Research Article

Gene Therapy (2003) 10, 1781–1790. doi:10.1038/sj.gt.3302067

CrmA gene expression protects mice against concanavalin-A-induced hepatitis by inhibiting IL-18 secretion and hepatocyte apoptosis

M Fujino1, M Kawasaki1, N Funeshima1, Y Kitazawa1, M Kosuga2, K Okabe3, M Hashimoto3, H Yaginuma3, K Mikoshiba3, T Okuyama2, S Suzuki1 and X-K Li1

  1. 1Department of Innovative Surgery, National Research Institute for Child Health and Development, Japan
  2. 2Department of Clinical Genetics and Molecular Medicine, National Center for Child Health and Development, Tokyo, Japan
  3. 3Laboratory for Developmental Neurobiology, RIKEN Brain Science Institute, Saitama, Japan

Correspondence: Dr X-K Li, Department of Innovative Surgery, National Research Institute for Child Health and Development, 3-35-31 Taishido, Setagaya-ku, Tokyo 154-8567, Japan

Received 20 September 2002; Accepted 7 March 2003.

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Abstract

Activated cytotoxic T-cell-mediated hepatocyte apoptosis via Fas/Fas-ligand and perforin/granzyme pathways are believed to involve the model of concanavalin A (ConA)-induced hepatitis. The purpose of the present study is to investigate whether the cytokine response modifier A (crmA) gene effectively inhibits the hepatocyte apoptosis of ConA-induced hepatitis. We examined survival rates, liver pathology, immune histological changes, and cytokine profiles from mice receiving the recombinant adenovirus vectors containing cre and/or crmA genes, transferred to the liver 3 days before ConA injection, and a crmA gene nonexpression control group. Injection of ConA into mice rapidly led to massive hepatocyte apoptosis, and infiltration of leukocytes, especially CD11b+ inflammatory cells. In contrast, liver damage was dramatically reduced in the mice that expressed the crmA gene. However, infiltration by CD4+ cells was not affected. The survival of the mice increased significantly to 100% in the treated group versus the control group. Furthermore, we demonstrated that interleukin (IL)-18 plays an important role in ConA-induced hepatitis, and that crmA expression significantly inhibited IL-18 secretion. Our results showed that the crmA gene effectively inhibits apoptosis induced by ConA hepatitis. This indicates a potential therapeutic usage of crmA for protection from cellular damage due to hepatitis.

Keywords:

hepatitis,concanavalin A, CrmA, interleukin-18, Adenovirus vector

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Introduction

T lymphocytes activated by viral antigens, autoantigens or allogenic stimulation are key effector cells for inducing liver damage during liver graft rejection or in all types of acute, chronic active hepatitis. Activated T cells exhibit direct cytotoxicity or release proinflammatory cytokines that mediate hepatocell ular death. Therefore, an ideal strategy for treating fulminant hepatitis would be to protect the hepatocytes against severe attack by the host's immune system by transducing antiapoptotic genes.

Recently, a new hepatitis model has been developed in which T-cell-dependent liver lesions were induced in mice by injecting them with the mitogenic plant lectin, concanavalin A (ConA).1 Further works have considered ConA-induced hepatitis to be an experimental mouse model of human autoimmune hepatitis.2 In this model, hepatocyte injury is associated with lymphocyte infiltration, suggesting immune reaction involvement. The assembly of activated CD4+ T cells in the liver results in a time-dependent release of interleukin (IL)-1beta, IL-2, IL-6, interferon (IFN)-gamma, and tumor necrosis factor (TNF)-alpha, resulting in Fas/Fas ligand (FasL) and/or perforin/granzyme-mediated liver cell death.3,4 Hence, hepatocyte apoptosis via cell-mediated cytotoxicity is believed to play an important role in ConA-induced hepatitis.5 The perforin-mediated pathway causes membrane damage and apoptosis of the target cell through granule exocytosis and release of potent cytolytic molecules, perforin and granzymes, from cytotoxic T lymphocyte (CTL) and natural killer (NK) cells.3 Furthermore, FasL-mediated lysis is affected by the interaction of membrane proteins expressed on cytolytic lymphocytes and on target cells and mediates target cell apoptosis.4 In addition, many reports confirmed that this model depends on the production of TNF-alpha and IFN-gamma, and the activation of T cells.6,7 Production of IFN-gamma from activated CD4+ T, CD8+ T lymphocytes, and NK cells depends on the availability of IFN-gamma-inducing cytokines: IL-12 and IL-18.8 IL-18, originally identified as an IFN-gamma-inducing factor, is a recently described cytokine. It is mainly produced by activated monocytes/macrophages and Kupffer cells, and shares structural similarities with IL-1beta. Both cytokines have a unique all-beta-pleated structure.9,10,11,12,13 IL-18 exerts several immunologic and regulatory functions; indeed, IL-18-deficient mice (IL-18-/-), despite normal IL-12 production, are unable to release IFN-gamma and demonstrate a defective NK and T helper 1 (Th1) activity in response to bacterial endotoxin (lipopolysaccharide, LPS).14 In vitro studies have shown that IL-18 potentiates IFN-gamma release induced by IL-12, upregulates FasL expression on NK cells, and enhances FasL-mediated Th1 cytotoxicity.9,10,11,12,13,14,15,16 Owing to its ability to induce TNF-alpha, IL-1beta, both CXC and CC chemokines, and the nuclear translation of nuclear factor-kappaB (NF-kappaB), IL-18 ranks with other proinflammatory cytokines as a likely contributor to systemic and local inflammation.9,10,11,12,13,14,15,16

