Essential role for neutrophil recruitment to the liver in concanavalin A-induced hepatitis

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.     

Curcumin protects mice against concanavalin A-induced hepatitis by inhibiting intrahepatic intercellular adhesion molecule-1 (ICAM-1) and CXCL10 expression

The effect of curcumin on liver injury caused by Concanavalin A (Con A) has not been carefully examined. This study was designed to evaluate the protective effect of curcumin on Con A-induced hepatitis in mice. Liver injured mice received curcumin by gavage at a dose of 200 mg/kg body weight before Con A intravenous administration. Curcumin was effective in reducing the elevated plasma levels of aminotransferases and the incidence of liver necrosis compared with Con A-injected control group. Enzyme-linked immunosorbent assay (ELISA) showed that curcumin suppressed proinflammatory cytokines such as tumor necrosis factor (TNF)-α, interferon (IFN)-γ, and interleukin (IL)-4 production in Con A-injected mice. The reduced severity of hepatitis in curcumin pretreated mice correlated with decrease in numbers of liver CD4(+) T cells but not CD8(+) T cells by immunohistochemical analysis. Furthermore, the expression levels of intercellular adhesion molecule-1 (ICAM-1) and the interferon-inducible chemokine CXCL10 in hepatic tissue were significantly decreased by curcumin pretreatment. In conclusion, curcumin pretreatment protects against T cell-mediated hepatitis in mice.     

Silymarin inhibits TNF-alpha-induced expression of adhesion molecules in human umbilical vein endothelial cells

Silymarin is known to have an anti-atherosclerotic activity, but the mechanism responsible for it remains unclear. Here, we demonstrate a possible mechanism involved in the anti-atherosclerotic activity of silymarin. Silymarin inhibited THP-1 cell adhesion to human umbilical vein endothelial cells (HUVECs). Silymarin also suppressed the TNF-alpha-induced protein and mRNA expression of adhesion molecules, such as VCAM-1, ICAM-1 and E-selectin, in HUVECs. Moreover, silymarin suppressed the TNF-alpha-induced DNA binding of NF-kappaB/Rel in HUVECs. Taken together, these results demonstrate that silymarin exerts an anti-atherosclerotic activity, at least in part, by inhibiting the expression of adhesion molecules.     

Short-form Ron receptor is required for normal IFN-gamma production in concanavalin A-induced acute liver injury

Abrogation of Ron receptor tyrosine kinase function results in defects in macrophage activation and dysregulated acute inflammatory responses in vivo. Several naturally occurring constitutively active alternative forms of Ron have been identified, including from primary human tumors and tumor cell lines. One of these alternative forms, short-form (SF) Ron, is generated from an alternative start site in intron 10 of the Ron gene that eliminates most of the extracellular portion of the receptor and is overexpressed in several human cancers. To test the physiological significance of SF-Ron in vivo, mice were generated that solely express the full-length form of Ron (FL-Ron). Our results show that elimination of the capacity to express SF-Ron in vivo leads to augmented production of IFN-gamma from splenocytes following stimulation ex vivo with either concanavalin A or anti-CD3/T cell receptor monoclonal antibody. Moreover, in a concanavalin A-induced murine model of acute liver injury, FL-Ron mice have increased production of serum INF-gamma and serum alanine aminotransferase levels and worsened liver histology and overall survival compared with wild-type control mice. Taken together, these results suggest for the first time that SF-Ron impacts the progression of inflammatory immune responses in vivo and further support a role for the Ron receptor and its various forms in liver pathophysiology.     

Preventive effects of soyasapogenol B derivatives on liver injury in a concanavalin A-induced hepatitis model

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.     

NF-kappaB, but not p38 MAP kinase, is required for TNF-alpha-induced expression of cell adhesion molecules in endothelial cells

