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.     

Co-appearance of autoantibody-producing B220 B cells with NKT cells in the course of hepatic injury

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.     

C-C chemokine ligand 2/monocyte chemoattractant protein-1 directly inhibits NKT cell IL-4 production and is hepatoprotective in T cell-mediated hepatitis in the mouse

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.     

Critical role of interleukin-17/interleukin-17 receptor axis in mediating Con A-induced hepatitis

Concanavalin A (Con A)-induced hepatitis is thought to be a T-cell-mediated disease with active destruction of liver cells. Interleukin (IL)-17 is a cytokine produced principally by CD4(+) T cells. However, whether IL-17/IL-17 receptor (IL-17/IL-17R)-mediated responses are involved in T-cell-mediated Con A-induced liver injury remains unclear. In this study, we found that IL-17 expression was highly elevated in liver tissues during Con A-induced hepatitis. The increased levels of IL-17 were paralleled with the severity of liver injury reflected by Alanine aminotransaminase and histological assay as well as the secretion of tumor necrosis factor (TNF)-α and IL-6. Blockage of IL-17 significantly ameliorated Con A-induced hepatitis, while overexpression of IL-17 systemically resulted in massive hepatocyte necrosis in mice. Furthermore, overexpression of an IL-17R immunoglobulin G1 fusion protein significantly attenuated liver inflammation after acute Con A treatment. High expression of IL-17R on Kupffer cells was also observed along with the production of cytokines including TNF-α and IL-6. Inhibition of Kupffer cells by gadolinium chloride completely prevented Con A-induced liver injury and cytokine release. Finally, IL-17-expressing CD4(+) T and natural killer T cells were greatly increased in Con A-injected mice compared with that in controls. Overall, our results indicate that IL-17R signaling is critically involved in the pathogenesis in Con A-induced hepatitis, and blockade of IL-17/IL-17R signaling pathway may represent a novel therapeutic intervention in human autoimmune-related 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.     

Sphingosine-1-phosphate receptor agonists suppress concanavalin A-induced hepatic injury in mice

T cell-mediated immune responses play a critical role in a variety of liver injuries including autoimmune hepatitis. Injection of concanavalin A (Con A) into mice mimics the histological and pathological phenotype of T cell-mediated hepatitis. Recent advances in host immune control of organ transplantation include the development of sphingosine-1-phosphate (S1P) receptor agonists such as FTY720, which alter lymphocyte homing but do not suppress host general immunity. Herein we examined the effect of the new S1P receptor agonist KRP-203 on the Con A-induced liver damage model. In normal liver lymphocytes of BALB/c mice, both FTY720 and KRP203 promoted lymphocyte sequestering from the liver to secondary lymph nodes and significantly reduced the number of liver lymphocytes (p<0.05). Based on this observation, KRP203 was employed in the Con A-induced hepatitis model. KRP203 markedly reduced the number of CD4(+) lymphocytes that infiltrate Con A-treated liver (p<0.05) and successfully reduced serum transaminase elevation (p=0.017), therefore protecting mice from Con A-induced liver injury. Interestingly this homing modulation less occurs in natural hepatic T cell homing through the chemokine receptor, CXCR4. Therefore, S1P receptor agonists preferentially target CXCR4(+)CD4(+) peripheral blood T lymphocytes and suppress the occurrence of Con A-induced hepatitis, suggesting their therapeutic usefulness against T cell-mediated hepatic injury.     

CD44-deficient mice exhibit enhanced hepatitis after concanavalin A injection: evidence for involvement of CD44 in activation-induced cell death

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.     

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.     

Galectin-9 Ameliorates Con A-Induced Hepatitis by Inducing CD4+CD25low/int Effector T-Cell Apoptosis and Increasing Regulatory T Cell Number

Background

T cell-mediated liver damage is a key event in the pathogenesis of many chronic human liver diseases, such as liver transplant rejection, primary biliary cirrhosis, and sclerosing cholangitis. We and other groups have previously reported that galectin-9, one of the β-galactoside binding animal lectins, might be potentially useful in the treatment of T cell-mediated diseases. To evaluate the direct effect of galectin-9 on hepatitis induced by concanavalin A (Con A) administration in mice and to clarify the mechanisms involved, we administered galectin-9 into mice, and evaluated its therapeutic effect on Con A-induced hepatitis.