Proteolytic activity of caspases can be blocked by CrmA, a cowpox virus-encoded serpin-like protease inhibitor.17 CrmA presumably enables the cowpox virus to inhibit apoptosis of infected cells and to block host inflammatory responses caused by IL-1beta, which is released from its inactive precursor form by caspase-mediated proteolysis.17,18 CrmA binds tightly to caspase-1, but at very high in vitro concentrations it can inhibit caspase-3, and may also act on other members of this family.19,20 Cells transfected with crmA have been shown to be resistant to apoptosis induction by granzyme B, a serine protease from cytotoxic cells whose substrate specificity resembles that of Ced-3-like cystein proteases.21,22 Previously, we demonstrated that the crmA gene could effectively decrease the activation of caspase-3, -8 and inhibit apoptosis in the anti-Fas antibody-induced lethal hepatitis model. However, except for the Fas/FasL pathway, for example, perforin and granzyme or the TNF-alpha/TNF-alpha receptor pathway in vivo, it is not known how crmA inhibits apoptosis. We undertook this study to investigate the protection effect of crmA against ConA-induced hepatitis. Our results show that the crmA gene effectively inhibits apoptosis, infiltration of leukocytes, and release of inflammatory cytokines induced by ConA hepatitis, and indicates a potential therapeutic use of crmA for protection from cellular damage due to hepatitis.

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Results

Recombinant adenovirus vector expressing the crmA gene using a Cre-loxP system

The on/off switch unit CALNLCrmA consisted of the CAG promoter, a stuffer, the authentic crmA gene, and a polyA signal (Figure 1a). The stuffer was made up of the neo-gene and another polyA sequence flanked by a pair of loxP sites to prevent downstream crmA gene expression (crmA – off structure). The CALNCrmA unit should initially express the neo-gene but not the crmA gene. However, after supplying a sufficient amount of functional Cre to the switching unit, the stuffer DNA is excised as circular DNA; then the CAG promoter and the crmA gene are joined together via a single loxP site. The resulting construction of the recombinant adenovirus expressed the crmA gene under the control of the CAG promoter (crmA – on construction).

Figure 1.
Figure 1 - Unfortunately we are unable to provide accessible alternative text for this. If you require assistance to access this image, please contact help@nature.com or the author

Recombinant adenovirus vector expressing the crmA gene using a Cre-loxP system. (a) Activation of the crmA gene in the CALNLCrmA unit by Cre recombinase. The on/off switch unit CALNLCrmA consisted of the CAG promoter, a stuffer, authentic crmA gene, and a polyA signal. CAG, the CAG promoter; Neo, neomycin-resistant gene; pA, polyadenylation signal; T7, T7-tag; CrmA, cytokine response modifier A; loxP, loxP sequence. Arrows show the orientation of the mRNA transcription. (b) Immunohistochemical and immunocytochemical study of CrmA. (b-a). Mouse liver expressing the T7-tag-fused CrmA protein, indicating that 80–90% of hepatocytes are positive after adenovirus infection. (b-b). Isolated mouse hepatocytes also were confirmed by an anti-T7-tag antibody. (c) Protein expression detected by immunoblotting demonstrated that CrmA is a 38 kDa protein and the T7-tag-positive control is 31 kDa. CrmA expression on either hepatocyte or nonparenchyma cells was detected by Western blotting. We injected 109 PFU of AxCALNLCrmA combined with 109 PFU of AxCANCre (CrmA+) or AxCALNLCrmA only (CrmA-) via the mouse tail vein.