In response to inflammation stimuli, tumor necrosis factor-alpha (TNF-alpha) induces expression of cell adhesion molecules (CAMs) in endothelial cells (ECs). Studies have suggested that the nuclear factor-kappaB (NF-kappaB) and the p38 MAP kinase (p38) signaling pathways play central roles in this process, but conflicting results have been reported. The objective of this study is to determine the relative contributions of the two pathways to the effect of TNF-alpha. Our initial data indicated that blockade of p38 activity by chemical inhibitor SB203580 (SB) at 10 microM moderately inhibited TNF-alpha-induced expression of three types of CAMs; ICAM-1, VCAM-1 and E-selectin, indicating that p38 may be involved in the process. However, subsequent analysis revealed that neither 1 microM SB that could completely inhibit p38 nor specific knockdown of p38alpha and p38beta with small interference RNA (siRNA) had an apparent effect, indicating that p38 activity is not essential for TNF-alpha-induced CAMs. The most definitive evidence to support this conclusion was from the experiments using cells differentiated from p38alpha knockout embryonic stem cells. We could show that deletion of p38alpha gene did not affect TNF-alpha-induced ICAM-1 and VCAM-1 expression when compared with wild-type cells. We further demonstrated that inhibition of NF-kappaB completely blocked TNF-alpha-induced expression of ICAM-1, VCAM-1 and E-selectin. Taken together, our results clearly demonstrate that NF-kappaB, but not p38, is critical for TNF-alpha-induced CAM expression. The inhibition of SB at 10 microM on TNF-alpha-induced ICAM-1, VCAM-1 and E-selectin is likely due to the nonspecific effect of SB.     

A novel accessory role of neutrophils in concanavalin A-induced hepatitis

Concanavalin A (Con A)-induced hepatitis has been investigated as a model of T cell-mediated liver injury, in which IFN-gamma plays an essential role by inducing apoptosis of liver cells. Since a large number of neutrophils infiltrate into the liver in the model, the role of neutrophils was investigated in this study. Con A hardly caused liver injury in neutrophil-depleted mice, as assessed as to the plasma alanine aminotransferase level as well as histochemistry. Neutrophil-depleted mice also failed to produce IFN-gamma. Intracellular IFN-gamma staining revealed that, among liver leukocytes, T and NK cells but not neutrophils are the main producers of IFN-gamma. Nylon wool-purified "T cells", however, failed to produce IFN-gamma in response to Con A in vitro, while the production was restored by the addition of neutrophils. Overall, this study suggests that neutrophils play a novel accessory role in IFN-gamma production in Con A-induced hepatitis.     

Hyperproduction of proinflammatory cytokines by WSX-1-deficient NKT cells in concanavalin A-induced hepatitis

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.     

Ginsenoside Rg1 attenuates concanavalin A-induced hepatitis in mice through inhibition of cytokine secretion and lymphocyte infiltration

The effect of ginsenoside Rg1 (Rg1) on hepatic damage caused by concanavalin A (Con A) has not been fully elucidated. This study was designed to evaluate the protective effect of Rg1 on Con A-induced hepatitis in mice and explore the potential mechanisms of this effect. C57BL/6 mice were divided randomly into the following four experimental groups: phosphate-buffered saline group, Rg1 group, Con A group, Con A + Rg1 group. Mice received Rg1 (20 mg/kg) 3 h before intravenous administration of Con A (15 mg/kg). Levels of alanine transaminase, aspartate transaminase and cytokine production were measured, the amount of phosphorylated IκBα and p65 were tested, the numbers of CD4⁺ and CD8⁺ T lymphocytes infiltrated in the blood, spleen and liver were calculated, intercellular adhesion molecule-1 (ICAM-1) and interferon-inducible chemokine-10 (CXCL-10) levels were measured and histological examination of the livers was conducted. Pretreatment with Rg1 markedly reduced the elevated levels of serum aminotransferase, ameliorated liver damage and suppressed proinflammatory cytokines secretion via inhibition NF-κB activity following Con A injection of mice. Furthermore, Rg1 administration reduced ICAM-1 and CXCL-10 mRNA expression in the liver as well as the number of CD4⁺ and CD8⁺ T lymphocytes infiltrating in the liver. Rg1 reduced the incidence of liver damage through inhibition of the proinflammatory response and suppressed the recruitment of CD4⁺ and CD8⁺ T lymphocytes to the liver. These data indicate that Rg1 represents a novel agent for the treatment of T lymphocyte-dependent liver injury.     

