the detailed immunopathological impact of MSC treatment on liver disease, particularly for adipose tissue derived stromal stem cells (ADSCs), has not been described. Here, we investigated the immuno-modulatory effect of ADSCs on hepatitis using an acute ConA C57BL/6 murine hepatitis model. i.v. administration of ADSCs simultaneously or 3 h post injection prevented and treated ConA-induced hepatitis. Immunohistochemical analysis revealed higher numbers of CD11b(+), Gr-1(+), and F4/80(+) cells in the liver of ConA-induced hepatitis mice was ameliorated after the administration of ADSCs. Hepatic expression of genes affected by ADSC administration indicated tissue regeneration-related biological processes, affecting myeloid-lineage immune-mediating Gr-1(+) and CD11b(+) cells. Pathway analysis of the genes expressed in ADSC-treated hepatic inflammatory cells revealed the possible involvement of T cells and macrophages. TNF-α and IFN-γ expression was downregulated in hepatic CD4(+) T cells isolated from hepatitis livers co-cultured with ADSCs. Thus, the immunosuppressive effect of ADSCs in a C57BL/6 murine ConA hepatitis model was dependent primarily on the suppression of myeloid-lineage cells and, in part, of CD4(+) T cells.

Topics: Adipose Tissue; Animals; Antigens, Differentiation; CD11b Antigen; CD4-Positive T-Lymphocytes; Cell- and Tissue-Based Therapy; Concanavalin A; Down-Regulation; Female; Hepatitis, Animal; Interferon-gamma; Liver; Lung; Macrophages; Male; Mesenchymal Stem Cell Transplantation; Mesenchymal Stem Cells; Mice; Mice, Inbred C57BL; Myeloid Cells; Receptors, Chemokine; Tumor Necrosis Factor-alpha

Negative feedback immune mechanisms are essential for maintenance of hepatic homeostasis and prevention of immune-mediated liver injury. We show here that scavenger receptor A (SRA/CD204), a pattern recognition molecule, is highly up-regulated in the livers of patients with autoimmune or viral hepatitis, and of mice during concanavalin A (Con A)-induced hepatitis (CIH). Strikingly, genetic SRA ablation strongly sensitizes mice to Con A-induced liver injury. SRA loss, increased mortality and liver pathology correlate with excessive production of IFN-γ and heightened activation of T cells. Increased liver expression of SRA primarily occurs in mobilized hepatic myeloid cells during CIH, including CD11b(+) Gr-1(+) cells. Mechanistic studies establish that SRA on these cells functions as a negative regulator limiting T-cell activity and cytokine production. SRA-mediated protection from CIH is further validated by adoptive transfer of SRA(+) hepatic mononuclear cells or administration of a lentivirus-expressing SRA, which effectively ameliorates Con A-induced hepatic injury. Also, CIH and clinical hepatitis are associated with increased levels of soluble SRA. This soluble SRA displays a direct T-cell inhibitory effect and is capable of mitigating Con A-induced liver pathology.. Our findings demonstrate an unexpected role of SRA in attenuation of Con A-induced, T-cell-mediated hepatic injury. We propose that SRA serves as an important negative feedback mechanism in liver immune homeostasis, and may be exploited for therapeutic treatment of inflammatory liver diseases.

Topics: Animals; Cells, Cultured; Chemical and Drug Induced Liver Injury; Concanavalin A; Hepatitis, Animal; Interferon-gamma; Liver; Lymphocyte Activation; Mice; Mice, Inbred C57BL; Mice, Knockout; Scavenger Receptors, Class A; T-Lymphocytes

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

In this study we explored the effects of microRNA let-7a on Con A-induced hepatitis and its possible mechanisms involved. We demonstrated that IL-6 and IL-17 expression were significantly upregulated in the liver following Con A treatment and IL-6 level was correlated with the IL-17 expression. To explore whether let-7a may have therapeutic effect on Con A-induced hepatitis, mice was infected with a lentiviral vector containing the let-7a sequence 7 days before Con A treatment. Significantly reduced Th17 cells and remarkably increased regulatory T cells frequency in the liver tissue were found as compared to control mice. It was accompanied by a significant decreased level of inflammatory cytokines as TNF-α, IL-6 and IFN-γ in the serum, and an decreased level of Th17 lineage-specific genes such as Il17a, Il17f, Il21 and Il23r. let-7a was further found to inhibit Th17 differentiation by downregulating IL-6 secretion. It may represent as a novel therapeutic strategy in treating immune-mediated inflammatory hepatitis.

Topics: Animals; Cell Differentiation; Cell Lineage; Concanavalin A; Hepatitis, Animal; Humans; Interleukin-17; Interleukin-6; Liver; Mice; MicroRNAs; Th17 Cells

Severe liver injury that occurs when immune cells mistakenly attack an individual's own liver cells leads to autoimmune hepatitis. In mice, acute hepatitis can be induced by concanavalin A (ConA) treatment, which causes rapid activation of CD1d-positive natural killer (NK) T cells. These activated NKT cells produce large amounts of cytokines, which induce strong inflammation that damages liver tissues. Here we show that PKC-θ(-/-) mice were resistant to ConA-induced hepatitis due to essential function of PKC-θ in NKT cell development and activation. A dosage of ConA (25 mg/kg) that was lethal to wild-type (WT) mice failed to induce death resulting from liver injury in PKC-θ(-/-) mice. Correspondingly, ConA-induced production of cytokines such as IFNγ, IL-6, and TNFα, which mediate the inflammation responsible for liver injury, were significantly lower in PKC-θ(-/-) mice. Peripheral NKT cells had developmental defects at early stages in the thymus in PKC-θ(-/-) mice, and as a result their frequency and number were greatly reduced. Furthermore, PKC-θ(-/-) bone marrow adoptively transferred to WT mice displayed similar defects in NKT cell development, suggesting an intrinsic requirement for PKC-θ in NKT cell development. In addition, upon stimulation with NKT cell-specific lipid ligand, peripheral PKC-θ(-/-) NKT cells produced lower levels of inflammatory cytokines than that of WT NKT cells, suggesting that activation of NKT cells also requires PKC-θ. Our results suggest PKC-θ is an essential molecule required for activation of NKT cell to induce hepatitis, and thus, is a potential drug target for prevention of autoimmune hepatitis.

Topics: Animals; Autoimmune Diseases; Concanavalin A; Hepatitis, Animal; Inflammation; Isoenzymes; Lymphocyte Activation; Mice; Mice, Knockout; Natural Killer T-Cells; Protein Kinase C; Protein Kinase C-theta

Dysregulation of signal transducer and activator of transcription (STAT) signaling is usually associated with intricate immune diseases and rebuilding the balance of STAT1 and STAT3 signaling is being explored as a useful approach for the treatment of these diseases. However, few chemicals have been reported to rebuild the balance of these two signalings for immune hepatitis therapy. In the present study, we found that fusaruside, a new kind of cerebroside isolated from an endophytic fungus Fusarium sp. IFB-121 in Quercus variabilis, significantly ameliorated concanavalin A (Con A)-induced T-cell-mediated fulminant hepatitis in mice, which was closely associated with the improvement of histopathological parameters, inhibition of activation of liver CD4(+) T cells and NKT cells, regulation of balance of Th1/Th2/Th17/Treg cytokines and protection of hepatocyte from apoptosis. Moreover, T-cell proliferation and activation was also notably inhibited by fusaruside in vitro. Furthermore, the protective effect of fusaruside was attributable to a novel regulatory mechanism through down-regulating STAT1 activation and T-bet expression in liver CD4(+) T cells and up-regulating STAT3 activation and Bcl-X(L) expression in hepatocytes. In conclusion, fusaruside exhibited its capability against T-cell-mediated liver injury in vivo, through rebuilding the balance of STAT1 and STAT3 signalings. These results suggest that fusaruside is potentially useful for the treatment of T-cell-mediated human liver disorders.

Topics: Animals; bcl-X Protein; CD4 Antigens; Cell Proliferation; Cerebrosides; Concanavalin A; Cytokines; Female; Fusarium; Hepatitis, Animal; Liver; Liver Failure, Acute; Lymphocyte Activation; Mice; Mice, Inbred C57BL; Natural Killer T-Cells; Signal Transduction; Spleen; STAT1 Transcription Factor; STAT2 Transcription Factor; T-Lymphocytes

Interleukin (IL)-33, a member of the IL-1 cytokine family, positively correlates with acute hepatitis and chronic liver failure in mice and humans. IL-33 is expressed in hepatocytes and is regulated by natural killer T (NKT) cells during concanavalin A (ConA)-induced acute liver injury. Here, we investigated the molecular mechanisms underlying the expression of IL-33 during acute hepatitis. The expression of IL-33 and its regulation by death receptor pathways was investigated after the induction of ConA-acute hepatitis in wildtype (WT), perforin(-/-) , tumor necrosis factor related apoptosis inducing ligand (TRAIL)(-/-) , and NKT cell-deficient (CD1d(-/-) ) mice. In addition, we used a model of acute liver injury by administering Jo2/Fas-antibody or D-galactosamine-tumor necrosis factor alpha (TNFα) in WT mice. Finally, the effect of TRAIL on IL-33 expression was assessed in primary cultured murine hepatocytes. We show that IL-33 expression in hepatocytes is partially controlled by perforin during acute liver injury, but not by TNFα or Fas ligand (FasL). Interestingly, the expression of IL-33 in hepatocytes is blocked during ConA-acute hepatitis in TRAIL-deficient mice compared to WT mice. In contrast, administration of recombinant murine TRAIL associated with ConA-priming in CD1d-deficient mice or in vitro stimulation of murine hepatocytes by TRAIL but not by TNFα or Jo2 induced IL-33 expression in hepatocytes. The IL-33-deficient mice exhibited more severe ConA liver injury than WT controls, suggesting a protective effect of IL-33 in ConA-hepatitis.. The expression of IL-33 during acute hepatitis is dependent on TRAIL, but not on FasL or TNFα.

Topics: Acute Lung Injury; Animals; Antibodies, Monoclonal; Antibodies, Monoclonal, Murine-Derived; Concanavalin A; Fas Ligand Protein; Galactosamine; Gene Expression; Hepatitis, Animal; Hepatocytes; Interleukin-33; Interleukins; Mice; Natural Killer T-Cells; Perforin; Primary Cell Culture; Receptors, Tumor Necrosis Factor, Type I; Receptors, Tumor Necrosis Factor, Type II; RNA, Messenger; TNF-Related Apoptosis-Inducing Ligand; Tumor Necrosis Factor-alpha

Con A-induced hepatitis has been used as a model of human autoimmune or viral hepatitis. During the process of identifying immunologically bioactive proteins in human plasma, we found that apolipoprotein A-II (ApoA-II), the second major apolipoprotein of high-density lipoprotein, inhibited the production of IFN-γ by Con A-stimulated mouse and human CD4 T cells. Con A-induced hepatitis was attenuated by the administration of ApoA-II. The beneficial effect of ApoA-II was associated with reduced leukocyte infiltration and decreased production of T cell-related cytokines and chemokines in the liver. ApoA-II inhibited the Con A-induced activation of ERK-MAPK and nuclear translocation of NFAT in CD4 T cells. Interestingly, exacerbated hepatitis was observed in ApoA-II-deficient mice, indicating that ApoA-II plays a suppressive role in Con A-induced hepatitis under physiological conditions. Moreover, the administration of ApoA-II after the onset of Con A-induced hepatitis was sufficient to suppress disease. Thus, the therapeutic effect of ApoA-II could be useful for patients with CD4 T cell-related autoimmune and viral hepatitis.

Topics: Animals; Apolipoprotein A-II; Autoimmune Diseases; CD4-Positive T-Lymphocytes; Cell Migration Inhibition; Cell Movement; Concanavalin A; Female; Gene Knockout Techniques; Growth Inhibitors; Hepatitis, Animal; Humans; Interferon-gamma; Mice; Mice, Inbred BALB C; Mice, Knockout

Suppressor of cytokine signaling-1 (SOCS1) has been shown to be an essential negative regulator of cytokine responses, including those of IFNγ, IL-2, IL-4 and IL-7. SOCS1 deficiency resulted in hyperactivation not only of T cells in general but also of NKT cells specifically. Consistent with previous reports, T- and NKT-cell-specific deletion of Socs1 in mice resulted in enhanced sensitivity to ConA-induced hepatitis. Compared with wild-type (WT) NKT cells, SOCS1-deficient NKT cells produced larger quantities of IFNγ in response to ConA and proliferated faster in response to IL-2 and IL-15. To our surprise, however, SOCS1-deficient NKT cells did not respond to the synthetic glycolipid ligand alpha-galactosylceramide (α-GalCer), though they did respond to sulfatide. α-GalCer-CD1d-tetramer-positive type I NKT [invariant NKT (iNKT)] cells were marginally detected in the periphery of SOCS1-conditional knockout (cKO) mice, suggesting that most of the SOCS1-deficient NKT cells at the periphery were type II NKT cells. Consistently, invariant Vα14 expression was much lower in SOCS1-deficient NKT cells than in WT NKT cells, indicating that iNKT cell homeostasis was abnormal in SOCS1-cKO mice. This reduction in iNKT cells was not observed in mice of an IFNγ-deficient background. These results suggest that SOCS1 is an important regulator of the balance between type I and type II NKT cells at the periphery.