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We detected the levels of liver-expressed CrmA by immune staining using an anti-T7-tag antibody, because AxCALNLCrmA expresses a fusion protein of T7-tag and CrmA. Figure 1b-a revealed that 80–90% of the hepatocytes are positive to T7-tag protein expression. For the control, we performed X-gal staining to identify E. coli beta-galactosidase activity in the gene-transfected livers, and compared the approximate percentage of the X-gal-positive cells to T7-tag CrmA-positive cells (data not shown). The expression level of the transfer gene on the isolated mouse hepatocytes was also confirmed by an anti-T7-tag antibody (Figure 1b-b). In addition, we confirmed crmA gene expression on either hepatocyte or non-parenchyma cells by Western blotting (Figure 1c).

CrmA protected ConA-induced hepatitis in mice

At 3 days after adenovirus administration, we injected the three groups of mice with a single dose of ConA, 1 mg/mouse, via the tail vein. Aside from the mice for the survival study, three mice from each group were killed 3, 6, and 18 h after ConA administration (Figure 2a). First, we examined the effect of CrmA on the survival of mice with ConA-induced hepatic failure (Figure 2b). When mice were administered 1mg/mouse of ConA, the mice began to die from 6 to 9 h after administration. In all, 90% of the mice from Group 2 and 100% of the mice from Group 3 died within 24 h. In contrast, expression of the crmA gene dramatically improved the survival of ConA-treated mice. Furthermore, this protection was dependent on CrmA expression levels that are regulated by AxCANCre from 107 to 109 PFU, indicating that adjustment of levels of the crmA gene expression could be achievable by the protective effect (data not shown).

Figure 2.
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CrmA protected mice from ConA-induced hepatitis. (a) Experimental design and sampling. (b) Effect of CrmA on the survival of mice with ConA-induced hepatic failure. (c) Serum AST and ALT levels decreased and were dependent on the crmA gene expression in the mouse liver. (d) Morphologic changes showed that the damage to the liver was dramatically reduced after CrmA expression. CrmA (-), 109 PFU of AxCALNLCrmA only; CrmA (+), 109 PFU of AxCALNLCrmA and 109 PFU of AxCANCre. **P<0.01 compared to the control sample, ConA(-).

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Serum aspartate aminotransferase (AST) and alanine aminotransferase (ALT) levels in the different groups showed that AST and ALT levels decreased and were dependent on crmA gene expression in the mouse livers (Figure 2c).

Histological examination of crmA nonexpressed livers at 18 h after ConA treatment showed many necrotic cells forming focal necrosis-like lesions (Figure 2d-a). In contrast, in crmA-expressed livers, morphologic changes produced a dramatic reduction in liver damage (Figure 2d-b), consistent with the low levels of serum ALT and AST seen in these mice.

CrmA prevention of ConA-induced hepatocyte apoptosis in mice

ConA treatment in Group 2 mice (AxCALNLCrmA adenovirus injected only) rapidly led to massive hepatocyte apoptosis and induced fulminant hepatic failure. Morphologic changes showed large areas of necrosis, disruption of the hepatic architecture, and apoptotic nuclei (Figure 2d-a). Extensive areas of DNA fragmentation were demonstrated by in situ 3' end labeling. In contrast, hepatic apoptosis was dramatically reduced in the mice expressing the crmA gene (Figure 3a). The apparent changes of internucleosomal DNA cleavage were confirmed after ConA injection, and were absent after CrmA expression (Figure 3b). Moreover, the presence of caspase-3, one of the key executioners of apoptosis, was confirmed by identifying its activated form, p17 subunits, and by a cleaved caspase-3 antibody (Figure 3c). In addition to in situ 3' end labeling and apparent internucleosomal DNA cleavage, CrmA expression resulted in a significant reduction of hepatocyte apoptosis.

Figure 3.
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CrmA protected against ConA-induced hepatocyte apoptosis in mice. (a) DNA fragmentation was demonstrated by the TUNEL method. At 18 h after injection of ConA, we observed a large number of TUNEL-positive cells in the CrmA-negative liver (CrmA(-)), but not in the CrmA-expressed liver (CrmA(+)). (b) The extent of the hepatocyte apoptosis was semiquantitatively measured by LM-PCR assay. The apparent changes of internucleosomal DNA cleavage were confirmed after ConA injection, and were absent after CrmA expression. (c) Activated caspase-3 form p17 was confirmed by identifying a cleaved caspase-3 antibody. CrmA (-), 109 PFU of AxCALNLCrmA only; CrmA (+), 109 PFU of AxCALNLCrmA and 109 PFU of AxCANCre. The data are representative of three separate experiments.

