Induction of thymocyte apoptosis by systemic administration of concanavalin A in mice: role of TNF-alpha, IFN-gamma and glucocorticoids

Administration of concanavalin A (Con A) is a well-established model of acute immune-mediated hepatitis. Here, we demonstrate that intravenous injection of Con A in mice induces profound thymic atrophy. Compared to liver damage, the kinetics of Con A-induced thymic atrophy is slower and more prolonged; the nadir in thymocyte number is reached 4 days after Con A injection, whereas peak transaminase levels are observed at 12-24 h. Marked alterations in the ratio of CD4+ and CD8+cells in the thymus and spleen and significantly increased rates of thymocyte and splenocyte apoptosis are observed. Neutralization of the cytokines TNF-alpha or IFN-gamma, which protects mice from Con A-induced hepatitis, prevents thymic atrophy as well as alterations in CD4+ and CD8+ cell numbers and apoptosis rates. However, neither TNF-alpha nor IFN-gamma are detectable in thymocyte lysates after Con A injection, whereas both cytokines are present in liver, spleen and serum. Administration of the glucocorticoid receptor antagonist mifepristone does not prevent thymic atrophy, thus ruling out a possible contribution of endogenous glucocorticoids. Con A-induced thymic atrophy is accompanied by down-regulation of Bcl-2 expression in the thymus, which is prevented by neutralization of TNF-alpha or IFN-gamma. These data demonstrate that the thymus is a critical target organ of Con A-induced inflammation; the effects of Con A on the thymus are mediated by extrathymic production of TNF-alpha and IFN-gamma, but not by glucocorticoids.     

Regulation of the development of acute hepatitis by IL-23 through IL-22 and IL-17 production

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.     

Bimodal role of endogenous interleukin-6 in concanavalin A-induced hepatitis in mice

Acute concanavalin A (Con A)-induced hepatitis in mice is an animal model for hepatic injury induced by activated T cells. The evolution of hepatic involvement can be followed from hour to hour by measuring serum transaminase levels. We investigated the possible role of endogenous interleukin-6 (IL-6) in this model. We found serum IL-6 levels and splenic IL-6 mRNA during Con A-induced hepatitis to be significantly lower in interferon-gamma (IFN-gamma)-deficient mice, which are resistant against the Con A-induced syndrome, than in wild-type ones, suggesting that systemic IL-6 production favors development of hepatic injury. However, IL-6-deficient mice proved to be more susceptible to the disease than wild-type mice, indicating that endogenous IL-6 plays a predominantly hepatoprotective role. Experiments in which wild-type mice were treated with anti-IL-6 antibodies, before or after Con A challenge, allowed us to reconcile these contrasting observations. The antibody injections resulted in a biphasic alteration of serum IL-6 levels, initial neutralization being followed by rebound increased levels due to accumulation of IL-6 in the form of antigen-antibody complexes. The effect of antibody on disease severity differed depending on the time of injection. Antibody injection at 2.5 h post Con A resulted in delayed disease manifestation, whereas treatment initiated before Con A resulted in accelerated disease. We conclude that endogenous IL-6 plays a bimodal role. IL-6 present before Con A challenge as well as that induced in the very early phase after Con A injection triggers hepatoprotective pathways. Continuation of IL-6 production beyond this early phase, by some other pathway, seems to be harmful to hepatocytes.     

MicroRNA let-7a Ameliorates Con A-Induced Hepatitis by Inhibiting IL-6-Dependent Th17 Cell Differentiation

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.     

Deficient liver regeneration after carbon tetrachloride injury in mice lacking type 1 but not type 2 tumor necrosis factor receptor.