Topics: Animals; Cell Count; Cell Proliferation; Concanavalin A; Hepatitis, Animal; Interferon-gamma; Killer Cells, Natural; Mice; Mice, Knockout; Mitogens; Signal Transduction; Suppressor of Cytokine Signaling Proteins; Survival Analysis

Interleukin-33 (IL-33) is thought to be released during cellular death as an alarming cytokine during the acute phase of disease, but its regulation in vivo is poorly understood. We investigated the expression of IL-33 in two mouse models of acute hepatitis by administering either carbon tetrachloride (CCl(4) ) or concanavalin A (ConA). IL-33 was overexpressed in both models but with a stronger induction in ConA-induced hepatitis. IL-33 was weakly expressed in vascular and sinusoidal endothelial cells from normal liver and was clearly induced in CCl(4) -treated mice. Surprisingly, we found that hepatocytes strongly expressed IL-33 exclusively in the ConA model. CD1d knock-out mice, which are deficient in NKT cells and resistant to ConA-induced hepatitis, no longer expressed IL-33 in hepatocytes following ConA administration. Interestingly, invariant NKT (iNKT) cells adoptively transferred into ConA-treated CD1d KO mouse restored IL-33 expression in hepatocytes. This strongly suggests that NKT cells are responsible for the induction of IL-33 in hepatocytes.

Topics: Acute Disease; Adoptive Transfer; Animals; Antigens, CD1d; Carbon Tetrachloride; Concanavalin A; Female; Flow Cytometry; Fluorescent Antibody Technique; Gene Expression; Hepatitis, Animal; Hepatocytes; Interleukin-1beta; Interleukin-33; Interleukin-6; Interleukins; Liver; Male; Mice; Mice, Inbred BALB C; Mice, Knockout; Natural Killer T-Cells; Reverse Transcriptase Polymerase Chain Reaction; Tumor Necrosis Factor-alpha

Platelets are reportedly causal in hepatitis. We previously showed that in mice, lipopolysaccharide (LPS) induces a reversible and macrophage-dependent hepatic platelet accumulation (HPA), including translocation of platelets into Disse spaces and their entry into hepatocytes. Concanavalin A (ConA), which induces hepatitis in mice via both T cells and macrophages, also induces HPA. Here, we examined the relationship between HPA and ConA-hepatitis. ConA-hepatitis and HPA were evaluated by serum transaminases, hepatic 5-hydroxytryptamine, and/or electron microscopy. Unlike LPS-induced HPA, ConA-induced HPA was only moderately dependent on phagocytic macrophages. Against expectations, platelet-depletion significantly exacerbated ConA-hepatitis, and anti-P-selectin antibody and P-selectin receptor blockade reduced both ConA-induced HPA and hepatitis. Prior induction of HPA by pretreatment with low-dose LPS powerfully reduced ConA-hepatitis. Such protection by LPS-pretreatment was not effective in mice depleted of phagocytic macrophages. In platelet-depleted mice, LPS-pretreatment severely exacerbated ConA-hepatitis. In mice depleted of both macrophages and platelets, neither ConA nor LPS-pretreatment+ConA induced hepatitis. In mice deficient in IL-1α and IL-1β (but not in TNFα), ConA-induced hepatitis was mild, and a protective effect of LPS was not detected. These results suggest that (i) there are causal and protective types of HPA, (ii) the causal type involves hepatic aggregation of platelets, which may be induced by platelet stimulants leaked from injured hepatocytes, (iii) the protective type is inducible by administration of prior low-dose LPS in a manner dependent on phagocytic (or F4/80-positive) macrophages, and (iv) IL-1 is involved in both the causal and protective types.

Topics: Animals; Antibodies, Monoclonal; Blood Platelets; Concanavalin A; Hepatitis, Animal; Interleukin-1; Interleukin-10; Lipopolysaccharides; Liver; Macrophages; Male; Mice; Mice, Inbred BALB C; Microscopy, Electron; P-Selectin; Phagocytosis; Platelet Aggregation; Platelet Membrane Glycoprotein IIb; Serotonin

IL-23 plays a critical role in the expansion of highly proinflammatory Th17 cells secreting IL-17 and IL-22. Recently, we demonstrated that Notch signaling drives IL-22 secretion through the aryl hydrocarbon receptor (AHR) and plays a protective role in Con A-induced hepatitis. In this study, we investigated the role of IL-23 in hepatitis using IL-23p19- and IL-17-deficient mice. In WT mice, the injection of Con A induced the upregulation of various cytokines, which included IL-23, IL-22, IL-17, IFN-γ and TNF-α. In IL-23p19-deficient mice, exacerbated hepatitis was observed and serum IL-22 and IL-17 levels were greatly reduced, whereas in IL-17-deficient mice, ameliorated hepatitis was observed. The injection of exogenous IL-22 protected p19-deficient mice from hepatitis, whereas the injection of exogenous IL-23 significantly increased the serum levels of not only IL-22 but also IL-17, and less effectively protected against hepatitis in IL-17-dependent and -independent manners. Finally, it was revealed that STAT3, STAT4 and Notch contributed to the production of both the cytokines, and that the AHR was important only for IL-22 production in response to Con A and IL-23 in liver mononuclear cells. These results suggest that IL-23 plays a protective role in hepatitis through IL-22 production and also a pathological role via IL-17-dependent and -independent mechanisms.

Topics: Animals; Concanavalin A; Cytokines; Hepatitis, Animal; Interleukin-17; Interleukin-22; Interleukin-23; Interleukin-23 Subunit p19; Interleukins; Mice; Mice, Inbred C57BL; Mice, Knockout; Receptors, Aryl Hydrocarbon; Receptors, Notch; Reverse Transcriptase Polymerase Chain Reaction; Signal Transduction; STAT3 Transcription Factor; STAT4 Transcription Factor; Th17 Cells

In this paper, we reported the synthesis of bifendate derivatives and evaluation of anti-inflammatory activity by detecting the production of the Nitric Oxide (NO) in the lipopolysaccharide(LPS)-stimulated RAW 264.7 cell lines. Among the newly derivatives, compound 7k was the most potent one and two other compounds (7e and 7f) also exhibited greater anti-inflammatory activity than bifendate. Further in vivo studies confirmed that 7k significantly and dose-dependently inhibited carrageenan-induced paw edema and decreased the serum levels of alanine aminotransaminase, and aspartate aminotransaminase in concanavalin A-induced hepatitis model. Histopathological evaluation demonstrated that 7k has better hepatoprotective effect on acute liver injury induced by concanavalin A than bifendate, suggesting that 7k is a potential drug candidate for the treatment of hepatic injuries.

Topics: Animals; Anti-Inflammatory Agents, Non-Steroidal; Biphenyl Compounds; Cell Line; Concanavalin A; Edema; Female; Hepatitis, Animal; Lipopolysaccharides; Liver; Macrophages; Male; Mice; Mice, Inbred BALB C; Nitric Oxide; Rats

Severe hepatic injury is induced by Concanavalin A (Con A) administration in mice, the major effector cells being CD4(+) T cells, NKT cells and macrophages. Since autologous lymphocyte subsets are associated with tissue damage, Con A-induced hepatic injury is considered to be autoimmune hepatitis. However, it has remained to be investigated how autoantibodies and B-1 cells are responsible for this phenomenon. In this study, it was demonstrated that autoantibodies which were detected using Hep-2 cells in immunofluorescence tests and using double-strand (ds) DNA in the ELISA method, appeared after Con A administration (a peak at day 14). Moreover, autoantibody-producing B220(low) cells (i.e., B-1 cells) also appeared at this time. Purified B220(low) cells were found to have a potential to produce autoantibodies. These results suggest that Con A-induced hepatic injury indeed includes the mechanism of autoimmune hepatitis.

Topics: Alanine Transaminase; Animals; Autoantibodies; B-Lymphocytes; Cell Line, Tumor; Concanavalin A; Enzyme-Linked Immunosorbent Assay; Female; Granulocytes; Hepatitis, Animal; Humans; Immunohistochemistry; Lymphocyte Count; Macrophages; Mice; Mice, Inbred C57BL; Natural Killer T-Cells; Spleen; Time Factors

Due to the high morbidity and mortality of fulminant hepatitis, early diagnosis followed by early effective treatment is the key for prognosis improvement. So far, little is known about the gene expression changes in the early stage of this serious illness. Identification of the genes related to the very early stage of fulminant hepatitis development may provide precise clues for early diagnosis.. Balb/C mice were used for ConA injection to induce fulminant hepatitis that was confirmed by pathological and biochemical examination. After a gene chip-based screening, the data of gene expression in the liver, was further dissected by ANOVA analysis, gene expression profiles, gene network construction and real-time RT-PCR. At the very early stage of ConA-triggered fulminant hepatitis, totally 1,473 genes with different expression variations were identified. Among these, 26 genes were finally selected for further investigation. The data from gene network analysis demonstrate that two genes, MPDZ and Acsl1, localized in the core of the network.. At the early stages of fulminant hepatitis, expression of twenty-six genes involved in protein transport, transcription regulation and cell metabolism altered significantly. These genes form a network and have shown strong correlation with fulminant hepatitis development. Our study provides several potential targets for the early diagnosis of fulminant hepatitis.

Topics: Algorithms; Analysis of Variance; Animals; Concanavalin A; Gene Expression Profiling; Hepatitis, Animal; Liver Failure, Acute; Mice; Mice, Inbred BALB C; Oligonucleotide Array Sequence Analysis

Increasing evidence suggests that TLRs are involved in the pathogenesis of liver diseases; however, the underlying mechanisms remain obscure. In this study, we found that treatment with CpG-oligodeoxynucleotide (ODN) promoted the accumulation and activation of murine hepatic NKT cells. Additional experiments showed that CpG-ODN preferred to act on CD4(+) NKT cells, while having less effect on CD4(-) NKT cells. The effect of CpG-ODN on liver NKT cells depended on the presence of Kupffer cells and IL-12. Meanwhile, CpG-ODN pretreatment aggravated liver injury and promoted the production of inflammatory cytokines in a Con A-induced fulminant hepatitis model via TLR9 activation. Collectively, our data demonstrate that TLR9 stimulation prefers to promote the accumulation and activation of hepatic CD4(+) NKT cells and suggest that TLR9 signaling might be involved in the pathogenesis of human hepatitis.

Topics: Acute Disease; Adjuvants, Immunologic; Animals; CD4-Positive T-Lymphocytes; Cell Movement; Concanavalin A; CpG Islands; Hepatitis, Animal; Kupffer Cells; Leukocytes, Mononuclear; Ligands; Lymphocyte Activation; Male; Mice; Mice, Inbred C57BL; Mice, Knockout; Natural Killer T-Cells; Oligodeoxyribonucleotides; Signal Transduction; Toll-Like Receptor 9

Selectively inducing apoptosis of activated T cells is essential for the clearance of pathogenic injurious cells and subsequent efficient resolution of inflammation. However, few chemicals have been reported to trigger apoptosis of activated T cells in the treatment of hepatitis without affecting quiescent T cells. In the present study, we found that fraxinellone, a small natural compound isolated from the root bark of Dictamnus dasycarpus, selectively facilitated apoptosis of concanavalin A (Con A)-activated CD4(+) T cells rather than those non-activated, by disrupting the mitochondrial transmembrane potential, decreasing the ratio of Bcl-2/Bax, and increasing cytochrome c release from the mitochondria to the cytosol. The enhancement in Fas expression and caspase-8 activity, truncation of Bid, and down-regulation of anti-apoptotic cellular FLICE-inhibitory protein expression by fraxinellone also suggested the participation of an extrinsic apoptosis pathway. Furthermore, fraxinellone significantly alleviated Con A-induced T-cell-dependent hepatitis in mice, which was closely associated with reduced serum transaminases, pro-inflammatory cytokines, and pathologic parameters. Consistent with the in vitro results, fraxinellone dramatically induced apoptosis of activated peripheral CD4(+) T cells in vivo, consequently resulting in less CD4(+) T-cell activation and infiltration to the liver. These results strongly suggest fraxinellone might be a potential leading compound useful in treating T-cell-mediated liver disorders in humans.