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Immunohistological examinations

To determine which types of cells infiltrating the liver were affected by CrmA transfection, we performed immunohistochemical analysis using cryostat sections of liver injected with ConA. Figure 4 shows that a large number of cells in the portal area were stained with an anti-mouse CD4 monoclonal antibody, whereas only a small number of cells exhibited the CD8+ phenotype. There was no difference between the livers with or without crmA expression. However, accumulation of CD11b-positive inflammatory cells was significantly decreased after crmA gene expression on the model ConA-induced hepatic livers.

Figure 4.
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Immunohistological examinations. A large number of cells in the portal area were stained with an anti-mouse CD4 monoclonal antibody, whereas only a small number of cells exhibited the CD8+ phenotype. There was no difference between subjects with or without crmA expression. However, CD11b-positive inflammatory cell accumulation was significantly decreased after the crmA gene expressed on the liver. CrmA (-), 109 PFU of AxCALNLCrmA only; CrmA (+), 109 PFU of AxCALNLCrmA and 109 PFU of AxCANCre. The data are representative of three separate experiments.

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CrmA expression protected hepatocytes from CTL cytotoxic attack

To test whether crmA expression prevents the adenovirus-infected hepatocytes from cell death induced by CTLs, we performed an in vitro CTL assay (Figure 5). T lymphocytes were isolated from mouse spleens that had been injected with the adenovirus vector 7 days before. These cells were cocultured with primary hepatocytes isolated from the mice, which had been already infected with both AxCALNLCrmA and AxCANCre (Group 1) or with AxCANCrmA only (Group 2). We observed a remarkable increase in AST and ALT concentrations in the medium when the effector T lymphocytes were cocultured with target hepatocytes of Group 2 at ratios of 10:1 and 50:1. However, the increase in the AST and ALT concentrations was significantly reduced when the T cells were cocultured with Group 1 hepatocytes. These results demonstrate that CrmA expression efficiently protects adenovirus-infected hepatocytes from CTL cytotoxic attack.

Figure 5.
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CrmA expression protected hepatocytes from CTL cytotoxic attack. A remarkable increase of AST and ALT concentrations in the medium occurred when the effector T lymphocytes, isolated from the spleen of the adenovirus vector-injected mice 7 days before, were cocultured with target hepatocytes of Group 2 at ratios of 10:1 and 50:1, while it was significantly reduced when the T cells were cocultured with Group 1 hepatocytes. CrmA (-), 109 PFU of AxCALNLCrmA only; CrmA (+), 109 PFU of AxCALNLCrmA and 109 PFU of AxCANCre. The data are representative of three separate experiments.

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Elevation of serum IL-18 and IFN-bold gamma cytokines level in ConA-induced mice

IL-18 has been shown to play a central role in LPS-induced liver injury in Propionibacterium acnes-primed mice.9,10,11,12,13 To test whether IL-18 was induced in ConA-induced hepatitis and the effect of the crmA expression, we examined and compared serum IL-18 levels after ConA administration. As shown in Figure 6a, serum IL-18 levels were increased 3, 6, and 18 h after ConA injection, indicating, as in LPS-induced liver injury, that IL-18 plays an important role in ConA-induced hepatitis. However, compared to the control group mice, after crmA expression, significantly lower levels of serum IL-18 were present at 3, 6, and 18 h. We also elevated the serum IFN-gamma levels, which were reported to increase levels of ConA-induced liver injury in different groups. We found that crmA expression inhibited serum IFN-gamma in the samples at 3, and 18 h, although there was no change at 6 h after ConA treatment (Figure 6b).

Figure 6.
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Elevation of serum IL-18 and IFN-gamma cytokine levels in ConA-induced mice. (a) Serum IL-18 levels were increased at 3, 6, and 18 h after ConA injection. However, after crmA expression, significantly lower levels of IL-18 were present. (b) Serum IFN-gamma levels were inhibited by CrmA expression at 3 and 18 h, although there was no measurable difference at 6 h after ConA treatment. CrmA (-), 109 PFU of AxCALNLCrmA only; CrmA (+), 109 PFU of AxCALNLCrmA and 109 PFU of AxCANCre. The data are representative of three separate experiments. **P<0.01 and *P<0.05 compared to the control sample, CrmA (-).