Methodology/Principal Findings

Galectin-9 was administrated i.v. to Balb/c mice 30 min before Con A injection. Compared with no treatment, galectin-9 pretreatment significantly reduced serum ALT and AST levels and improved liver histopathology, suggesting an ameliorated hepatitis. This therapeutic effect was not only attributable to a blunted Th1 immune response, but also to an increased number in regulatory T cells, as reflected in a significantly increased apoptosis of CD4⁺CD25^low/int effector T cells and in reduced proinflammatory cytokine levels.

Conclusion/Significance

Our findings constitute the first preclinical data indicating that interfering with TIM-3/galectin-9 signaling in vivo could ameliorate Con A-induced hepatitis. This strategy may represent a new therapeutic approach in treating human diseases involving T cell activation.     

TH1 and TH2 cytokine inhibition by 3,5-bis(trifluoromethyl)pyrazoles,a novel class of immunomodulators

In order to discover novel immunomodulators for application in treating autoimmune diseases, a stable Jurkat transfectant was constructed in which luciferase reporter gene is driven by a full-length IL-2 promotor. A chemical library was screened to identify compounds that inhibited luciferase expression in Jurkat transfectants stimulated with PMA and ionomycin. A class of compounds (bis-trifluoromethyl pyrazole, BTPs) was identified from this screen. BTPs were shown to inhibit anti-CD3 and anti-CD28 antibody-induced IL-2 secretion, mixed lymphocyte reaction, and Con A-induced T cell proliferation in normal human peripheral blood T cells. In addition, mRNA levels of IL-4, IL-5, IL-9, IL-10, IL-13, IL-15, and IFN-gamma were markedly inhibited by BTPs in peripheral blood mononuclear cells stimulated by Con A as determined by multi-probe RNA protection assay. Furthermore, IL-2, IL-4, IL-5, and IFN-gamma secretion by Hut 78 cells or CD3(+) T cells stimulated with PMA plus ionomycin or anti-CD3 antibody plus PMA were inhibited in a concentration-dependent manner by BTPs. Therefore, BTPs inhibit a wide spectrum of cytokine production including TH1 and TH2 type cytokines. Taken together, these compounds may be useful for treating autoimmune diseases and organ transplant rejection.     

IL-6 prevents T cell-mediated hepatitis via inhibition of NKT cells in CD4+ T cell- and STAT3-dependent manners

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.     

Heterogeneity of eosinophil chemotactic factors produced by splenic T cells of Schistosoma japonicum-infected mice

Spleen cells of Schistosoma japonicum-infected mice produced eosinophil chemotactic factors (ECF-Ls) upon stimulation with soluble egg antigen preparation (SEA) and Con A, while spleen cells from uninfected mice produced ECF-L upon stimulation with Con A but not with SEA. Depletion of CD4⁺ T cells, but not of CD8⁺ T cells, almost completely removed Con A-induced ECF-L production. In contrast, depletion of CD8⁺ T cells completely abolished SEA-induced ECF-L production while depletion of CD4⁺ T cells did not, indicating that CD4⁺ CD8⁻ T cells and CD4⁻CD8⁺ T cells play essential roles for the production of Con A-induced ECF-L and SEA-induced ECF-L, respectively. Con-A-induced ECF-L had a high affinity to Con A-Sepharose but not to Procion Red agarose. In contrast, SEA-induced ECF-L bound to Procion Red agarose, but not to Con A-Sepharose. A gel permission HPLC analysis revealed that the apparent molecular weight of Con A-induced ECF-L and SEA-induced ECF-L was 16 kDa and 35 kDa, respectively. Both Con A-induced ECF-L and SEA-induced ECF-L had a similar isoelectric point (pI 3.5–3.6). These results indicate that selective stimulation of either CD4⁺ or CD8⁺ T cells of S. japonicum-infected mice produces heterogeneous ECF-L.     

Vγ4 γδ T cell-derived IL-17A negatively regulates NKT cell function in Con A-induced fulminant hepatitis