Topics: Animals; Apoptosis; Benzofurans; Concanavalin A; Female; Hepatitis, Animal; Lymphocyte Activation; Membrane Potentials; Mice; Mice, Inbred C57BL; Mitochondrial Membranes; T-Lymphocytes

The liver is an organ with paradoxic immunologic properties and is known for its tolerant microenvironment, which holds important implications for hepatic diseases. The molecular basis for this local immune suppression, however, is poorly understood. In this study, we aimed to determine the role of liver sinusoidal endothelial cell lectin (LSECtin), a recently identified member of the dendritic cell-specific ICAM-3 grabbing nonintegrin (DC-SIGN) family, in the regulation of hepatic T-cell immune response.. The regulation of T-cell effector function by LSECtin was determined by co-stimulated T cells with anti-CD3/CD28 monoclonal antibody and LSECtin protein, or co-culture of T-cell receptor transgenic T cells with mouse LSECs in vitro. We generated LSECtin knockout mice and prepared recombinant LSECtin protein and complementary DNA plasmids to analyze the role of LSECtin in hepatic T-cell immune regulation in vivo.. We showed that LSECtin specifically recognized activated T cells and negatively regulated their immune responses. In mice with T-cell-mediated acute liver injury, the lack of LSECtin accelerated the disease owing to an increased T-cell immune response, whereas the exogenous administration of recombinant LSECtin protein or plasmid ameliorated the disease via down-regulation of T-cell immunity.. Our results reveal that LSECtin is a novel regulator of T cells and expose a crucial mechanism for hepatic T-cell immune suppression, perhaps opening up a new approach for treatment of inflammatory diseases in the liver.

Topics: Animals; Binding Sites; Cell Proliferation; CHO Cells; Coculture Techniques; Concanavalin A; Cricetinae; Cricetulus; Disease Models, Animal; Endothelial Cells; Genetic Therapy; Hepatitis, Animal; HL-60 Cells; Humans; Immune Tolerance; Jurkat Cells; Lectins, C-Type; Liver; Lymphocyte Activation; Mice; Mice, Inbred BALB C; Mice, Knockout; Polysaccharides; Recombinant Fusion Proteins; Signal Transduction; T-Lymphocytes; Transfection; U937 Cells

T cells play central roles in liver diseases, but the regulatory mechanism by cytokine signaling is not well understood. In the present study, we explored the role of SOCS3 in T cells in concanavalin A (ConA)-induced hepatitis. Mice with T-cell-specific overexpression of SOCS3 (SOCS3-cTg) showed reduced hepatic damage and improved mice survival relative to the control, an event that was associated with decreased apoptotic signals Fas and pStat1. Expression of Th1-cytokines/chemokines was decreased in SOCS3-cTg liver with reduced expression of T-bet, a Th1-transcription factor. Flow cytometric analysis of the liver lymphocytes demonstrated that activated CD4(+) T cells, cytotoxic T cells and natural killer T cells were significantly decreased in SOCS3-cTg liver with decreased expression of perforin and granzyme B, injurious molecules for hepatocyte damage. These results suggest that forced expression of SOCS3 in T cells prevents ConA-induced liver injury by inhibiting several phases of Th1 responses.

Topics: Animals; Apoptosis; Blotting, Western; Caspases; Concanavalin A; DNA Fragmentation; Granzymes; Hepatitis, Animal; Histocytochemistry; Male; Mice; Mice, Inbred C57BL; Mice, Transgenic; Perforin; Reverse Transcriptase Polymerase Chain Reaction; RNA; Specific Pathogen-Free Organisms; Suppressor of Cytokine Signaling 3 Protein; Suppressor of Cytokine Signaling Proteins; Survival Analysis; T-Lymphocytes

The integrity of lipid rafts in cell membranes is important for signal transduction.. To determine the distinct effects of beta-glycolipids on the composition of lipid rafts in natural killer T (NKT) cells and on the level of expression of flotillin-2, leukocyte-specific protein tyrosine kinase (Lck), and STAT1-associated pathways.. The effects of glycolipids were determined by composition analysis of the raft domains, FACS analysis of the distribution patterns for the raft ganglioside, GM1, and fluorescence microscopy of raft patching. To evaluate the effects of the immune environment on glycolipid-associated alteration of lipid rafts, hepatitis was induced by an intravenous injection of concanavalin A (ConA) in mice treated with various glycolipids.. The administration of beta-glucosylceramide, beta-lactosylceramide, and a combination of both significantly altered GM1 content in lymphocyte membranes in an environment-dependent manner. These effects were associated with altered expression levels of flotillin-2, Lck, and STAT1, and with a significant decrease in intrahepatic CD8+ lymphocyte trapping and the alleviation of ConA-induced hepatitis. The administration of alpha-glycolipids failed to induce similar effects.. The alteration in the expression levels of flotillin-2, Lck, and STAT1 that occurs concomitantly with changes in lipid raft composition and structure following the administration of beta-glycolipids in ConA-induced hepatitis is microenvironment-dependent and is associated with decreased intrahepatic CD8(+) T lymphocyte trapping and amelioration of immune-mediated hepatitis.

Topics: Animals; Concanavalin A; Glycolipids; Hepatitis, Animal; Killer Cells, Natural; Liver; Lymphocyte Specific Protein Tyrosine Kinase p56(lck); Male; Membrane Microdomains; Membrane Proteins; Mice; Mice, Inbred C57BL; Spleen; T-Lymphocytes, Cytotoxic

Tumor necrosis factor-alpha (TNF-alpha) is critically involved in a wide variety of inflammatory pathologies, such as hepatitis, via the TNF receptor-1 (TNFR1). To develop TNFR1-targeted anti-inflammatory drugs, we have already succeeded in creating a TNFR1-selective antagonistic mutant TNF-alpha (R1antTNF) and shown that R1antTNF efficiently inhibits TNF-alpha/TNFR1-mediated biological activity in vitro. In this study, we examined the therapeutic effect of R1antTNF in acute hepatitis using two independent experimental models, induced by carbon tetrachloride (CCl(4)) or concanavalin A (ConA). In a CCl(4)-induced model, treatment with R1antTNF significantly inhibited elevation in the serum level of ALT (alanine aminotransferase), a marker for liver damage. In a ConA-induced T-cell-mediated hepatitis model, R1antTNF also inhibited the production of serum immune activated markers such as IL-2 and IL-6. These R1antTNF-mediated therapeutic effects were as good as or better than those obtained using conventional anti-TNF-alpha antibody therapy. Our results suggest that R1antTNF may be a clinically useful TNF-alpha antagonist in hepatitis.

Topics: Alanine Transaminase; Animals; Carbon Tetrachloride; Cell Line; Concanavalin A; Cytokines; Disease Models, Animal; Female; Hepatitis, Animal; Humans; Mice; Mice, Inbred BALB C; Receptors, Tumor Necrosis Factor, Type I; Tumor Necrosis Factor-alpha

Tetrandrine (TET) is the major pharmacologically active compound of Chinese herb Stephania tetrandra S Moore, which has been used traditionally for the treatment of rheumatic disorders, silicosis and hypertension. Concanavalin A (ConA)-induced hepatitis (CIH) is a T-cell-dependent hepatitis and a well-established animal model for studying the mechanisms and therapy of immune-mediated hepatotoxicity. The aim of this study was to investigate whether TET could protect mice from CIH. C57BL/6 mice were injected with ConA to induce CIH pretreated with or without TET. Liver injury was assessed biochemically and histologically. Levels of plasma cytokines and the expressions of chemokine messenger RNA (mRNA) in the liver were determined. We found that pretreatment of mice with TET markedly reduced plasma transaminase release and the severity of liver damage. We further investigated the mechanisms of the protective effects of TET. When CIH-induced mice pretreated with TET, the increases of plasma concentrations of TNF-alpha, IFN-gamma, IL-12 and IL-4 were dramatically attenuated; at the same time, IFN-inducible protein-10 and macrophage inflammatory protein-1alpha expressions in liver were decreased. Furthermore, TET inhibited NF-kappaB activity, the critical transcriptional factor of the above mentioned inflammatory cytokines, by preventing the activation of IkappaBalpha kinasealpha (IKKalpha) and then inhibiting phosphorylation of IkappaBalpha to stabilize IkappaBalpha in intrahepatic leukocytes. In conclusion, TET is able to prevent T-cell-mediated liver injury in vivo. The beneficial effect may depend on suppressing the production of various inflammatory mediators in the liver through inhibiting of NF-kappaB activation.

Topics: Animals; Apoptosis; Benzylisoquinolines; Cells, Cultured; Chemokine CCL3; Chemokine CXCL10; Concanavalin A; Cytokines; Drugs, Chinese Herbal; Enzyme Induction; Hepatitis, Animal; Hepatocytes; I-kappa B Kinase; Immunosuppressive Agents; Mice; Mice, Inbred C57BL; NF-kappa B; Phytotherapy; Signal Transduction; Stephania tetrandra; Transcriptional Activation

The recent discovery of bacterial receptors such as NOD2 that contribute to crosstalk between innate and adaptive immune systems in the digestive tract constitutes an important challenge in our understanding of liver injury mechanisms. The present study focuses on NOD2 functions during liver injury. NOD2, TNF-alpha and IFN-gamma mRNA were quantified using real-time PCR in liver samples from patients and mice with liver injury. We evaluated the susceptibility of concanavalin A (ConA) challenge in NOD2-deficient mice (Nod2-/-) compared to wild-type littermates. We tested the effect of muramyl dipeptide (MDP), the specific activator of NOD2, on ConA-induced liver injury in C57BL/6 mice. We studied the cellular distribution and the role of NOD2 in immune cells and hepatocytes. We demonstrated that NOD2, TNF-alpha and IFN-gamma were upregulated during liver injury in mice and humans. Nod2-/- mice were resistant to ConA-induced hepatitis compared to their wild-type littermates, through reduced IFN-gamma production by immune cells. Conversely, administration of MDP exacerbated ConA-induced liver injury. MDP was a strong inducer of IFN-gamma in freshly isolated human PBMC, splenocytes and hepatocytes. Our study supports the hypothesis that NOD2 contributes to liver injury via a regulatory mechanism affecting immune cells infiltrating the liver and hepatocytes. Taken together, our results indicate that NOD2 may represent a new therapeutic target in liver diseases.

Topics: Acetylmuramyl-Alanyl-Isoglutamine; Adjuvants, Immunologic; Animals; Apoptosis; Case-Control Studies; Cell Separation; Cells, Cultured; Concanavalin A; Hepatitis, Animal; Hepatocytes; Humans; Immunity, Innate; Interferon-gamma; Kinetics; Leukocytes, Mononuclear; Liver; Mice; Mice, Inbred C57BL; Mice, Knockout; Nod2 Signaling Adaptor Protein; RNA, Messenger; Spleen; Tumor Necrosis Factor-alpha; Up-Regulation

Acute liver failure is associated with significant mortality. However, the underlying pathophysiological mechanism is not yet fully understood. Suppressor of cytokine signaling-1 (SOCS1), which is a negative-feedback molecule for cytokine signaling, has been shown to be rapidly induced during liver injury. Here, using liver-specific SOCS1-conditional-knockout mice, we demonstrated that SOCS1 deletion in hepatocytes enhanced concanavalin A (ConA)-induced hepatitis, which has been shown to be dependent on activated T and natural killer T (NKT) cells. Although serum cytokine level and NKT cell activation were similar in wild-type (WT) and SOCS1-deficient mice after ConA treatment, proapoptotic signals, including signal transducers and activators of transcription 1 (STAT1) and Jun-terminal kinase (JNK) activation, were enhanced in SOCS1-deficient livers compared with those in WT livers. SOCS1-deficient hepatocytes had higher expression of Fas antigen and were more sensitive to anti-Fas antibody-induced apoptosis than were WT hepatocytes. Furthermore, SOCS1-deficient hepatocytes were more sensitive to tumor necrosis factor (TNF)-alpha-induced JNK activation and apoptosis. These data indicate that SOCS1 is important to the prevention of hepatocyte apoptosis induced by Fas and TNF-alpha. In contrast, SOCS1 overexpression in the liver by adenoviral gene transfer prevented ConA-induced liver injury.. These findings indicate that SOCS1 plays important negative roles in fulminant hepatitis and that forced expression of SOCS1 is therapeutic in preventing hepatitis.