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Discussion

ConA induced acute hepatitis in mouse that is a model of T-lymphocyte-mediated liver injury. ConA-induced hepatitis is also thought to be a model of immunologically induced hepatocyte injury, and its histological features resemble those of viral- or drug-induced acute hepatitis in humans.1,7,25,26,27,28,29,30 In this mode, hepatocyte cell death induced by ConA is caused by necrosis and apoptosis. Hence, apoptosis and Th1 cytokine release were thought to be key factors. Several cytotoxic effector molecules are considered to be involved in the development of ConA-induced hepatitis. First is the perforin–granzyme system that is known to induce liver injury.3 Indeed, perforin knockout mice fail to develop ConA-induced hepatitis,3 suggesting that the perforin-granzyme system is essential for inducing lymphocyte-mediated hepatitis. Although the involvement of the Fas/FasL system in the induction of ConA-induced hepatitis is controversial,3,4 the system is likely to be one of the effector mechanisms for hepatocyte injury, particularly for T-cell-mediated hepatocyte injury. In fact, this hepatitis has been reported to be significantly milder in Fas-mutant lpr/lpr mice, and it is completely abrogated in gld/gld mice where FasL is defective or in mice pretreated with anti-FasL antibodies.4 In addition, normal hepatocytes express a significant amount of Fas, and the hepatocyte expression level of Fas mRNA is increased upon ConA stimulation.4 We also know that the stimulation of Fas on hepatocytes by anti-Fas antibodies causes severe damage to hepatocytes by apoptotic cell death.31 Earlier results showed that liver necroapoptosis induced by ConA is mediated by TNF-alpha and IFN-gamma.1,4,5,7,25,26,27,28,29,30,32 On TNF-alpha receptor crosstalk, the death domain of the TNF-alpha receptor 1 associates with TNF receptor 1-associated protein (TRADD), causing Fas-associated death domain (FADD) recruitment. FADD in turn activates upstream caspase-8, and the effector caspases involved in apoptosis, caspase-9 and -3.32,33,34,35 Therefore, this model is more suitable for investigating the action of crmA in the activated lymphocyte-induced hepatocyte apoptosis in hepatitis.

To explore the participation of CrmA expression using AxCALNLCrmA with a Cre-mediated switching system, we attempted to induce fulminant hepatitis by administrating ConA intravenously into the mice transduced with or without the crmA gene. Injection of ConA into the control mice resulted in rapid animal death by inducing massive hepatocyte apoptosis and necrosis (Figures 2b,d). Similarly, ConA-induced hepatotoxicity was recognizable by morphological evaluation, and DNA fragmentation, as well as by determination of highly elevated activities of serum AST and ALT (Figures 2c, d and 3). However, when we transduced CrmA, at which point the expression rate was approximately 80–90% of the whole hepatocyte (Figure 1b), no mouse death was induced with ConA injection (Figure 2b). Histological changes in the liver accompanied by ConA injection were prevented by CrmA transduction and serum AST and ALT also decreased dramatically in CrmA-transducted mice (Figures 2c,d).

The CrmA protein is the crmA gene encoding a 38 kDa protein whose amino-acid sequence is similar to those of members of the serpin superfamily.36 It expresses early during infection and is found in the cytoplasm of the infected cell. The CrmA protein presumably enables the cowpox virus to escape by host immune system.37 Initially, the CrmA protein was demonstrated to decrease the inflammatory response to viral infection by inhibiting caspase-1.37 The protein also inhibits caspase-8 and downstream caspases.36,37 In this study, we found that in CrmA-transfected mice, DNA fragmentation in the liver cell and increase of TUNEL-positive cells, accompanied by ConA injection, were inhibited (Figures 3a, b). Similarly, CrmA transfection inhibited the formation of an active structure (17 kDa) of caspase-3 from its proform (32 kDa) in the livers of ConA-injected mice (Figure 3c). Activated caspase-3 is one of the key proteins that directly activates the DNA fragmentation factor.38 Since CrmA behaves as a broad caspase inhibitor, it may prevent activation of caspases involved in liver cell death. In line with this view, a previous report36 showed that CrmA directly prevented activation of caspase-8 and cell death in mammalian cells. The inhibition of caspase-3 activation may not be induced by the direct effect of CrmA, but by the inhibition of upstream caspases, including caspases-1, -4, and -8.39 Therefore, the antiapoptotic activity of CrmA was initially achieved by the inhibition of caspase-8. This suggests that Fas-mediated and TNF-alpha-mediated apoptosis was completely inhibited by expressing the crmA gene, which is markedly different from the antiapoptotic action of Bcl-2.40 CrmA has also been reported to inhibit perforin/granzyme-B-mediated apoptosis.22,41 Furthermore, caspase-1 is known to be a key caspase in perforin/granzyme-B-mediated apoptosis, while Acetyl-Asp-Glu-Val-Asp-aldehyde (Ac-YVAD-CHO), a caspase-1-specific inhibitor, blocks perforin/granzyme-B-induced apoptosis.42 In the present study, in addition to improving the survival rate in mice with ConA-induced hepatitis, the induction of apoptosis and activation of caspase-3 were inhibited in CrmA-transfected mice. Therefore, in mice transfected with CrmA, caspase-3 activation inhibition may be one of the factors that improves the survival rate and prevents apoptosis.