Con A-induced fulminant hepatitis is a well-known animal model for acute liver failure. However, the role of γδ T cells in this model is undefined. In this report, using TCR δ(-/-) mice, we demonstrated a protective role of γδ T cells in Con A-induced hepatitis model. TCR δ(-/-) mice showed significantly decreased levels of IL-17A and IL-17F in the Con A-treated liver tissue, and reconstitution of TCR δ(-/-) mice with wild-type (Wt), but not IL-17A(-/-), γδ T cells significantly reduced hepatitis, strongly suggesting a critical role of IL-17A in mediating the protective effect of γδ T cells. Interestingly, only Vγ4, but not Vγ1, γδ T cells exerted such a protective effect. Furthermore, depletion of NKT cells in TCR δ(-/-) mice completely abolished hepatitis, and NKT cells from Con A-challenged liver tissues of TCR δ(-/-) mice expressed significantly higher amounts of proinflammatory cytokine IFN-γ than those from Wt mice, indicating that γδ T cells protected hepatitis through targeting NKT cells. Finally, abnormal capacity of IFN-γ production by NKT cells of TCR δ(-/-) mice could only be downregulated by transferring Wt, but not IL-17(-/-), Vγ4 γδ T cells, confirming an essential role of Vγ4-derived IL-17A in regulating the function of NKT cells. In summary, our report thus demonstrated a novel function of Vγ4 γδ T cells in mediating a protective effect against Con A-induced fulminant hepatitis through negatively regulating function of NKT cells in an IL-17A-dependent manner, and transferring Vγ4 γδ T cells may provide a novel therapeutic approach for this devastating liver disease.     

A selective adenosine A2A receptor agonist, ATL-146e, prevents concanavalin A-induced acute liver injury in mice

BACKGROUND AND AIMS: Concanavalin A (Con A) activates T lymphocytes and induces CD4+ T cell-mediated hepatic injury in mice. Pro-inflammatory cytokines, such as tumor necrosis factor-alpha (TNF-alpha), interferon-gamma (IFN-gamma), and interleukin-6 (IL-6), are critical mediators in this experimental model. Activation of adenosine A2A receptors reduces the production of various pro-inflammatory cytokines and suppresses T cell activation. A selective adenosine A2A receptor agonist (ATL-146e) has been shown to be a potent inhibitor of inflammation by increasing intracellular cyclic AMP (cAMP) in leukocytes. The aim of the present study was to determine whether ATL-146e could ameliorate Con A-induced hepatic injury, reduction of pro-inflammatory cytokine production. METHODS: Balb/c mice were injected with 25mg/kg Con A with or without a single injection of ATL-146e (0.5-50 microg/kg), 5 min prior to Con A administration. Liver enzymes, histology, and serum levels of tumor necrosis factor-alpha, interferon-gamma, and interleukin-6 were examined. We also assessed the effects of ATL-146e on pro-inflammatory cytokine production with CD4+ T cell. RESULTS: Pretreatment with ATL-146e significantly reduced serum levels of liver enzymes (P<0.001). The serum pro-inflammatory cytokines were all increased after Con A administration and reduced to near normal levels by ATL-146e. ATL-146e also inhibited CD4+ T cell pro-inflammatory cytokine production. CONCLUSION: A selective adenosine A2A receptor agonist, ATL-146e, can prevent concanavalin A-induced hepatic injury that is presumably mediated by its anti-inflammatory properties.     

Nephronectin is upregulated in acute and chronic hepatitis and aggravates liver injury by recruiting CD4 positive cells

Nephronectin (Npnt) is an extracellular matrix protein known to play a critical role in kidney development; however, its physiological role in the liver remains elusive. Here we show that Npnt expression is upregulated in mouse models of both acute and chronic hepatitis induced by Concanavalin A (Con A) and 3,5-diethocarbonyl-1,4-dihydrocollidine (DDC), respectively. In both models, Npnt was localized in inflammatory foci and was mainly secreted from mesenchymal cells and in part by cholangiocytes. Interestingly, ectopic expression of Npnt in hepatocytes induced the development of granuloma-like cell clusters mainly composed of CD4⁺ T cells or NKT cells but did not induce apparent hepatitis. Furthermore, we found that Npnt exacerbated the Con A-induced acute hepatitis. These results indicate that Npnt plays an important role in the initiation of hepatitis by recruiting CD4⁺ T cells or NKT cells into the foci of inflammation. In addition, we reveal that Npnt expression is also upregulated in human hepatitis. Therefore, Npnt may be a potential therapeutic target for acute and chronic hepatitis.     

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.     