Topics: Acute Disease; Animals; Apoptosis; Concanavalin A; Disease Models, Animal; Enzyme-Linked Immunosorbent Assay; Flow Cytometry; Hepatitis, Animal; Liver; Liver Failure; Lymphocytes; Mice; Mice, Inbred C57BL; Mice, Knockout; Reverse Transcriptase Polymerase Chain Reaction; RNA; Suppressor of Cytokine Signaling 1 Protein; Suppressor of Cytokine Signaling Proteins

Sodium tanshinone IIA sulfonate (STS) is a water-soluble derivative of tanshinone IIA, the main pharmacologically active component of Salvia miltiorrhiza. The aim of this study was to investigate the effect of STS on concanavalin A (ConA)-induced hepatitis (CIH) in mice, an experimental model of immune-mediated liver injury.. C57BL/6 mice pretreated with STS released much less alanine transaminase into plasma in response to ConA challenge and had reduced inflammatory infiltration and hepatocyte apoptosis in the liver compared with control mice pretreated with vehicle solutions. Thus, STS protected mice from CIH. In STS-pretreated mice induced with CIH, we found abrogated tumor necrosis factor-alpha and interferon (IFN)-gamma production. Moreover, mRNA expressions of IFN-inducible protein-10 and macrophage inflammatory protein-1alpha in these mice were decreased. The mechanism of anti-inflammatory effects of STS may be attributed to its modulation of crucial inflammatory signaling pathways, including NF-kappaB and IFN-gamma/STAT1.. In conclusion, STS was capable of protecting mice from immune-mediated liver injury in vivo, and the protection was associated with its suppressive effect on the production of important inflammatory mediators through modulating NF-kappaB and IFN-gamma/STAT1 signaling pathways.

Topics: Animals; Concanavalin A; Hepatitis, Animal; Immunosuppressive Agents; Injections, Intraperitoneal; Interferon-gamma; Liver; Male; Mice; Mice, Inbred C57BL; NF-kappa B; Phenanthrenes; Phytotherapy; Salvia miltiorrhiza; Signal Transduction; STAT1 Transcription Factor; Tumor Necrosis Factor-alpha

The detrimental impact of opioid agonist on the clinical management of inflammatory diseases remains elusive. Given the anti-inflammatory properties of the mu-opioid receptor (MOR) agonists at the intestinal barrier, we hypothesised that MOR activation might also dampen acute hepatic inflammation and cell death-major determinants in the pathogenesis of liver diseases.. The expression of MOR in liver biopsy specimens and peripheral blood mononuclear cells of untreated patients with chronic hepatitis C virus infection and controls, primary hepatocytes and cell lines was determined by quantitative PCR, immunoblotting and/or immunohistochemistry. The effects of peripheral MOR agonist (d-Ala2,NMe-Phe4,Gly5-ol (DAMGO)) and/or antagonist (naloxone methiodide) were explored in two models of acute hepatitis in mice. MOR-deficient mice were used to evaluate the essential regulatory role of MOR during carbon tetrachloride (CCl(4))-induced hepatitis. The role of DAMGO in cell death was investigated using terminal deoxynucleotidyl transferase-mediated dUTP nick-end labelling (TUNEL) analysis and quantification of lactate dehydrogenase release.. The key role of MOR in the prevention of acute hepatic inflammation and cell death in vivo and in vitro is reported. Whereas MOR gene expression increased transiently in the model of acute liver injury and TNFalpha-treated HepG2 cells, an impaired expression of MOR mRNA in human chronic hepatitis C samples was found. Furthermore, preventive administration of the selective MOR agonist DAMGO enhanced hepatoprotective-signalling pathways in vivo that were blocked by using naloxone methiodide. Consistently, genetic and pharmacological inhibition of MOR enhanced the severity associated with experimental hepatotoxin-induced hepatitis. Finally, treatment with DAMGO was shown to prevent cell death in vitro in HepG2 cells in a MOR-dependent manner and to prevent concanavalin A- and CCl(4)-induced cell death in vivo, providing a possible explanation for the anti-inflammatory role of MOR activation in the liver.. The results indicate that MOR agonists may prevent acute hepatitis and hold promising therapeutic use to maintain remission in both chronic inflammatory bowel and liver diseases.

Topics: Acute Disease; Animals; Biopsy; Carbon Tetrachloride; Cell Death; Concanavalin A; Enkephalin, Ala(2)-MePhe(4)-Gly(5)-; Gene Expression; Hepatitis; Hepatitis C, Chronic; Hepatitis, Animal; Hepatocytes; Humans; Male; Mice; Mice, Inbred BALB C; Mice, Inbred C57BL; Receptors, Opioid, mu; Reverse Transcriptase Polymerase Chain Reaction; RNA, Messenger; Tumor Cells, Cultured; Tumor Necrosis Factor-alpha; Up-Regulation

NKT cells are responsible for hepatitis induced either by concanavalin A (Con-A) or alpha-galactosylceramide (alpha-GalCer), and they are also profoundly involved in the generalised Shwartzman reaction (GSR) induced by consecutive injections of interleukin (IL)-12 and lipopolysaccharide (LPS). In the present study, using NC/Nga (NC) mice and SJL mice lacking the Vbeta(+)8 gene, we examined the role of Vbeta(+)8+NKT cells in hepatitis models and in the GSR. The absence of Vbeta(+)8+NKT cells in the liver mononuclear cells (MNC) was confirmed by the alpha-GalCer/CD1d/Ig dimer. Unexpectedly, other dimer+NKT cells including Vbeta7(+)NKT cells in these mice were found to decrease in comparison to that of C57BL/6 mice. No significant hepatocyte injury was observed after alpha-GalCer or Con-A administration in either mice. The serum interferon (IFN)-gamma, IL-4 and tumour necrosis factor (TNF) levels did not increase in these mice after alpha-GalCer injection, however these cytokines substantially increased after Con-A administration, thus suggesting that the roles of NKT cells differ between the two hepatitis models. However, in GSR, although neither mice showed lower IFN-gamma levels after a priming IL-12 injection, they showed TNF levels comparable to those in normal mice after LPS injection, and thus resulted in a decreased but substantial mortality. Although liver MNC from IL-12-injected SJL mice showed an impaired antitumour cytotoxicity, liver MNC of NC mice exhibited a greater antitumour cytotoxicity than that of C57BL/6 mice because liver NK cells proportionally increased in NC mice. These results confirm the critical role that Vbeta8(+)NKT cells play in both liver and multi-organ injury.

Topics: Animals; Concanavalin A; Galactosylceramides; Hepatitis, Animal; Interferon-gamma; Interleukin-4; Killer Cells, Natural; Mice; Mice, Inbred BALB C; Mice, Inbred C57BL; Mice, Knockout; Receptors, Antigen, T-Cell, alpha-beta; Shwartzman Phenomenon; T-Lymphocyte Subsets; Tumor Necrosis Factor-alpha

IL-12 is essential for invariant NKT (iNKT) cells because it can maintain a functionally active population and promote a cytokine profile that is assumed to be mainly of the pro-Th1 type. We used the murine concanavalin A (Con A)-induced hepatitis model, in which iNKT cells, IL-12, IL-4, and IFN-gamma are equally requisite, to reevaluate this issue. We demonstrate that IL-12 interacts directly with iNKT cells, contributes to their recruitment to the liver, and enhances their IL-4 production, which is essential for disease onset. IL-12-deficient mice were less susceptible to experimental hepatitis and their iNKT cells produced less IL-4 than their wild-type counterpart. A normal response could be restored by IL-12 injection, revealing its importance as endogenous mediator. In accordance with this observation, we found that iNKT cells expressed the IL-12R constitutively, in contrast to conventional T cells. Furthermore, the physiological relevance of our data is supported by the lower susceptibility to disease induction of NOD mice, known for their inherent functional and numerical abnormalities of iNKT cells associated with decreased iNKT cell-derived IL-4 production and low IL-12 secretion. Taken together, our findings provide the first evidence that IL-12 can enhance the immune response through increased IL-4 production by iNKT cells, underscoring once more the functional plasticity of this subset.

Topics: Animals; Concanavalin A; Cytokines; Disease Models, Animal; Hepatitis, Animal; Interleukin-12; Interleukin-12 Receptor beta 1 Subunit; Interleukin-4; Killer Cells, Natural; Mice; Mice, Mutant Strains; T-Lymphocyte Subsets; Th1 Cells

To explore the gene expressions of LTC4 synthase homologs in concanavalin A (Con A)-induced mouse hepatitis and regulation role of cyclosporine A (Cs A) treatment.. Male Balb/c mouse liver injury model was developed by iv injection of Con A (20 mg/kg) and protected by Cs A pretreatment (150 mg/kg) before Con A administration. Blood samples were collected at indicated times after Con A treatment with or without Cs A pretreatment. Liver damage was assessed by serum transaminase ALT and AST measurement and histological evaluation. Meantime, three LTC4 synthase homolog gene expressions were determined by RT-PCR.. Serum ALT and AST upregulation were accompanied with histological damage at 2 h after Con A administration, and further aggravated at 8 h. mGST2 gene expression increased 1.7 fold at 2 h and 1.9 fold at 8 h, while the expression of LTC4 S and mGST3 changed little. Pretreatment with Cs A prevented mouse liver from injury by Con A and partly inhibited the mGST2 gene expression upregulation.. Administration of Con A in mouse lead to a significant increase of mGST2 gene expression without any significant effect on LTC4 S and mGST3 mRNA levels. Cs A pretreatment results in protection of liver damage, whereas fails to fully inhibit the increase of mGST2 gene expression.

Topics: Alanine Transaminase; Animals; Aspartate Aminotransferases; Chemical and Drug Induced Liver Injury; Concanavalin A; Cyclosporine; Gene Expression Regulation, Enzymologic; Glutathione Transferase; Hepatitis, Animal; Immunosuppressive Agents; Injections, Intravenous; Isoenzymes; Male; Mice; Mice, Inbred BALB C; Reverse Transcriptase Polymerase Chain Reaction

To shed light on the structure-activity relationship, various soyasapogenol B derivatives were synthesized and evaluated for preventive effects on liver injury in the concanavalin A (Con A)-induced hepatitis model in mice. Con A injection into mice induces some pathophysiology of human liver disease such as autoimmune or viral hepatitis. Two hydroxyl groups on the A ring of soyasapogenol B are required for amelioration of liver damage. Modification of the C-22 hydroxyl moiety with an acyloxy or alkyloxy group, or removal of the hydroxyl group, resulted in a greatly enhanced percentage of alleviation. Among the series of soyasapogenol B derivatives examined, six compounds exhibited preventive effects on liver damage.

Topics: Animals; Concanavalin A; Crystallography, X-Ray; Disease Models, Animal; Hepatitis, Animal; Liver; Liver Function Tests; Male; Mice; Mice, Inbred BALB C; Nuclear Magnetic Resonance, Biomolecular; Oleanolic Acid; Saponins

Diphenyl dimethyl bicarboxylate (DDB) is a hepatoprotectant and used in the treatment of chronic viral hepatitis patients in China. The aim of the present paper was to investigate the effect of DDB on liver injury mediated by immune response in concanavalin A (Con A)-treated mice. A dose of Con A 30 mg/kg was injected via the tailvein to induce liver injury in mice. Serum alanine transaminase (ALT), aspartate aminotransferase (AST), total bile acid (TBA), total bilirubin (TBIL) and tumor necrosis factor alpha (TNF-alpha) level as well as liver TNF-alpha mRNA expression were determined. The following results were obtained: (1) Prior oral administration of DDB 150 mg/kg markedly reduced the elevated serum ALT, TBA and TBIL levels, and the liver lesions in Con A-treated mice; (2) DDB significantly inhibited the elevation of serum TNF-alpha and liver TNF-alpha mRNA expression 2 h after Con A injection; (3) DDB significantly inhibited hepatocyte nuclear DNA fragmentation 12 h after Con A injection; (4) DDB dose-dependently prevented the direct DNA damage induced by CuSO(4)-Phen-Vit C-H(2)O(2) system in vitro, and the ex vivo experiment also showed that the administration of DDB reduced the susceptibility of mouse liver nuclei DNA to CuSO(4)-Phen-Vit C-H(2)O(2) system. These results suggest that DDB could directly protect hepatocyte DNA from oxidative damage, and inhibit TNF-alpha mRNA expression in liver tissue, which resulted in prevention of liver damage induced by Con A in mice.