IL-37 overexpression enhances the therapeutic effect of endometrial regenerative cells in concanavalin A-induced hepatitis

Background aimsMesenchymal stromal cells and immunosuppressive factor IL-37 can both suppress concanavalin A (Con A)-induced hepatitis in mice. Endometrial regenerative cells (ERCs), novel types of mesenchymal-like stromal cells, possess powerful immunomodulatory effects and are effective in treating various diseases. The aim of this study was to explore the effects of ERCs in suppressing Con A-induced hepatitis and determine whether IL-37 overexpression could enhance the therapeutic effect of ERCs in this process.MethodsERCs were extracted from the menstrual blood of healthy female volunteer donors. The IL-37 gene was transferred into ERCs, and the expression of IL-37 in cells was detected by western blot and enzyme-linked immunosorbent assay. Hepatitis was induced by Con A in C57BL/6 mice that were randomly divided into groups treated with phosphate-buffered saline, ERCs, IL-37 or ERCs transfected with the IL-37 gene (IL-37-ERCs). Cell tracking, liver function, histopathological and immunohistological changes, immune cell proportions and levels of cytokines were measured 24 h after Con A administration.ResultsCompared with ERC or IL-37 treatment, IL-37-ERCs further reduced levels of liver enzymes (alanine aminotransferase and aspartate aminotransferase) and improved histopathological changes in the liver. In addition, IL-37-ERC treatment further reduced the proportions of M1 macrophages and CD4⁺ T cells and increased the proportion of regulatory T cells. Moreover, IL-37-ERC treatment resulted in lower levels of IL-12 and interferon gamma, and higher level of transforming growth factor beta.ConclusionsThe results of this study suggest that ERCs can effectively alleviate Con A-induced hepatitis. Furthermore, IL-37 overexpression can significantly enhance the therapeutic efficacy of ERCs by augmenting the immunomodulatory and anti-inflammatory properties of ERCs. This study may provide a promising strategy for treatment of T-cell-dependent hepatitis.     

Effects of thalidomide on the expression of adhesion molecules in rat liver cirrhosis

This study was to evaluate the effects of thalidomide on expression of adhesion molecules in liver cirrhosis. The cirrhosis was induced in Wistar rats by intraperitoneal injection of CCl(4), and thalidomide (10 mg/kg/day or 100 mg/kg/day) was given by intragastric administration for 8 weeks. Liver histopathology and immunohistochemistry were significantly improved and the expressions of ICAM-1, VCAM-1, E-selectin, and TNF-alpha mRNA and protein were decreased significantly in rats treated with a high dose of thalidomide. Close positive correlation was observed in the expression of the TNF-alpha mRNA and that of ICAM-1, VCAM-1, and E-selectin mRNA, respectively. These results indicate that thalidomide exerts its effect on the downregulation of adhesion molecules via TNF-alpha signaling pathway to inhibit liver fibrosis.     

Roles of platelets and macrophages in the protective effects of lipopolysaccharide against concanavalin A-induced murine hepatitis

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.     

Apolipoprotein A-II suppressed concanavalin A-induced hepatitis via the inhibition of CD4 T cell function

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.     

Temporal changes in the expression and distribution of adhesion molecules during liver development and regeneration

We have compared by immunocytochemistry and immunoblotting the expression and distribution of adhesion molecules participating in cell-matrix and cell-cell interactions during embryonic development and regeneration of rat liver. Fibronectin and the fibronectin receptor, integrin alpha 5 beta 1, were distributed pericellularly and expressed at a steady level during development from the 16th day of gestation and in neonate and adult liver. AGp110, a nonintegrin fibronectin receptor was first detected on the 17th day of gestation in a similar, nonpolarized distribution on parenchymal cell surfaces. At that stage of development haemopoiesis is at a peak in rat liver and fibronectin and receptors alpha 5 beta 1 and AGp110 were prominent on the surface of blood cell precursors. During the last 2 d of gestation (20th and 21st day) hepatocytes assembled around lumina. AGp110 was initially depolarized on the surface of these acinar cells but then confined to the lumen and to newly-formed bile canaliculi. At birth, a marked increase occurred in the canalicular expression of AGp110 and in the branching of the canalicular network. Simultaneously, there was enhanced expression of ZO-1, a protein component of tight junctions. On the second day postpartum, presence of AGp110 and of protein constituents of desmosomes and intermediate junctions, DGI and E-cadherin, respectively, was notably enhanced in cellular fractions insoluble in nonionic detergents, presumably signifying linkage of AGp110 with the cytoskeleton and assembly of desmosomal and intermediate junctions. During liver regeneration after partial hepatectomy, AGp110 remained confined to apical surfaces, indicating a preservation of basic polarity in parenchymal cells. A decrease in the extent and continuity of the canalicular network occurred in proliferating parenchyma, starting 24 h after resection in areas close to the terminal afferent blood supply of portal veins and spreading to the rest of the liver within the next 24 h. Distinct acinar structures, similar to the ones in prenatal liver, appeared at 72 h after hepatectomy. Restoration of the normal branching of the biliary tree commenced at 72 h. At 7 d postoperatively acinar formation declined and one-cell-thick hepatic plates, as in normal liver, were observed.     