Furthermore, in this study, we showed that CrmA expression prevented cytotoxic T lymphocyte attack on target cells (Figure 5). To confirm that CrmA expression is effective in protecting target cells from cytotoxic stimulation by effector cells, we performed CTL assay using adenovirus-infected hepatocytes both expressing and not expressing CrmA for target cells. Hepatocyte lysis was induced in adenovirus-infected hepatocytes by coculturing T cells stimulated by adenoviral infection. However, hepatocyte lysis was efficiently prevented by primary hepatocyte CrmA expression. These results demonstrate that exogenous CrmA expression protects hepatocytes from elimination by T-cell-mediated cytotoxic (eg Fas/FasL and perforin/granzyme) stimulation.

In addition to the contribution of perforin and FasL as effector molecules for cell-mediated hepatitis via apoptosis, various cytokines including TNF-alpha, IL-18, IL-1, and IFN-gamma produced by CD4 T cells and macrophages were reported to play crucial roles in the development of ConA-induced hepatitis.43 Since IFN-gamma drives cultured hepatocytes to apoptosis, enhances CTL differentiation, upregulates FasL and IL-2R expression on T lymphocytes,8 and mediates CD4/CD8-dependent fulminant hepatitis in transgenic mice expressing the hepatitis B surface antigen,44 this cytokine bodes well as a key liver damage mediator in mice challenged with ConA. IFN-gamma release from T lymphocytes and NK cells in response to mitogenic and antigenic stimulation is regulated by the availability of IFN-gamma-releasing cytokines, IL-12 and IL-18.8,9,10,11,12,13,45 Although in vitro studies have documented that IL-12 and IL-18 act synergistically to release IFN-gamma,45 Takeda et al14 recently showed a defective Th1-like response and NK cell activity in IL-18-/- mice. Since splenic lymphocytes derived from IL-18-/- mice release a normal amount of IL-12 but are unable to produce IFN-gamma in response to bacterial endotoxin, and because exogenous IL-18 fully restores this activity, this IFN-gamma-inducing factor has emerged as a key regulatory factor in modulating Th1-like cytokines.14

Previous in vivo studies have shown that IL-18, released in the liver by resident macrophages (Kupffer cells), plays a critical role in the development of liver damage induced by LPS in Propionibacterium acnes-sensitized mice.9,10,11,12,13 Recently, Fiorucci et al43 reported that IL-18 is critically involved in modulating Th1-like cytokines IFN-gamma and TNF-alpha release in mice injected with ConA. Like IL-1beta, IL-18 is synthesized as a 24-kDa bioinactive precursor (pro-IL-18) that lacks a typical signal peptide.11,12 Pro-IL-18, as pro-IL-1beta, is devoid of biological activity, and precursor amino acids must be cleaved to generate the 18-kDa bioactive molecules.9,10,11,12,13,14,15,16 The IL-1beta-converting enzyme (ICE), which processes pro-IL-1beta, is also required to process pro-IL-18.19,46,47,48,49,50,51 Caspase-1 denotes the original ICE and has the greatest specificity for cleaving pro-IL-1beta and pro-IL18.50,51 In support of the role of caspase-1 in IL-18 maturation, caspase-1 knockout mice (caspase-1-/-) have been found to be resistant to endotoxic shock and unable to release IL-1beta, IFN-gamma, and IL-18 in response to bacterial endotoxin.52 However, evidence increasingly indicates that the requirement of caspase-1 for IL-1beta and IL-18 production is stimulus dependent. Caspase-1-/- mice released a normal amount of IL-1beta and IFN-gamma when stimulated with ConA.53 Although caspase-1 has greater specificity for pro-IL-18, other caspases, that is, caspase-3-like proteases, are able to process pro-IL-1beta and pro-IL-18 in response to antigenic stimulation or FasL, providing an alternative pathway for cytokine generation.53,54,55 Consistent with the above reports, we demonstrated the release of IL-18 and IFN-gamma in response to ConA-induced hepatitis. In addition to inhibiting apoptosis in the liver, mice transduced with CrmA inhibited IFN-gamma and IL-18 production induced by ConA (Figure 6). Since CrmA prevents activation of broad caspases, IL-18 activation via caspase, for example, caspase-1, may be inhibited in ConA-induced hepatitis. It seems clear that besides its ability to prevent activation of death pathways, this molecule modulates cytokine release.