Topics: Adjuvants, Immunologic; Animals; Chemical and Drug Induced Liver Injury; Concanavalin A; Dioxoles; Disease Models, Animal; DNA Damage; Dose-Response Relationship, Drug; Drugs, Chinese Herbal; Gene Expression; Hepatitis, Animal; Hepatocytes; Liver; Liver Function Tests; Male; Mice; Mice, Inbred ICR; Protective Agents; RNA, Messenger; Tumor Necrosis Factor-alpha

Imatinib exerts potent antileukemic effects in vitro and in vivo. Despite its well known antitumor activity, the potential of imatinib for the treatment of inflammatory diseases remains elusive so far. Our current report provides strong evidence that imatinib has potent antiinflammatory effects. It potently inhibits LPS- and Con A-induced TNF-alpha production by human myeloid cells in vitro (peripheral blood mononuclear cells, CD14-selected monocytes, and monocyte-derived macrophages). Of note, the production of the antiinflammatory cytokine IL-10 was not significantly regulated by imatinib. In line with this observation, phosphorylation of IkappaB and subsequent DNA binding of NF-kappaB, which is critically involved in TNF-alpha, but not IL-10 expression, was reduced by imatinib. Using several murine models of acute hepatitis, we could corroborate our in vitro findings, as imatinib prevented macrophage- and TNF-alpha-dependent inflammatory damage of the liver induced by injection of either Con A or d-galactosamine/LPS by inhibition of hepatic TNF-alpha production. Of note, d-galactosamine/TNF-induced hepatitis was not affected, showing that imatinib does not directly inhibit TNF-alpha-induced hepatocellular cell death. These findings suggest a potent antiinflammatory role of imatinib by modulation of TNF-alpha production in monocytes/macrophages. This observation might be of therapeutic value for the treatment of TNF-mediated diseases.

Topics: Animals; Benzamides; Cell Death; Cells, Cultured; Chemical and Drug Induced Liver Injury; Concanavalin A; Female; Hepatitis, Animal; Humans; I-kappa B Proteins; Imatinib Mesylate; Inflammation; Interleukin-10; Lipopolysaccharides; Macrophages; NF-kappa B; Phosphorylation; Piperazines; Protein Kinase Inhibitors; Pyrimidines; Tumor Necrosis Factor-alpha

Leukocyte infiltration into the liver is paramount to the development of liver injury in hepatitis. Hepatitis occurring after the administration of Con A in mice is felt to be a T lymphocyte-mediated disease. In this study, we report that neutrophils are the key initiators of lymphocyte recruitment and liver injury caused by Con A. The objectives of this study were to investigate the involvement of neutrophils in Con A-induced hepatitis in vivo via intravital microscopy. After Con A administration, we observed a significant increase in leukocyte rolling flux, a decrease in rolling velocity, and an increase in leukocyte adhesion to the hepatic microvasculature. Fluorescence microscopy identified that within 4 h of Con A administration only a minority of the recruited leukocytes were T lymphocytes. Furthermore, immunohistochemistry showed a significant increase in neutrophils recruited to the liver post-Con A treatment in association with liver cell damage, as reflected by elevated serum alanine aminotransferase levels. Using flow cytometry, we observed that Con A could bind directly to neutrophils, which resulted in a shedding of L-selectin, an increase in beta(2)-integrin expression, and the production of reactive oxidants. Following neutrophil depletion, a significant inhibition of Con A-induced CD4+ T lymphocyte recruitment to the liver resulted and complete reduction in hepatic injury, as assessed by serum alanine aminotransferase levels. In summary, the present data support the concept that neutrophils play an important and previously unrecognized role in governing Con A-induced CD4+ T cell recruitment to the liver and the subsequent development of hepatitis.

Topics: Animals; Antibodies, Monoclonal; CD4-Positive T-Lymphocytes; Cell Movement; Concanavalin A; Down-Regulation; Hepatitis, Animal; Injections, Intravenous; Liver; Mice; Mice, Inbred BALB C; Mice, Nude; Microscopy, Fluorescence; Microscopy, Video; Neutropenia; Neutrophil Activation; Neutrophil Infiltration; Neutrophils; Oxidants; Protein Binding; Reactive Oxygen Species

The TNF-related apoptosis-inducing ligand (TRAIL) induces apoptosis of tumor cells but not most normal cells. Its role in hepatic cell death and hepatic diseases is not clear. In vitro studies suggest that murine hepatocytes are not sensitive to TRAIL-induced apoptosis, indicating that TRAIL may not mediate hepatic cell death. Using two experimental models of hepatitis, we found that hepatic cell death in vivo was dramatically reduced in TRAIL-deficient mice and mice treated with a blocking TRAIL receptor. Although both TRAIL and its death receptor 5 were constitutively expressed in the liver, TRAIL expression by immune cells alone was sufficient to restore the sensitivity of TRAIL-deficient mice to hepatitis. Thus, TRAIL plays a crucial role in hepatic cell death and hepatic inflammation.

Topics: Animals; Apoptosis Regulatory Proteins; Concanavalin A; Gene Expression Regulation; Hepatitis, Animal; Immunity, Innate; Inflammation; Listeriosis; Membrane Glycoproteins; Mice; Mice, Inbred BALB C; Mice, Knockout; Receptors, Tumor Necrosis Factor; TNF-Related Apoptosis-Inducing Ligand; Tumor Necrosis Factor-alpha

Administration of Con A induces liver injury that is considered to be an experimental model for human autoimmune or viral hepatitis, where immunopathology plays roles mediated by activated lymphocytes, especially NK1.1+ CD3+ NKT cells, and inflammatory cytokines, including IFN-gamma and IL-4. In the present study we investigated the role of WSX-1, a component of IL-27R, in Con A-induced hepatitis by taking advantage of WSX-1 knockout mice. WSX-1-deficient mice were more susceptible to Con A treatment than wild-type mice, showing serum alanine aminotransferase elevation and massive necrosis in the liver. Although the development of NKT cells appeared normal in WSX-1 knockout mice, purified NKT cells from the knockout mice produced more IFN-gamma and IL-4 than those from wild-type mice in response to stimulation with Con A both in vitro and in vivo. In addition, hyperproduction of proinflammatory cytokines, including IL-1, IL-6, and TNF-alpha, was observed in the knockout mice after Con A administration. These data revealed a novel role for WSX-1 as an inhibitory regulator of cytokine production and inflammation in Con A-induced hepatitis.

Topics: Animals; Cell Differentiation; Concanavalin A; Cytokines; Genetic Predisposition to Disease; Hepatitis, Animal; Inflammation Mediators; Injections, Intravenous; Interferon-gamma; Interleukin-4; Killer Cells, Natural; Mice; Mice, Inbred C57BL; Mice, Knockout; Receptors, Cytokine; Receptors, Interleukin; T-Lymphocyte Subsets; Up-Regulation

The hepatoprotective effect of IL-6 on various forms of liver injury including T cell-mediated hepatitis has been well documented, and it is believed that induction of antiapoptotic proteins is an important mechanism. In this study, we provide evidence suggesting an additional mechanism involved in the protective role of IL-6 in T cell-mediated hepatitis. In NKT cell-depleted mice, Con A-induced liver injury is diminished; this can be restored by the adoptive transfer of liver mononuclear cells or NKT cells from wild-type mice, but not from IL-6-treated mice. In vitro IL-6 treatment inhibits the ability of mononuclear cells to restore Con A-induced liver injury in NKT-depleted mice, whereas the same treatment does not inhibit purified NKT cells from restoring the injury. The addition of CD3(+) T cells or CD4(+) T cells can restore the inhibitory effect of IL-6 on purified NKT cells, whereas the addition of CD3(+) T cells from CD4-deficient mice fails to restore this inhibitory effect. The expression of IL-6R was detected in 52.6% of hepatic CD3(+) T cells and 32.7% of hepatic CD4(+) T cells, but only in 3.9% of hepatic NK and 1.5% of hepatic NKT cells. Finally, treatment with IL-6 induces STAT3 activation in hepatic lymphocytes and hepatic T cells, and blocking such activation abolishes the inhibitory effect of IL-6 on hepatic lymphocytes to restore liver injury. Taken together, these findings suggest that in addition to its antiapoptotic abilities, as previously well documented, IL-6/STAT3 inhibits NKT cells via targeting CD4(+) T cells and consequently prevents T cell-mediated hepatitis.

Topics: Adoptive Transfer; Animals; Antibodies, Monoclonal; Antigens; Antigens, Surface; CD4-Positive T-Lymphocytes; Cell Death; Concanavalin A; Cytotoxicity Tests, Immunologic; DNA-Binding Proteins; Hepatitis, Animal; Hepatocytes; Humans; Immunosuppressive Agents; Injections, Intravenous; Interleukin-6; Killer Cells, Natural; Lectins, C-Type; Leukocytes, Mononuclear; Lymphocyte Activation; Lymphocyte Depletion; Male; Mice; Mice, Inbred BALB C; Mice, Inbred C57BL; Mice, Knockout; NK Cell Lectin-Like Receptor Subfamily B; Proteins; Receptors, Interleukin-6; Recombinant Proteins; Signal Transduction; STAT3 Transcription Factor; T-Lymphocyte Subsets; Trans-Activators

We have previously shown that IFN-gamma/STAT1 plays an essential role in concanavalin A (ConA)-induced T cell hepatitis via activation of apoptotic signaling pathways. Here we demonstrate that IFN-gamma/STAT1 also plays a crucial role in leukocyte infiltration into the liver in T cell hepatitis. After injection of ConA, leukocytes were significantly infiltrated into the liver, which was suppressed in IFN-gamma(-/-) and STAT1(-/-) mice. Disruption of the IFN regulatory factor-1 (IRF-1) gene, a downstream target of IFN-gamma/STAT1, abolished ConA-induced liver injury and suppressed leukocyte infiltration into the liver. Additionally, ConA injection induced expression of a wide variety of chemokines and adhesion molecules in the liver. Among them, expression of ICAM-1, VCAM-1, monokine induced by IFN-gamma (Mig), CC chemokine ligand-20, epithelial cell-derived neutrophil-activating peptide (ENA)-78, IFN-inducible T cell-alpha chemoattractant (I-TAC), and IFN-inducible protein-10 (IP-10) was markedly attenuated in IFN-gamma(-/-), STAT1(-/-), and IRF-1(-/-) mice. In primary mouse hepatocytes, Kupffer cells, and endothelial cells, in vitro treatment with IFN-gamma activated STAT1, STAT3, and IRF-1, and induced expression of VCAM-1, ICAM-1, Mig, ENA-78, I-TAC, and IP-10 mRNA. Induction of these chemokines and adhesion molecules was markedly diminished in STAT1(-/-) and IRF-1(-/-) hepatic cells compared with wild-type hepatic cells. These findings suggest that in addition to induction of apoptosis, previously well documented, IFN-gamma also stimulated hepatocytes, sinusoidal endothelial cells, and Kupffer cells partly via an STAT1/IRF-1-dependent mechanism to produce multiple chemokines and adhesive molecules responsible for promoting infiltration of leukocytes and, ultimately, resulting in hepatitis.

Topics: Animals; Cell Adhesion Molecules; Chemokines; Concanavalin A; DNA-Binding Proteins; Endothelial Cells; Eosinophils; Hepatitis, Animal; Hepatocytes; Inflammation Mediators; Interferon Regulatory Factor-1; Interferon-gamma; Kupffer Cells; Liver; Male; Mice; Mice, Inbred C57BL; Mice, Knockout; Neutrophil Infiltration; Phosphoproteins; Signal Transduction; STAT1 Transcription Factor; T-Lymphocytes; Trans-Activators

Both osteopontin (OPN) and natural killer T (NKT) cells play a role in the development of immunological disorders. We examined a functional link between OPN and NKT cells. Concanavalin A (Con A)-induced hepatitis is a well-characterized murine model of T cell-mediated liver diseases. Here, we show that NKT cells secrete OPN, which augments NKT cell activation and triggers neutrophil infiltration and activation. Thus, OPN- and NKT cell-deficient mice were refractory to Con A-induced hepatitis. In addition, a neutralizing antibody specific for a cryptic epitope of OPN, exposed by thrombin cleavage, ameliorated hepatitis. These findings identify NKT cell-derived OPN as a novel target for the treatment of inflammatory liver diseases.

Topics: Amino Acid Motifs; Animals; Blotting, Western; Cell Movement; Concanavalin A; Electrophoresis, Polyacrylamide Gel; Hepatitis, Animal; Immunohistochemistry; Integrins; Killer Cells, Natural; Liver; Lymphocyte Activation; Male; Mice; Models, Immunological; Osteopontin; Reverse Transcriptase Polymerase Chain Reaction; Sialoglycoproteins; Signal Transduction; T-Lymphocyte Subsets; Thrombin

T cell-mediated liver diseases are associated with elevated serum levels of C-C chemokine ligand 2 (CCL2)/monocyte chemoattractant protein-1 (MCP-1). However, the extent to which the actions of CCL2/MCP-1 contribute to the pathogenesis of T cell-mediated hepatitis remains incompletely understood. Con A-induced hepatitis is a liver-specific inflammation mediated by activated T cells and is driven by an up-regulation of the hepatic expression of TNF-alpha, IFN-gamma, and IL-4. The present study examined the role of CCL2/MCP-1 in the pathogenesis of T cell-mediated hepatitis induced by Con A administration in the mouse. We demonstrate a novel hepatoprotective role for CCL2/MCP-1 during Con A-induced hepatitis, because CCL2/MCP-1 neutralization strikingly enhanced hepatic injury, both biochemically and histologically, after Con A administration. Furthermore, CCL2/MCP-1 neutralization was associated with a significant reduction in the hepatic levels of TNF-alpha and IFN-gamma, but with a significant increase in hepatic IL-4 levels. Moreover, IL-4 production and CCR2 expression by Con A-stimulated CD3(+)NK1.1(+) T cells was significantly reduced by rMCP-1 treatment in vitro. In summary, we propose that CCL2/MCP-1 fulfills a novel anti-inflammatory role in T cell-mediated hepatitis by inhibiting CD3(+)NK1.1(+) T cell-derived IL-4 production through direct stimulation of its specific receptor CCR2. These findings may have direct clinical relevance to T cell-mediated hepatitis.