Signaling by tumor necrosis factor type 1 receptor (TNFR-1) is required for the initiation of liver regeneration after partial hepatectomy. Using knockout mice that lack either TNFR-1 or TNFR-2, we determined whether signaling through TNF receptors is important for liver injury and hepatocyte proliferation induced by carbon tetrachloride (CCl4). Lack of TNFR-1 inhibited hepatocyte DNA synthesis after CCl4 injection. At 44 hours after the injection, replication of hepatocytes in TNFR-1 was 50% to 90% lower than in wild-type (WT) animals, depending on the dose injected. In WT animals, hepatocyte replication was essentially completed by 4 days after CCl4 injection, but replication at a low level persisted in TNFR-1 mice for at least 2 weeks. TNFR-1 knockout mice had little detectable NF-kappa B and STAT3 binding during the first 5 hours after CCl4, high plasma TNF, and reduced levels of plasma interleukin (IL)-6 and liver IL-6 mRNA. Injection of IL-6 30 minutes before CCl4 administration corrected the deficiency of hepatocyte replication at 44 hours and restored STAT3 binding to normal levels. In contrast, mice lacking TNFR-2 did not differ significantly from WT mice in NF-kappa B and STAT3 binding, IL-6 and TNF levels, or hepatocyte replication. Although AP-1 binding was induced in WT TNFR-1 and TNFR-2 knockout mice, binding in TNFR-2 knockouts was lower than in WT mice. C/EBP binding was much lower in TNFR-1 and TNFR-2 knockout mice than in WT mice. As assessed by morphological analysis and alanine aminotransferase levels, the acute injury caused by CCl4 appeared to be similar in the three groups of animals, but subsequent regeneration was impaired in mice lacking TNFR-1. We conclude that a TNFR-1 signaling pathway involving NF-kappa B, IL-6, and STAT3 is an important component of the hepatocyte mitogenic response induced by CCl4 injury in mouse liver.     

经肝动脉同种异体脂肪干细胞移植治疗小鼠自身免疫性肝炎的实验研究

目的: 探讨同种异体脂肪干细胞经肝动脉移植对刀豆素A(Con A)诱导的小鼠自身免疫性肝炎的治疗价值。方法: Con A注射法建立小鼠自身免疫性肝炎模型。将40只小鼠随机分为4组:阴性对照组、经肝动脉移植组、经尾静脉移植组(移植脂肪干细胞2×10⁶)和阳性对照组。阳性对照组应用环磷酰胺腹腔注射。观察移植前后肝脏组织病理病变情况、血清炎症指标和肝功能指标的改变。结果: 经肝动脉移植组和经尾静脉移植组行脂肪干细胞移植后全部存活,未发生明显并发症;经肝动脉移植组血清炎症指标(IFN-γ、IL-4和IL-5)及血清天冬氨酸氨基转移酶、丙氨酸氨基转移酶及碱性磷酸酶明显下降(P<0.05);经尾静脉移植组血清炎症指标及血清肝功能指标有所下降,但无显著差异。经肝动脉移植组及阳性对照组病理学上改善最明显,肝内炎症细胞浸润及肝细胞坏死均明显减少。结论: 同种异体脂肪干细胞移植有助于改善Con A介导的小鼠自身免疫性肝炎,经肝动脉途径可提高脂肪干细胞肝移植的效能。     

Concanavalin A-induced hepatitis in mice is prevented by interleukin (IL)-10 and exacerbated by endogenous IL-10 deficiency