CD4+ T cells have been shown to be critical for the development of ConA hepatitis by antibody-dependent depletion of CD4+ cells.1 However, in this study, although the number of CD11b-positive cells that infiltrated the control livers after injection with ConA was markedly decreased in the livers that expressed the CrmA gene (Figure 4), CD4+ T-cell infiltration was unaltered in CrmA-transfected mice (compared to the control animals). Some previous reports help us understand our results. Ishiwata et al56 showed that mice treated with propagermanium (the organic germanium compound) were resistant to ConA-induced hepatitis, while CD4+ T-cell liver infiltration remained unchanged. Recently, Wolf et al57 indicated that TNFR1-/- and TNFR2-/- mice were resistant to ConA-induced hepatitis, while infiltration of CD4+ T cells in liver also remained unchanged. Furthermore, adhesion molecule blockage partially inhibited CD4+ T cell liver infiltration, whereas the development of liver disease remained unaffected. It is conceivable that antibody-dependent depletion of CD4+ T cells also depleted the CD4+ proportion of liver-resident Valpha14 NK T cells, which have been shown to be central for the onset of ConA-induced liver disease.58,59,60 Hence, it seems likely that ConA-induced stimulation of local immune cells, that is, NK T cells in concert with Kupffer cells and thymus-derived T cells, which have been shown to be abundant in the parenchymal space,61 is sufficient to induce hepatocellular damage. Recruitment of CD4+ cells from the circulatory system may contribute to the elimination of harmful activated intrahepatic lymphocytes (ie T cells, NK T cells, or NK cells).62

In this study, we demonstrated a potent protective effect of CrmA expression on the liver damage induced by ConA. CrmA inhibited hepatitis by preventing apoptosis induced by CTL cytotoxicity, CD11b+ cell infiltration, and the decline of IL-18 and IFN-gamma release. These results suggest that crmA gene transfection may be a potent therapy in clinical patients undergoing immune-mediated fulminant hepatitis.

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Materials and methods

Mice

Male Balb/c mice, aged 10–12 weeks, were purchased from the Shizuoka Laboratory Animal Center (Shizuoka, Japan). All mice used were maintained under specific pathogen-free conditions in our animal facility. Animal care was in accordance with the guidelines of National Research Institute for Child Health and Development.

Generation of recombinant adenovirus expression crmA genes

Recombinant adenovirus vector AxCALNLCrmA was constructed based on the previously described COS-TPC method.23 In brief, a cosmid containing a Cre-mediated switching expression cassette pAxCALNLCrmA was constructed by cloning cDNA for crmA at a unique SwaI site of pAxCALNLw. 293 cells were cotransfected with cosmid pAxCALNLCrmA and the adenovirus DNA–terminal protein complex digested at several sites with EcoT22I. A recombinant adenovirus AxCALNLCrmA was generated through homologous recombination in the 293 cells. A recombinant adenovirus expressing a modified cre gene, AxCANCre, was also previously described.23

Experimental design and sampling

Mice were divided into three groups: Group 1, treated with AxCALNLCrmA and AxCANCre adenovirus; Group 2, treated with AxCALNLCrmA only; and Group 3, no treatment. At 3 days after adenovirus administration, all groups of mice were injected with a single dose of ConA (Sigma, St Louis, MO, USA), 1 mg/mouse, via the tail vein.

Aside from the mice used for the survival study, three mice from each group were killed 3, 6, and 18 h after ConA administration. Liver blocks up to 1 cm3 were embedded in OCT compound (Tissue-Tek, Elkhart, IN, USA) and snap-frozen in isopentane. We cut 6 mum frozen sections in a cryostat for DNA fragmentation analysis and immunohistology. A second part of the liver was immediately snap-frozen for subsequent molecular analysis and a third part was fixed in 10% neutral buffered formalin for routine histology. Mouse serum was also taken at the same points for transaminase measurement.

In situ assay for DNA fragmentation

As in previous experiments, we used the Apop Tag™ Plus Kit (Oncor, Gaithersburg, MD, USA) to detect DNA fragmentation.24 In brief, cryosections were fixed in 10% neutral buffered formalin in a coplin jar. The sections were quenched in 0.5–1% hydrogen peroxide in PBS for 5 min at room temperature and incubated at 37°C for 1 h with deoxynucleotidyl transferase (TdT) and digoxigenin-conjugated dUTP in 38 mul of reaction buffer. The reaction was terminated with a prewarmed working strength stop/wash buffer for 30 min at 37°C. To visualize the incorporated dUTP, the sections were incubated with peroxide-conjugated anti-digoxigenin antibody for 30 min at room temperature, then washed three times and further incubated with 3,3'-diaminobenzidine (DAB) substrate working solution for 3–6 min at room temperature. The reaction was terminated by washing with distilled water, and the sections were counterstained with hematoxylin and mounted. The negative controls were prepared by substituting PBS for the TdT enzyme in the reaction mixture.