CD4+ CD25+ Foxp3+ regulatory T cells protect against T cell-mediated fulminant hepatitis in a TGF-beta-dependent manner in mice

Regulatory T cells (Tregs), which are characterized by expression of CD4, CD25, and Foxp3, play a crucial role in the control of immune responses to both self and non-self Ags. To date, there are only limited data on their role in physiological and pathological hepatic immune responses. In this study, we examined the role of hepatic Tregs in immune-mediated liver injury by using the murine Con A-induced hepatitis model. Con A treatment was associated with an increased number of Foxp3(+) Tregs in liver but not in spleen. Moreover, the expression levels of Foxp3, CTLA-4, glucocorticoid-induced TNF receptor, as well as the frequency of CD103 of Tregs were increased after Con A injection, being significantly higher in liver than in spleen. Depleting CD25(+) cells aggravated liver injury, whereas adoptively transferring CD25(+) cells or Tregs reduced liver injury in Con A-treated recipients. Con A treatment induced elevated serum levels and hepatic mononuclear mRNA expressions of TGF-beta, which were reduced by Tregs depletion. In addition, anti-TGF-beta mAbs blocked the suppressive function of Tregs from Con A-treated mice in vitro. Finally, TGF-beta receptor II dominant-negative mice, whose T cells express a dominant negative form of TGFbetaRII and therefore cannot respond to TGF-beta, had a higher mortality rate and severer liver injury than normal mice injected with the same dose of Con A. These results indicate that CD4(+)CD25(+) Tregs play an important role in limiting the liver injury in Con A-induced hepatitis via a TGF-beta-dependent mechanism.     

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.     

Lyt-2+ cells. Requirements for concanavalin A-induced proliferation and interleukin 2 production

The requirements for inducing Lyt-2+ T cell proliferation in response to concanavalin A (Con A) were examined. Purified Lyt-2+ or L3T4+ spleen cells of C57BL/6 origin were stimulated with Con A and syngeneic macrophages (MO) in the presence of monoclonal antibodies to T cell markers or to polymorphic determinants on major histocompatibility complex molecules, and assessed for the ability to proliferate and to produce interleukin (IL) 2. alpha I-Ab failed to inhibit the Con A response of Lyt-2+ cells at dilutions that significantly inhibited the response of L3T4+ cells. In contrast, alphaKb/Db or alpha Lyt-2.2 specifically inhibited the response of Lyt-2+ cells, but not L3T4+ cells. The ability of alpha Kb/Db and of alpha Lyt-2.2 to inhibit the response of Lyt-2+ cells was dependent upon the concentration of Con A. These data demonstrate that optimal triggering of T cell subsets to proliferate and to produce IL-2 in response to Con A requires interactions with the appropriate restricting major histocompatibility complex molecule. The role of accessory cells in Lyt-2+ Con A-induced proliferation and IL-2 production was also investigated. Purified Lyt-2+ cells and purified L3T4+ cells failed to respond to Con A in the absence of MO. IL-1 reconstituted the response when MO were limiting, but failed to restore the response of either Lyt-2+ or L3T4+ cells when T cells were rigorously purified to remove all MO. These results demonstrate that triggering Lyt-2+ T cells, like L3T4+ T cells, requires accessory cells, and that this does not merely reflect a requirement for IL-1 production. Thus, Con A-induced proliferation and IL-2 production by Lyt-2+ T cells requires intimate contact with accessory cells and interactions dependent upon the class I-restricting element.     

P2X7 receptors regulate NKT cells in autoimmune hepatitis

Adenine nucleotides induce danger signals in T cells via purinergic receptors, raising the question whether they exert similar effects on innate immunity. Here we show that micromolar concentrations of nicotinamide adenine dinucleotide (NAD) induce a rapid increase of annexin V staining in NKT cells in vitro, a response that requires expression of P2X(7)Rs. Consistent with this result, treatment of mice with NAD causes a temporary decrease of NKT cells in the liver and protects from Con A- and alpha-galactosylceramide-induced hepatitis, both of which require functional NKT cells. Resistance to liver injury is associated with decreased cytokine production by NKT cells in NAD-treated mice. In contrast, when NAD is injected into Con A- or alpha-galactosylceramide-primed mice, liver injury is exacerbated and cytokine production by NKT cells is increased. This effect is caused by P2X(7)R-mediated stimulation of activated NKT cells. In agreement, mice lacking P2X(7)Rs on lymphocytes suffer reduced liver injury, and animals lacking ADP-ribosyltransferase, the enzyme that uses NAD to attach ADP-ribosyl groups to cell surfaces, are also resistant to Con A-induced hepatitis. These results prompt the conclusion that engagement of P2X(7)Rs on NKT cells inhibits naive, while stimulating activated cells, resulting in suppression or stimulation of autoimmune hepatitis.