One single intra-venous (i.v.) injection of Concanavalin A (Con A) into mice provokes a cell-mediated immunoinflammatory hepatitis. We have presently evaluated the immunopharmacological effects of exogenous interleukin (IL)-10 and the role of endogenous IL-10 in this model by using exogenous IL-10, anti-IL-10 monoclonal antibody (mAb) and mice with disrupted IL-10 gene (IL-10 KO mice). Whilst exogenous IL-10 administered in a prophylactic (1 h prior to Con A) and even "early" therapeutic fashion (30 min after Con A) reduced the elevation of transaminase activities in plasma in a dose-dependent manner, observed in control mice, these biochemical markers of liver injury were significantly increased both in IL-10 KO mice as well as in those receiving anti-IL-10 mAb. Interestingly, doses of Con A lower than 20 mg/kg that were only capable of inducing slight serological signs of hepatitis in mice, exerted marked hepatitic effects when administered to either anti-IL-10 mAb-treated mice or to IL-10 KO mice. The disease modulating effects of exogenous IL-10 and either genetical or pharmacologically-induced IL-10 deficiency were associated with profound and opposite modifications of the Con A-induced increase in the circulating levels of IFN-gamma and TNF-alpha. Relative to control animals, the blood levels of these cytokines were diminished in IL-10-treated mice and augmented in both IL-10 KO mice and anti-IL-10 mAb-treated mice. These results prove the physiological antiinflammatory role of endogenous IL-10 in Con A induced hepatitis and the beneficial effects of IL-10 treatment to prevent this condition.     

Blockade of IL-33 ameliorates Con A-induced hepatic injury by reducing NKT cell activation and IFN-γ production in mice

IL-33, a recently described member of the IL-1 family, has been identified as a cytokine endowed with pro-Th2 type functions. To date, there are only limited data on its role in physiological and pathological hepatic immune responses. In this study, we examined the role of IL-33 in immune-mediated liver injury by exploring the model of concanavalin A (Con A)-induced hepatitis. We observed that the level of IL-33 expression in the liver was dramatically increased at 12?h after Con A injection. Meanwhile, ST2L, the receptor of IL-33, was significantly up-regulated in lymphocytes including T and natural killer T (NKT) cells, especially in NKT cells. Moreover, administration of recombinant IL-33 exacerbated Con A-induced hepatitis, while pretreatment of IL-33-blocking antibody or psST2-Fc plasmids showed a protective effect probably by inhibiting the activation of late stage of T cells and NKT cells and also decreasing the production of the cytokine IFN-??. Furthermore, depletion of NKT cells abolished the protective effect of IL-33-blocking antibody, and IL-33 failed to exacerbate Con A-induced hepatitis in IFN-??^?/? mice. These data suggested the critical roles of NKT cells and IFN-?? in the involvement of IL-33 in Con A-induced hepatitis. Blockade of IL-33 may represent a novel therapeutic strategy through IL-33/ST2L signal to prevent immune-mediated liver injury.     

Leptin deficiency, not obesity, protects mice from Con A-induced hepatitis

Leptin-deficient ob/ob mice are protected from Con A-induced hepatitis. However, it is unclear whether leptin deficiency or obesity itself is responsible for this protection. To address this question, wild-type (WT) obese mice with high serum leptin levels were generated by injection of gold thioglucose (WT GTG). Both Con A-injected WT and WT GTG mice developed hepatitis, whereas no hepatic damage was observed in ob/ob mice. Moreover, TNF-alpha and IFN-gamma levels as well as expression of the activation marker CD69 were elevated in liver mononuclear cells of WT and WT GTG mice, but not in ob/ob mice following administration of Con A. The liver of WT and WT GTG mice had the same percentage of NK T cells, a lymphocyte population involved in Con A-induced hepatitis. This population decreased equally in both WT and WT GTG mice after Con A injection. In contrast, the liver of ob/ob mice contained 50% less NK T cells compared to WT and WT GTG mice. Furthermore, no decrease in NK T cells was observed in Con A-injected ob/ob mice. We conclude that leptin-deficiency, not obesity, is responsible for protection from Con A-induced hepatitis.     