Essential role of neutrophil mobilization in concanavalin A-induced hepatitis is based on classic IL-6 signaling but not on IL-6 trans-signaling

Neutrophil depleted mice are protected from concanavalin A-mediated hepatitis, showing that neutrophils are critical for cellular liver damage. Interleukin-6 has pro- and anti-inflammatory properties and mediates neutrophil recruitment in diseases such as rheumatoid arthritis. In classic signaling, interleukin-6 binds to the membrane-bound interleukin-6-receptor and initiates signaling via gp130. In interleukin-6 trans-signaling, the agonistic soluble interleukin-6-receptor can form a soluble interleukin-6/interleukin-6-receptor complex and stimulate cells which only express gp130 but no interleukin-6-receptor. Interleukin-6 trans-signaling was shown to be important for liver regeneration and development of liver adenomas. Here, we show that blocking classic interleukin-6 signaling but not interleukin-6 trans-signaling reduced concanavalin A-induced liver damage in mice, with reduced liver STAT3 phosphorylation and liver neutrophil accumulation. However, the level of neutrophil-attracting chemokine KC is only reduced by inhibition of interleukin-6 trans-signaling. Analysis of circulating neutrophils after concanavalin A challenge revealed that classic interleukin-6 signaling is required for the mobilization of blood neutrophils. Reduced neutrophil infiltration was accompanied by increased levels of hepatoprotective monocyte chemoattractant protein-1 and reduced level of hepatodestructive interleukin-4. Abrogated classic interleukin-6 signaling in concanavalin A-mediated hepatitis exhibited liver-protective effects indicating that interleukin-6 classic but not interleukin-6 trans-signaling is responsible for liver damage. Classic interleukin-6 signaling is required to mount an efficient neutrophilia during concanavalin A-induced immune response, which might have clinical implications in the regard that blocking global interleukin-6 signaling pathways is a treatment option in different chronic inflammatory diseases.     

Cell-cell adhesion in the embryonic chick: Partial purification of liver adhesion molecules from liver membranes

Polyspecific xenoantisera specific for embryonic chick liver cells have been used to develop an assay for the detection of embryonic chick liver adhesion molecules (LAM). The neutralization of the antiaggregation immune sera and subsequent semiquantitative assessment of the remaining activity is the basis for this assay. Embryonic chick liver membrane proteins have been solubilized using a number of detergents and the LAM activity has been partially purified using sucrose-gradient sedimentation, isoelectric focusing, gel filtration, affinity chromatography, and preparative polyacrylamide gel electrophoresis procedures. The isolated LAM appears to be an intrinsic, trypsin-resistant, membrane glycoprotein of approximately 65,000 daltons. The 65K preparation of LAM is an effective immunogen in generating an aggregation blocking xenoantiserum specific for embryonic chick liver cells. Purification of LAM to homogeneity has been hampered by an apparent lability by self-association or loss by adsorption of the more purified LAM antigen(s).     

CDK4 regulation by TNFR1 and JNK is required for NF-kappaB-mediated epidermal growth control

Nuclear factor kappaB (NF-kappaB) mediates homeostatic growth inhibition in the epidermis, and a loss of NF-kappaB function promotes proliferation and oncogenesis. To identify mechanisms responsible for these effects, we impaired NF-kappaB action in the epidermis by three different genetic approaches, including conditional NF-kappaB blockade. In each case, epidermal hyperplasia was accompanied by an increase in both protein levels and tissue distribution of the G1 cell cycle kinase, CDK4. CDK4 up-regulation required intact TNFR1 and c-Jun NH2-terminal kinase (JNK) function. Cdk4 gene deletion concomitant with conditional NF-kappaB blockade demonstrated that CDK4 is required for growth deregulation. Therefore, epidermal homeostasis depends on antagonist regulation of CDK4 expression by NF-kappaB and TNFR1/JNK.