Topics: Animals; Cell Movement; Cells, Cultured; Chemokine CCL2; Concanavalin A; Cytokines; Down-Regulation; Hepatitis, Animal; Immune Sera; Inflammation Mediators; Injections, Intraperitoneal; Injections, Intravenous; Interleukin-4; Killer Cells, Natural; Lymphocytes; Male; Mice; Mice, Inbred C57BL; Receptors, CCR2; Receptors, Chemokine; T-Lymphocyte Subsets; Up-Regulation

We earlier found that a rat monoclonal antibody (mAb) RE2 can induce rapid death of murine activated, but not resting, lymphocytes and lymphocyte cell lines, in a complement-independent manner, a cell death differing from typical apoptosis or necrosis. We here found that this cell death is independent of pathways involving Fas, caspase, and phosphoinositide-3 kinase. With the advantage of producing human B cell line transfectants with stable expression of human/mouse xeno-chimeric MHC class I genes, we found that RE2 epitope resides on the murine class I alpha2 domain. However, the alpha3 domain plays a key role in transducing the death signal, which mediates extensive aggregation of the MHC class I-integrin-actin filament system, giving rise to membrane blebs and pores. In mouse models with T/NKT cell activation-associated fulminant hepatitis, administration of mAb RE2 almost completely inhibited the development of liver cell injuries. Taken collectively, this form of cell death may be involved in homeostatic immune regulation, and induction of this form of cell death using the mAbs may be potentially therapeutic for subjects with immunological diseases mediated by activated lymphocytes.

Topics: Animals; Antibodies, Monoclonal; Cell Death; Cell Line; Concanavalin A; Disease Models, Animal; Epitopes; Genes, MHC Class I; Hepatitis, Animal; Histocompatibility Antigens Class I; Humans; Liver; Lymphocyte Activation; Lymphocytes; Major Histocompatibility Complex; Mice; Mice, Inbred C57BL; Protein Structure, Tertiary; Rats; Recombinant Fusion Proteins

T cell-mediated immune responses are implicated in the pathogenesis of a variety of liver disorders; however, the underlying mechanism remains obscure. Con A injection is a widely accepted mouse model to study T cell-mediated liver injury, in which STAT6 is rapidly activated. Disruption of the IL-4 and STAT6 gene by way of genetic knockout abolishes Con A-mediated liver injury without affecting IFN-gamma/STAT1, IL-6/STAT3, or TNF-alpha/NF-kappaB signaling or affecting NKT cell activation. Infiltration of neutrophils and eosinophils in Con A-induced hepatitis is markedly suppressed in IL-4 (-/-) and STAT6(-/-) mice compared with wild-type mice. IL-4 treatment induces expression of eotaxins in hepatocytes and sinusoidal endothelial cells isolated from wild-type mice but not from STAT6(-/-) mice. Con A injection induces expression of eotaxins in the liver and elevates serum levels of IL-5 and eotaxins; such induction is markedly attenuated in IL-4(-/-) and STAT6(-/-) mice. Finally, eotaxin blockade attenuates Con A-induced liver injury and leukocyte infiltration. Taken together, these findings suggest that IL-4/STAT6 plays a critical role in Con A-induced hepatitis, via enhancing expression of eotaxins in hepatocytes and sinusoidal endothelial cells, and induces IL-5 expression, thereby facilitating recruitment of eosinophils and neutrophils into the liver and resulting in hepatitis.

Topics: Adoptive Transfer; Animals; Cell Movement; Cells, Cultured; Chemokine CCL11; Chemokines, CC; Concanavalin A; DNA-Binding Proteins; Eosinophils; Hepatitis, Animal; Immune Sera; Injections, Intravenous; Interferon-gamma; Interleukin-4; Interleukin-5; Killer Cells, Natural; Leukocytes; Leukocytes, Mononuclear; Liver; Lymphocyte Activation; Male; Mice; Mice, Inbred BALB C; Mice, Inbred C57BL; Mice, Knockout; Neutrophil Infiltration; NF-kappa B; Signal Transduction; Spleen; STAT1 Transcription Factor; STAT3 Transcription Factor; STAT6 Transcription Factor; T-Lymphocyte Subsets; Trans-Activators; Tumor Necrosis Factor-alpha; Up-Regulation

The therapeutic potential of cannabinoids has been described previously for several inflammatory diseases, but the molecular mechanisms underlying the anti-inflammatory properties of cannabinoids are not well understood. In this study, we investigated the mechanism of action of a novel synthetic cannabinoid, [(+)(6aS,10aS)-6,6-Dimethyl-3-(1,1-dimethylheptyl)-1-hydroxy-9-(1H-imidazol-2-ylsulfanylmethyl]-6a,7,10,10a-tetrahydro-6H-dibenzo[b,d]pyran (PRS-211,092) that has no psychotropic effects but exhibits immunomodulatory properties. Treatment with PRS-211,092 significantly decreased Concanavalin A-induced liver injury in mice that was accompanied by: 1) promotion of early gene expression of interleukin (IL)-6 and IL-10 that play a protective role in this model; 2) induction of early gene expression of the suppressors of cytokine signaling (SOCS-1 and 3), followed by 3) inhibition of several pro-inflammatory mediators, including IL-2, monocyte chemoattractant protein-1 (MCP-1), IL-1beta, interferon-gamma, and tumor necrosis factor alpha. Based on these results, we propose a mechanism by which PRS-211,092 stimulates the expression of IL-6, IL-10 and the SOCS proteins that, in turn, negatively regulates the expression of pro-inflammatory cytokines. Negative regulation by PRS-211,092 was further demonstrated in cultured T cells, where it inhibited IL-2 production and nuclear factor of activated T cells activity. These findings suggest that this cannabinoid derivative is an immunomodulator that could be developed as a potential drug for hepatitis as well as for other short- or long-term inflammatory diseases.

Topics: Adjuvants, Immunologic; Animals; Cannabinoids; Concanavalin A; Cytokines; Female; Hepatitis, Animal; Mice; Mice, Inbred BALB C

Topics: Adenosine; Animals; Concanavalin A; Disease Models, Animal; Hepatitis, Animal; Hepatitis, Autoimmune; Liver; Mice; Phenethylamines; Purinergic P1 Receptor Agonists

Anorexia that develops in chronic hepatitis is associated with cytokine expression in the brain. Treatment of mice with concanavalin A (12.5 mg/kg, i.v.) elevated the plasma alanine aminotransferase activity at 8.5 h after treatment. Tumor necrosis factor-alpha (TNF-alpha) and interleukin-1beta mRNA expression was induced at 6 and 24 h after concanavalin A treatment in both the liver and brain. Treatment of mice with concanavalin A reduced the body weight at 24 h after treatment and this decreased body weight was accompanied by a decreased food intake. Glycyrrhizin (200 mg/kg, i.p.) inhibited the concanavalin A-induced elevation of plasma alanine aminotransferase activity, however, it did not inhibit the concanavalin A-induced decreased body weight. The present results indicate that treatment of mice with concanavalin A caused the development of anorexia and that this anorexia might develop independently of the induction of hepatitis.

Topics: Alanine Transaminase; Animals; Anorexia; Body Weight; Brain; Chemical and Drug Induced Liver Injury; Concanavalin A; Disease Models, Animal; Eating; Female; Glycyrrhizic Acid; Hepatitis, Animal; Interleukin-1; Liver; Mice; Mice, Inbred BALB C; RNA, Messenger; Tumor Necrosis Factor-alpha

The effects of coumarin derivatives, osthole, imperatorin, Pd-Ia, Pd-II and Pd-III, on mice concanavalin A (Con A) (0.2 mg/mouse, i.v.)-induced hepatitis were studied. At the dose of 200 mg/kg (i.p.), these coumarins inhibited more than 90% of the Con A-induced elevation of plasma alanine aminotransferase activity, but glycyrrhizin (200 mg/kg, i.p.) caused only 45% inhibition. At the dose of 100 mg/kg (i.p.), osthole produced the strongest inhibition among these coumarins. The inhibitory activity of osthole is lost when its 7-methoxy group is replaced by a 7-hydroxy group to form osthenol. The present results showed that coumarin derivatives inhibited Con A-induced hepatitis, with osthole being the most inhibitory.

Topics: Alanine Transaminase; Animals; Anticoagulants; Concanavalin A; Coumarins; Female; Furocoumarins; Glycyrrhizic Acid; Hepatitis, Animal; Liver; Mice; Mice, Inbred BALB C

Administration of Con A induces severe injury to hepatocytes in mice and is considered to be a model for human hepatitis. In the current study, we investigated the role of CD44 in Con A-induced hepatitis. Intravenous administration of Con A (20 mg/kg) caused 100% mortality in C57BL/6 CD44-knockout (KO) mice, although it was not lethal in C57BL/6 CD44 wild-type (WT) mice. Administration of lower doses of Con A (12 mg/kg body weight) into CD44 WT mice induced hepatitis as evident from increased plasma aspartate aminotransferase levels accompanied by active infiltration of mononuclear cells and neutrophils, and significant induction of apoptosis in the liver. Interestingly, CD44 KO mice injected with similar doses of Con A exhibited more severe acute suppurative hepatitis. Transfer of spleen cells from Con A-injected CD44 KO mice into CD44 WT mice induced higher levels of hepatitis when compared with transfer of similar cells from CD44 WT mice into CD44 WT mice. The increased hepatitis seen in CD44 KO mice was accompanied by increased production of cytokines such as TNF-alpha, IL-2 and IFN-gamma, but not Fas or Fas ligand. The increased susceptibility of CD44 KO mice to hepatitis correlated with the observation that T cells from CD44 KO mice were more resistant to activation-induced cell death when compared with the CD44 WT mice. Together, these data demonstrate that activated T cells use CD44 to undergo apoptosis, and dysregulation in this pathway could lead to increased pathogenesis in a number of diseases, including hepatitis.

Topics: Adjuvants, Immunologic; Adoptive Transfer; Animals; Apoptosis; B-Lymphocytes; Cell Division; Concanavalin A; Fas Ligand Protein; fas Receptor; Female; Hepatitis, Animal; Hyaluronan Receptors; Immunity, Innate; Injections, Intravenous; Interferon-gamma; Interleukin-2; Leukocyte Count; Ligands; Liver; Lymphocyte Activation; Lymphocyte Count; Macrophages; Membrane Glycoproteins; Mice; Mice, Inbred C57BL; Mice, Knockout; RNA, Messenger; Spleen; T-Lymphocytes; Tumor Necrosis Factor-alpha; Up-Regulation

We have previously shown that interferon-inducible protein-10 (IP-10), a chemokine for activated lymphocytes, was specifically induced in the liver of Concanavalin A (Con A)-treated mice. The aim of this study was to investigate the time course profile and cell-type-specific hepatic production of monokine induced by interferon-gamma (MIG), a chemokine which shares its receptor and most of its activity with IP-10, in Con A-treated mice and to compare them with those of IP-10.. Hepatic mRNA expression of MIG and IP-10 was studied by means of Northern blot analysis and in situ hybridization in Con A-treated mice. The levels of MIG and IP-10 in the serum and culture supernatants of murine hepatoma-, hepatic sinusoidal endothelial cell-, hepatic stellate cell- and macrophage-derived cell lines were determined by means of specific enzyme-linked immunosorbent assays.. The serum level of MIG slowly reached a maximum at 12 h after Con A injection and remained elevated for a long time, whereas that of IP-10 reached a maximum at 3 h and declined quickly, a finding supported by Northern blot analysis. Using in situ hybridization, the mRNA of MIG as well as IP-10 was found to be expressed in hepatocytes and hepatic non-parenchymal cells. Similar to IP-10, MIG was produced by hepatoma-, hepatic sinusoidal endothelial cell-, hepatic stellate cell- and macrophage-derived cell lines in vitro.. Although both MIG and IP-10 were produced by hepatocytes and hepatic non-parenchymal cells in Con A-treated mice, the time course profile of MIG was distinguishable from that of IP-10. The fact that hepatic MIG and IP-10 were produced sequentially in this hepatitis model may suggest that a non-redundant role is played by these two chemokines in the process of hepatic necro-inflammation.