CCL3/MIP-1alpha is pro-inflammatory in murine T cell-mediated hepatitis by recruiting CCR1-expressing CD4(+) T cells to the liver

T cell-mediated hepatitis is associated with significant morbidity and mortality worldwide. Levels of C-C chemokine ligand 3/macrophage inflammatory protein-1alpha (CCL3/MIP-1alpha) are elevated in the serum of patients with T cell-mediated liver diseases, but its role is not fully understood. Con A-induced hepatitis is a murine liver-specific inflammation mediated by activated T cells and is driven by an up-regulation of the hepatic expression of IFN-gamma. In this study, we have used CCL3/MIP-1alpha gene-deficient mice to examine the role of CCL3/MIP-1alpha in the pathogenesis of Con A-induced hepatitis. We demonstrate a novel pro-inflammatory role for CCL3/MIP-1alpha since CCL3/MIP-1alpha deficiency significantly attenuated hepatic injury, both biochemically and histologically. Moreover, the recruitment of CCR1-expressing CD4(+) T cells to the liver after Con A treatment was strikingly attenuated by CCL3/MIP-1alpha deficiency. Correspondingly, hepatic IFN-gamma produced by the recruited CD4(+) T cells was significantly reduced by CCL3/MIP-1alpha deficiency during Con A-induced hepatitis. Furthermore, treatment of mice with a dual CCR1/CCR5 peptide antagonist, methionylated RANTES, also markedly reduced hepatic injury and decreased the numbers of CD4(+) T cells within the liver producing IFN-gamma during Con A-induced hepatitis. These findings demonstrate that blockade of the CCL3/MIP-1alpha-CCR1 pathway may represent a novel therapeutic target for treating T cell-mediated liver diseases.     

A comparison between hormone levels and T lymphocyte function in young and old rats.

There is growing evidence that changes in hormone secretion during aging can alter some functions of the thymus. In order to identify further relationships between changes in endocrine and thymic function during aging we measured the circulating levels of prolactin (PRL), growth hormone (GH) thyrotropin (TSH) thyroxine (T4) and triiodothyronine (T3), and determined their relation to a number of T lymphocyte functional indices in 20 young (4 months) and 20 old (28-30 months) Long-Evans male rats. Half of the young and old rats were chronically cannulated in order to obtain sequential plasma samples for measuring PRL, GH and TSH by RIA. Total T4 and T3 were measured by RIA in the trunk serum of all animals. Thymus and spleen cell cultures from each rat were established and used to assess the ability of thymocytes to respond to mitogens and lymphokines as well as to measure the levels of IL-2 production by splenic lymphocytes. Mean plasma GH and serum T4 were significantly lower in old as compared to young rats (P less than 0.05 and P less than 0.001 for GH and T4, respectively), while PRL, TSH and T3 did not show significant age changes. Thymic cell number showed a 100-fold decrease in old as compared to young animals (10.6 +/- 3.0) x 10(6) vs. (898 +/- 98) x 10(6) cells, respectively) whereas no age change was detected in spleen cell numbers. The proliferative response of thymocytes exposed to Con A, IL-1, Con A + IL-1 or Con A + IL-2, was consistently higher in young than in old animals while the capacity of splenocytes to release IL-2 in response to Con A was not statistically different in young and old rats. The age changes in serum T4 showed a significant correlation with those in thymocyte count as well as with those in IL-1 + Con A- and IL-2 + Con A-induced thymocyte proliferation. Plasma GH showed a significant inverse correlation with splenocyte IL-2 release. Our results suggest, although do not prove, that the lower T4 and GH secretion that typically develops in old rats may play a causal role in some of the changes that occur in thymic-dependent functions during aging.     

T淋巴细胞内三磷酸肌醇传递ConA诱导的增殖信号

本文研究了不同浓度ConA诱导的人外周血T淋巴细胞内1-磷酸肌醇(IP_1),2-磷酸肌醇(IP_2),3-磷酸肌醇(IP_3)的变化。结果显示,不同剂量ConA刺激早期,细胞内IP_3、IP_2及IP_1水平均有所增高,尤以IP_3水平增高最为显著,与对照组比较增加率为107％,但各剂量组间无差异。PHA慢性处理后IL—2R表达阳性的T细胞,ConA不引起IP_3及相关产物的变化,但用抗IL-2R的单克隆抗体(抗Tac McAb)封闭T细胞的IL—2R后,ConA刺激可使细胞内IP_3水平明显增加。这提示:IP_3可能作为第二信使介导了ConA诱导的细胞增殖分化,但IL-2R表达可抑制丝裂原信息经IP_3途径传递,这种抑制作用可为抗Tac McAb所消除。     

Estrogen therapy for hepatectomy patients with poor liver function?