Topics: Animals; Blotting, Northern; Cell Line; Chemokine CXCL10; Chemokine CXCL9; Chemokines, CXC; Concanavalin A; Enzyme-Linked Immunosorbent Assay; Hepatitis, Animal; In Situ Hybridization; Intercellular Signaling Peptides and Proteins; Interferon-gamma; Liver; Male; Mice; Mice, Inbred C57BL; Time Factors

Lymphocyte infiltration is a manifest feature of hepatitis. To reveal the main site and mechanism of lymphocyte adhesion/extravasation in the hepatic vasculature during inflammation, we morphometrically and histologically analyzed these events in relation to adhesion molecule expression using a murine model of T-cell mediated hepatitis induced by concanavalin A (Con A). Although lymphocyte adhesion was restricted to the sinusoids in untreated mice, it increased in all the segments of porto-sinusoidal-hepatic venous system 8 hours after Con A injection; the number of adhering lymphocytes per unit vascular circumference was the largest in the sublobular veins, relatively large in the central veins and small hepatic veins, and relatively small in the sinusoids and negligible in the portal veins. At 20 hours, extravascular lymphocytes showed similar distribution to lymphocyte adhesion at 8 hours except in the portal veins, around which they were possibly accumulated by the translocation of extrasinusoidal lymphocytes. E-selectin and vascular cell adhesion molecule-1 (VCAM-1) were transiently expressed at 4 to 6 hours, whereas P-selectin and intercellular adhesion molecule-1 were not changed between 0 and 48 hours. In particular, E-selectin expression coincided with that of lymphocyte adhesion in distribution. Lymphocyte attachment was inhibited by pretreatment with anti-E-selectin monoclonal antibody (MAb) or anti-VCAM-1 MAb, and expression of E-selectin and VCAM-1 was suppressed by pretreatment with anti-tumor necrosis factor-alpha (TNF-alpha) MAb. Electron microscopically, lymphocytes were trapped by endothelial lamellipodia and traversed the endothelium by diapedesis. These results indicate that lymphocyte adhesion/transmigration preferentially takes place in the sublobular veins in association with TNF-alpha-induced endothelial activation, i.e., E-selectin and VCAM-1 expression and lamellipodia formation.

Topics: Animals; Antibodies, Monoclonal; Cell Adhesion; Cell Adhesion Molecules; Cell Movement; Concanavalin A; E-Selectin; Female; Hepatic Veins; Hepatitis, Animal; Kinetics; Lymphocytes; Mice; Mice, Inbred BALB C; Microscopy, Electron; P-Selectin; Portal System; Vascular Cell Adhesion Molecule-1

The present study was carried out to analyze the effects of soyasapogenol A on the liver injury mediated by the immune response in concanavalin A-induced hepatitis in mice. Soyasapogenol A reduced the number of infiltrating inflammatory cells in the liver and significantly lowered the elevated level of plasma tumor necrosis factor-alpha (TNF-alpha) 2 h after concanavalin A treatment, and then markedly reduced the elevated plasma alanine aminotransferase activity and decreased the number of apoptotic bodies in the liver parenchymal cells but not in the sinusoidal cells at 24 h. Since the effect of soyasapogenol A on the elevated plasma TNF-alpha level was not appreciable compared to the preventive effect of soyasapogenol A on the elevated plasma alanine aminotransferase level, these results suggest that soyasapogenol A directly prevents apoptosis of hepatocytes, and secondly, inhibits the elevation of plasma TNF-alpha, which consequently resulted in the prevention of liver damage in the concanavalin A-induced hepatitis model.

Topics: Alanine Transaminase; Animals; Apoptosis; Aspartate Aminotransferases; Concanavalin A; Cytokines; Hepatitis, Animal; Liver; Liver Function Tests; Male; Mice; Mice, Inbred BALB C; Oleanolic Acid

Natural killer T (NKT) cells constitute a distinct subpopulation of T cells with a unique antigen specificity, prompt effector functions, and an unusual tissue distribution. NKT cells are especially abundant in the liver, but their physiological function in this organ remains unclear. In the present study, we examined the possible contribution of NKT cells to a murine model of hepatitis induced by i.v. injection of Con A. CD1-deficient mice lacking NKT cells were highly resistant to Con A-induced hepatitis. Adoptive transfer of hepatic NKT cells isolated from wild-type mice, but not from FasL-deficient gld mice, sensitized CD1-deficient mice to Con A-induced hepatitis. Furthermore, adoptive transfer of hepatic mononuclear cells from wild-type mice, but not from CD1-deficient mice, sensitized gld mice to Con A-induced hepatitis. Upon Con A administration, hepatic NKT cells rapidly up-regulated cell surface FasL expression and FasL-mediated cytotoxicity. At the same time, NKT cells underwent apoptosis leading to their rapid disappearance in the liver. These results implicated FasL expression on liver NKT cells in the pathogenesis of Con A-induced hepatitis, suggesting a similar pathogenic role in human liver diseases such as autoimmune hepatitis.

Topics: Adoptive Transfer; Alanine Transaminase; Animals; Antigens, CD1; Apoptosis; Aspartate Aminotransferases; Concanavalin A; Cytotoxicity, Immunologic; Fas Ligand Protein; Hepatitis, Animal; Humans; Killer Cells, Natural; Liver; Lymphocyte Transfusion; Male; Membrane Glycoproteins; Mice; Mice, Inbred C57BL; Mice, Knockout

Aminoguanidine is an inhibitor of the inducible form of nitric oxide synthase (iNOS). In the present study, the effect of aminoguanidine on concanavalin A-induced hepatitis was examined. Treatment of mice with concanavalin A (10 mg/kg, i.v.) induced interferon-gamma and iNOS mRNA expression in the liver before the elevation of plasma alanine aminotransferase activity. Immunohistochemical study showed the induction of iNOS protein expression in the area of necrosis. Aminoguanidine (1, 3 and 10 mg/kg, i.p.) inhibited the concanavalin A-induced elevation of alanine aminotransferase activity. Aminoguanidine (10 mg/kg, i.p.) did not inhibit concanavalin A-induced interleukin-2, interferon-gamma, tumor necrosis factor-alpha or iNOS mRNA expression in the liver. The plasma nitrite/nitrate level was elevated at 6 and 24 h after concanavalin A treatment. The elevation of nitrite/nitrate was inhibited by aminoguanidine (10 mg/kg, i.p.). From these results, we conclude that nitric oxide formed by iNOS may be involved in the development of concanavalin A-induced hepatitis.

Topics: Alanine Transaminase; Animals; Concanavalin A; Female; Guanidines; Hepatitis, Animal; Interferon-gamma; Liver; Mice; Mice, Inbred BALB C; Nitrates; Nitric Oxide Synthase; Nitric Oxide Synthase Type II; Nitrites; RNA, Messenger; Tumor Necrosis Factor-alpha

Administration of concanavalin A (Con A) leads to acute hepatitis that involves T-cell activation and inflammatory mediator production in mice and rats. We examined the role of CH-100, a Chinese herbal medicine previously trialed in human hepatitis C, in the prevention of Con A-related, T-cell-mediated, acute liver injury in rats.. Female Wistar rats were fed 40% ethanol, 2% sucrose, or isocaloric sucrose for 8 weeks. At the same time, these animals were fed either the Chinese herbal medicine CH-100 (4 tablets/kg body weight/ day) or placebo in chow daily. Blood from the tail vein was collected for endotoxin (lipopolysaccharide) assay at 0, 4, and 8 weeks of ethanol consumption. Twenty-four hours after injection of Con A (20 mg/kg body weight) or phosphate-buffered saline, blood from the tail vein was collected for alanine aminotransferase and tumor necrosis factor (TNF)-alpha assays. Liver-associated CD4+ T cells were isolated from liver perfusates and then cultured with Con A (5 microg/ml) at 37 degrees C for 24 hr. Supernatants were harvested for TNF-alpha assay. The proportion of CD4+ T cells in blood and liver perfusates was measured. Liver samples were collected for histopathological analysis.. Lipopolysaccharide levels were significantly reduced in CH-100-treated ethanol-fed rats compared with placebo-treated rats. After Con A injection, alanine aminotransferase levels were lower at 12 and 24 hr in herb-treated rats compared with placebo-treated rats. Furthermore, serum TNF-alpha levels were lower in ethanol-fed rats on herbal treatment. A significant decrease in TNF-alpha production by liver-associated CD4+ T cells in culture was observed in CH-100-treated ethanol-fed rats. CH-100 treatment was associated with a decreased percentage of CD4+ cells in both blood and liver perfusate in all groups. Herb-treated rats displayed markedly less hepatic necrosis and a reduced CD4+ T-cell infiltrate in portal areas than did placebo-fed rats.. The results demonstrate that CH-100 modified the T-cell response to Con A injection. The effect was more marked in ethanol-fed rats, which suggests a possible role for CH-100 in treating alcoholic liver disease.

Topics: Alanine Transaminase; Animals; CD4-Positive T-Lymphocytes; Central Nervous System Depressants; Concanavalin A; Drugs, Chinese Herbal; Ethanol; Female; Hepatitis, Animal; Humans; Lipopolysaccharides; Liver Diseases, Alcoholic; Mice; Rats; Rats, Wistar; Tumor Necrosis Factor-alpha

The administration of concanavalin A (Con A) to mice induces cytokine-dependent hepatitis. In the present study, the effect of glycyrrhizin on Con A-induced hepatitis was examined. Treatment of mice with Con A (0.2 mg/mouse, i.v.) induced elevation of the plasma transaminase activities at 24 h. Mice were treated with glycyrrhizin (100, 200 and 400 mg/kg, i.p.), and glycyrrhizin at the doses of 200 and 400 mg/kg inhibited the Con A-induced elevation of the plasma transaminase activities. At 1 h after Con A treatment, interferon-gamma, tumor necrosis factor-alpha, interleukin-2 and interleukin-6 proteins were released into the plasma. Although treatment with glycyrrhizin at 200 mg/kg inhibited Con A-induced hepatitis, it did not affect the release of any of these Con A-induced cytokines into the plasma. The present results clearly show that glycyrrhizin inhibited Con A-induced hepatitis without affecting cytokine expression.

Topics: Alanine Transaminase; Animals; Anti-Inflammatory Agents, Non-Steroidal; Aspartate Aminotransferases; Concanavalin A; Cytokines; Female; Glycyrrhizic Acid; Hepatitis, Animal; Interferon-gamma; Interleukin-2; Interleukin-6; Mice; Mice, Inbred BALB C; Tumor Necrosis Factor-alpha

Heparin has been noted to inhibit inflammation independent of its known anti-coagulant activity. In the present study, we examined the ability of heparin and low molecular weight heparin to prevent immune-mediated, concanavalin A-induced liver damage.. Mice were pretreated with either heparin or low molecular weight heparin (Fragmin) prior to their inoculation with concanavalin A (10 mg/kg). Liver enzymes, liver histology, and the serum levels of tumor necrosis factor-a, interleukin-6, and interleukin-10 were examined in the control and treated mice.. The histopathologic damage in the liver, and the concanavalin A-induced release of aminotransferases, tumor necrosis factor-a, and interleukin-6 were significantly inhibited in mice pretreated with low molecular weight heparin, whereas the serum levels of the anti-inflammatory cytokine interleukin-10 were increased (p<0.01). Interestingly, maximal inhibition was obtained with low low molecular weight heparin doses (5 and 1 microg/mouse, p<0.001), while higher doses were less effective. Concanavalin A-induced liver injury was not prevented by pretreatment of the mice with heparan sulphate (p<0.001), which although it is structurally similar to heparin possesses neither anti-inflammatory nor anti-coagulant properties.. This study demonstrates the efficacy of low molecular weight heparin in preventing immune-mediated liver damage in mice.