Estrogen is well known to promote liver regeneration after partial hepatectomy. Administration of estradiol prior to partial hepatectomy also induces increased activity of DNA synthesis. Endogenous aromatase plays a key role in the conversion of testosterone to estradiol. The aromatase activity was induced by IL-6, which is a key factor for liver regeneration. It has been reported that IL-6 interacts with gp80/130 receptor and regulates the STAT1/3 pathway to induce DNA synthesis in hepatocyte. The IL-6 induced aromatase activity results in increased serum estradiol level. This corresponded well with observation that estradiol was elevated after partial hepatectomy. Therefore, it is very likely that estradiol and IL-6 synergize in stimulation of hepatocyte proliferation during liver regeneration. We propose that a short-term estradiol treatment may be beneficial for patients with poor liver function after hepatectomy.     

FXR通过调节促甲状腺素胚胎因子减轻Con A诱导的自身免疫性肝炎病变

 目的：观察法尼酯衍生物X受体（farnesoid X receptor,FXR)-促甲状腺素胚胎因子（thyrotropin embryonic factor,TEF）通路在自身免疫性肝炎模型小鼠肝损害中的作用,探讨FXR-TEF通路改善自身免疫性肝炎的部分可能机制。方法：检测FXR在伴刀豆球蛋白A (concanavalin A, Con A) 诱导的肝炎（Con A-induced hepatitis, CIH）小鼠肝脏的表达;检测FXR激活对TEF表达的影响;观察C57BL/6小鼠和鹅去氧胆酸（chenodeoxycholic acid,CDCA）激活FXR的CIH小鼠肝脏病理、肝脏酶学及炎症因子变化。结果：FXR在CIH小鼠中低表达;CDCA激活FXR的C57BL/6小鼠TEF表达上调;FXR被激活的CIH小鼠的肝损害较轻,FXR激活可减轻肝脏炎症因子释放。结论：CDCA激活FXR能减轻CIH引起的肝功能损害和炎症反应。FXR激活使TEF上调。FXR可能是自身免疫性肝炎的保护因素,其保护作用可能是通过TEF来实现的。激活FXR可能成为治疗自身免疫性肝炎的一个途径。     

Production of a monoclonal antibody that defines the alpha-subunit of the feline IL-2 receptor.

A mAb, termed 9F23, to feline Con A-stimulated PBMC was prepared to characterize feline IL-2R. 9F23 was identified by FACS studies, which showed that the antigen was expressed at a high density on Con A-induced feline T cell blasts while 9F23 binding was not detected on nonactivated PBMC or the Crandell feline kidney cell line CRFK. Chemical crosslinking of 125I-IL-2 to membrane IL-2Rs on Con A-stimulated feline PBMC under the low-affinity condition resulted in detection of a major 65-kDa band. 9F23 specifically immunoprecipitated the IL-2.IL-2R alpha complex in a cell extract; in contrast, neither anti-human IL-2R alpha H48 nor anti-mouse IL-2R alpha 7D4 reacted with the complex. Moreover, immunoprecipitation with 9F23 of the extract from surface-iodinated Con A-stimulated PBMC showed a major 50-55 kDa band. Furthermore, 9F23 had no effect on either IL-2-driven proliferation of the Con A-stimulated PBMC or IL-2 binding. Finally, the expression of feline IL-2R alpha on Con A-stimulated PBMC was up-regulated by addition of exogenous IL-2. Thus, 9F23 defines an epitope different from the IL-2 binding site on the alpha-subunit of feline IL-2R.