Topics: Alanine Transaminase; Animals; Concanavalin A; Heparin; Heparin, Low-Molecular-Weight; Hepatitis, Animal; Interleukin-10; Interleukin-6; Liver; Mice; Mice, Inbred BALB C; Tumor Necrosis Factor-alpha

Concanavalin A activates T lymphocytes and causes T cell-mediated hepatic injury in mice. Tumor necrosis factor alpha is a critical mediator in this experimental model. T-cell-mediated liver injury involves the migration of immune cells, notably CD4+ T lymphocytes, into liver tissue. Pentoxifylline is a strong suppressor of tumor necrosis factor alpha release and prevents leukocyte adherence to vascular endothelium and down-regulates the expression of intercellular adhesion molecule-1 in monocytes. In this study, we examined the efficacy of pentoxifylline as a potential therapeutic compound for the treatment of concanavalin A hepatitis.. Balb/c mice were injected with 12 mg/kg concanavalin A with or without a single injection of pentoxifylline (5-300 mg/kg), 2 h prior to concanavalin A administration. Liver damage was evaluated by determining serum levels of liver enzymes and tumor necrosis factor alpha, and hepatic histopathology compared to mice treated with concanavalin A only. We also assessed the effects of pentoxifylline on the adhesive properties of T lymphocytes to fibronectin, as a paradigm for immune cell-extracellular matrix interactions required for migration. Pretreatment with pentoxifylline significantly reduced serum levels of liver enzymes (3800+/-650 vs 150+/-28 IU/l) and tumor necrosis factor alpha (710+/-105 vs 113+/-15 pg/ml) with no evidence of inflammation in histopathologic examination compared to control mice treated with concanavalin A. Pentoxifylline also inhibited the binding of murine T cells to fibronectin. All the effects of pentoxifylline were dose-dependent.. These results indicate that high doses of pentoxifylline can prevent concanavalin A hepatitis by suppression of tumor necrosis factor alpha release and inhibition of T cells adhesion to extracellular matrix.

Topics: Animals; Cell Adhesion; Concanavalin A; Extracellular Matrix; Fibronectins; Hepatitis, Animal; Lactulose; Male; Mice; Mice, Inbred BALB C; Pentoxifylline; Receptors, Tumor Necrosis Factor; T-Lymphocytes; Tumor Necrosis Factor-alpha; Vasodilator Agents

Concanavalin A (Con A)-induced hepatitis is an experimental hepatitis model in which hepatic injury is caused by the action of cytokines produced by T cells. Using IFN-gamma-deficient mice, we previously demonstrated that IFN-gamma plays a central role in Con A-induced hepatitis. Here, we show that development of the disease is completely suppressed in gld/gld mice, in which Fas ligand is defective. In contrast, suppression of the disease in Ipr/Ipr mice was incomplete, since a small amount of the fas mRNA was produced in these mice. The data indicate that activation of the Fas/Fas ligand system is a necessary step in the development of Con A-induced hepatitis. Furthermore, we found that not only fas but also caspase-1 expression was reduced in IFN-gamma-deficient mice. Since caspase-1 is an integral component of Fas signal transduction, these observations suggest that IFN-gamma-induced activation of both fas and caspase-1 expression causes enhancement of hepatocyte apoptosis resulting in the development of hepatitis.

Topics: Animals; Apoptosis; Concanavalin A; Fas Ligand Protein; fas Receptor; Hepatitis, Animal; Interferon-gamma; Membrane Glycoproteins; Mice; Point Mutation; T-Lymphocytes

It has been shown that synthetic non-peptidic analogues of Arg-Gly-Asp, a major cell adhesive ligand of extracellular matrix, prevented an increase in serum aminotransferase activity, as a manifestation of concanavalin A induced liver damage in mice. This study examined the effects of an Arg-Gly-Asp mimetic on liver histology and cytokine release in response to concanavalin A administration, and the efficacy of soluble receptor of tumour necrosis factor (TNF) alpha in preventing hepatitis in this model of liver injury.. Mice were pretreated with either the Arg-Gly-Asp mimetic SF-6,5 or recombinant soluble receptor of TNF alpha before their inoculation with 10 mg/kg concanavalin A. Liver enzymes, histology, and the serum values of TNF alpha and interleukin (IL)6 were examined.. The histopathological damage in the liver, and the concanavalin A induced release of TNF alpha and IL6 were significantly inhibited by the synthetic Arg-Gly-Asp mimetic (p < 0.001). Liver injury, manifested by the increase in serum aminotransferase and cytokines, as well as by histological manifestations of hepatic damage, was effectively prevented by pretreatment of the mice with the soluble TNF receptor (p < 0.001).. This study confirms the efficacy of a synthetic Arg-Gly-Asp mimetic and soluble TNF receptor in the prevention of immune mediated liver damage in mice.

Topics: Alanine Transaminase; Animals; Aspartate Aminotransferases; Concanavalin A; Guanidines; Hepatitis, Animal; Interleukin-6; Male; Mice; Mice, Inbred BALB C; Receptors, Tumor Necrosis Factor; Recombinant Proteins; Tumor Necrosis Factor-alpha; Valerates

Con A-induced hepatitis (Con A-hepatitis) is a hepatitis model in which hepatic injury is supposed to be caused by cytokines from activated T cells. To elucidate the pathogenesis of this disease, we analyzed the roles of IFN-gamma and TNF-alpha using deficient mice of these cytokines. Development of hepatitis was reduced significantly in IFN-gamma(-/-) mice, while susceptibility of TNF-alpha(-/-) mice was not changed. Interestingly, apoptotic cell death was observed in the affected livers of control or TNF-alpha(-/-) mice, but not in those of IFN-gamma(-/-) mice. Fas mRNA expression was increased in the livers of hepatitis mice, but less abundantly in those of IFN-gamma(-/-) mice. Since apoptosis of liver cells was rarely observed in Con A-treated lpr/lpr mice, involvement of the Fas-Fas ligand system in this apoptotic process was suggested. These observations suggest that IFN-gamma plays a central role in Con A-hepatitis by activating Fas-induced apoptosis of liver cells.

Topics: Animals; Apoptosis; Chimera; Concanavalin A; fas Receptor; Gene Expression Regulation; Genetic Engineering; Hepatitis, Animal; Immune Tolerance; Interferon-gamma; Liver; Male; Mice; Mice, Inbred C57BL; Mice, Knockout; Plant Lectins; Tumor Necrosis Factor-alpha

Fas has been implicated in liver damage. The aim of this study was to investigate the role of its ligand to induce hepatocyte death and liver damage in T cell-dependent hepatitis.. Fas ligand-mediated lysis of primary hepatocytes from C57BL/6 wild-type, Fas ligand-deficient gld, and Fas-deficient lpr mice and concanavalin A-induced hepatitis in these mice were assessed.. Freshly isolated hepatocytes from wild-type or gld mice, but not those from lpr mice, were susceptible to Fas ligand-mediated lysis. When concanavalin A was intravenously administered into wild-type mice, they developed acute hepatic injury with massive degenerative changes in hepatocytes. In contrast, both gld and lpr mice had lower aminotransferase levels with milder histological changes. Reverse-transcription polymerase chain reaction and flow cytometric analysis showed that Fas ligand was induced in the liver shortly after the concanavalin A injection and was predominantly expressed on intrahepatic T cells. Administration of monoclonal antibody neutralizing mouse Fas ligand could reduce the aminotransferase increase.. The results indicate that Fas ligand plays a role in the T cell-dependent hepatitis induced by concanavalin A administration.

Topics: Alanine Transaminase; Animals; Antibodies, Monoclonal; Aspartate Aminotransferases; Cells, Cultured; Concanavalin A; DNA Primers; Fas Ligand Protein; fas Receptor; Female; Hepatitis, Animal; Liver; Membrane Glycoproteins; Mice; Mice, Inbred C57BL; Mice, Knockout; Mice, Nude; Mice, SCID; Polymerase Chain Reaction; T-Lymphocytes

The significance of tumor necrosis factor receptor 1 (TNFR1) for TNF function in vivo is well documented, whereas the role of TNFR2 so far remains obscure. In a model of concanavalin A (Con A)-induced, CD4+ T cell-dependent experimental hepatitis in mice, in which TNF is a central mediator of apoptotic and necrotic liver damage, we now provide evidence for an essential in vivo function of TNFR2 in this pathophysiological process. We demonstrate that a cooperation of TNFR1 and TNFR2 is required for hepatotoxicity as mice deficient of either receptor were resistant against Con A. A significant role of TNFR2 for Con A-induced hepatitis is also shown by the enhanced sensitivity of transgenic mice overexpressing the human TNFR2. The ligand for cytotoxic signaling via both TNF receptors is the precursor of soluble TNF, i.e. transmembrane TNF. Indeed, transmembrane TNF is sufficient to mediate hepatic damage, as transgenic mice deficient in wild-type soluble TNF but expressing a mutated nonsecretable form of TNF developed inflammatory liver disease.

Topics: Animals; Antigens, CD; Concanavalin A; Hepatitis, Animal; Immunity, Innate; Interferon-gamma; Liver Failure, Acute; Male; Membrane Proteins; Mice; Mice, Inbred C57BL; Mice, Inbred CBA; Mice, Knockout; Receptors, Tumor Necrosis Factor; Receptors, Tumor Necrosis Factor, Type I; Receptors, Tumor Necrosis Factor, Type II; Tumor Necrosis Factor-alpha

A single intravenous injection of concanavalin A (Con A) induces T-cell activation and an acute hepatitis in mice. This study investigated the role of interferon gamma (IFN-gamma) in the pathogenesis of this hepatitis model. Striking increases in the plasma levels of various cytokines, including tumor necrosis factor (TNF), interleukin-2 (IL-2), and IFN-gamma, were detected before the increase in plasma aminotransferase levels induced by Con A injection. TNF levels peaked within 2 hours, whereas IFN-gamma levels peaked at 6 hours after Con A injection. In contrast to a sharp peak of TNF levels, high IFN-gamma levels were detected for a more prolonged period. Passive immunization with anti-IFN-gamma monoclonal antibody (MAb) conferred a dose-dependent protection against liver injury in this model. This protection was observed when anti-IFN-gamma MAb was administered at least 30 minutes before Con A injection but not when given 1 hour after Con A injection. The protection from Con A-induced hepatitis was also induced by administration of rIL-6 before Con A injection. rIL-6 treatment induced significant albeit incomplete inhibition of IFN-gamma and TNF production, whereas this regimen did not affect IL-2 production. Despite striking protective effects of rIL-6 or anti-IFN-gamma MAb, comparable levels of cellular (both T cell and polymorphonuclear cell) infiltration were detected in liver sections from animals untreated, or treated with either rIL-6 or anti-IFN-gamma MAb. Moreover, electron microscopic examination showed that infiltrating T cells exhibited a blastoid appearance in all groups. These results indicate that IFN-gamma plays a critical role in the development of Con A-induced acute hepatitis and suggest that IL-6 administration can regulate the manifestation of hepatitis through mechanisms including the reduced production of inflammatory cytokines such as IFN-gamma.

Topics: Animals; Antibodies, Monoclonal; Concanavalin A; Cytokines; Disease Models, Animal; Female; Hepatitis, Animal; Interferon-gamma; Interleukin-2; Interleukin-6; Lymphocyte Activation; Mice; Mice, Inbred BALB C; Microscopy, Electron; Recombinant Proteins; T-Lymphocytes; 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

This study investigates the molecular mechanisms underlying the induction of and protection from T cell activation-associated hepatic injury. When BALB/c mice were given a single intravenous injection of concanavalin A (Con A) (> or = 0.3 mg/mouse), they developed acute hepatic injury as assessed by a striking increase in plasma transaminase levels within 24 h. Histopathologically, only the liver was injured while moderate infiltration of T cells and polymorphonuclear cells occurred in the portal areas and around the central veins. The induction of hepatic injury was dependent on the existence as well as the activation of T cells, as untreated BALB/c nu/nu mice or BALB/c mice pretreated with a T cell-specific immunosuppressive drug, FK506, failed to develop disease. Significant increases in the levels of various cytokines in the plasma were detected before an increase in plasma transaminase levels. Within 1 h after Con A injection, tumor necrosis factor (TNF) levels peaked, this being followed by production of two other inflammatory cytokines, interleukin 6 (IL-6) and IL-1. Passive immunization with anti-TNF but not with anti-IL-1 or anti-IL-6 antibody, conferred significant levels of protection. Moreover, administration of rIL-6 before Con A injection resulted in an IL-6 dose-dependent protection. A single administration of a given dose of rIL-6 completely inhibited the release of transaminases, whereas the same regimen induced only 40-50% inhibition of TNF production. More than 80% inhibition of TNF production required four consecutive rIL-6 injections. These results indicate that: (a) TNFs are critical cytokines for inducing T cell activation-associated (Con A-induced) hepatitis; (b) the induction of hepatitis is almost completely controlled by rIL-6; and (c) rIL-6 exerts its protective effect through multiple mechanisms including the reduction of TNF production.

Topics: Animals; Antibodies; Concanavalin A; Female; Hepatitis, Animal; Humans; Immunohistochemistry; Interleukin-6; Liver; Lymphocyte Activation; Mice; Mice, Inbred BALB C; T-Lymphocytes; Tumor Necrosis Factor-alpha