Ligase-mediated PCR assay

We investigated the presence of internucleosomal liver DNA cleavage with a commercially available ligase-mediated PCR (LM-PCR) assay kit (Apoalert, CLONTECH Laboratories Inc., Palo Alto, CA, USA), enabling semiquantitative measurement of the extent of apoptosis. In brief, DNA was isolated from tissue samples using the kit according to the manufacturer's instructions. DNA purity and concentration were determined by electrophoresis through a 0.8% agarose gel containing ethidium bromide, followed by observation under ultraviolet illumination, as well as by measuring absorbance at 260/280 nm. Dephosphorylated adaptors were ligated to 5' phosphorylated blunt ends with T4 DNA ligase (during 16 h at 16°C), then used as primers in LM-PCR under the following conditions: hot start (72°C for 8 min, Taq polymerase added after 3 min), 25 cycles (94°C for 15 s, and 72°C for 180 s); and postcycling (72°C for 15 min). To confirm that equal amounts of DNA were used for PCR, we performed an internal control that consisted of DNA amplification with En-2 primer pairs. Amplified DNA was subjected to gel electrophoresis on a 1.2% agarose gel containing ethidium bromide.

Immunohistological examination

Liver samples harvested on the third day after adenovirus administration were snap-frozen in liquid nitrogen and stored at -80°C until they were sectioned on a cryostat. The sections were air-dried and fixed in acetone at -20°C overnight, followed by air-drying for 1 h. The mouse monoclonal T7-Tag antibody-conjugated alkaline phosphatase (Novagen, Madison, WI, USA) was diluted to 1:50 in PBS solution containing 2% bovine serum albumin and 0.1% sodium azide. Color was developed with a Vector Red detection reagent kit (Vector Laboratories, Inc., Burlingame, CA, USA). Finally, we counterstained the sections with hematoxylin (Sigma).

For CD4, CD8, and CD11b staining, we prepared 6 mum cryosections from the two different group's livers at 18 h after administration of ConA, and incubated them for 1 h with rat anti-mouse monoclonal antibody for CD4 (L3T4, BD PharMingen, San Diego, CA, USA), CD8 (Ly-2, BD PharMingen), and CD11b (Ly-40, Serotec, Oxford, UK) in a PBS solution containing 2% bovine serum albumin and 0.1% sodium azide. After 1 h, the secondary antibody, goat anti-rat IgG-conjugated alkaline phosphatase (SC2021, Santa Cruz Biotechnology, Inc., Santa Cruz, CA, USA) diluted at 1 : 100 in the above working solution, was added and incubated for another hour. We developed the color with the alkaline phosphatase detection reagent kit (Vector Laboratories, Inc.) and counterstained with hematoxylin.

Western blot analysis for detecting the CrmA and cleaved caspase-3

We prepared cell lysates and extracted tissue proteins with a 100 mul 4 times SDS sample buffer, and boiled 10 mul of the protein lysates for 5 min. Protein concentration was determined with a DC protein assay kit (Bio-RAD, Hercules, CA, USA) using bovine serum albumin as a standard. Proteins were separated by 10% SDS-PAGE, then transferred to nitrocellulose filters. We blocked the filters with a TBST buffer (10 mM Tris-HCl, pH 8.0, 0.15 M NaCl, 0.05% Tween 20) containing 5% skimmed milk and incubated them for 1 h with a cleaved caspase-3 (17 kDa) antibody (Cell Signaling, Beverly, MA, USA) at 1:1000. We used horseradish peroxidase (HRP)-conjugated anti-rabbit antibodies (Sigma) at 1 : 1000 as a second antibody. We washed the filters in the TBST buffer four times for 10 min between each step and then detected the immune complexes by the ECL chemiluminescence method (Amersham, Bucking-Hampshire, UK). The CrmA detection method was previously described.23

Cytotoxic T-lymphocyte assay

Mice infected with both AxCALNCrmA and AxCANCre (Group1), or AxCALNCrmA only (Group2), were killed 7 days after the adenovirus vector administration. The spleen T cells were isolated using Lympholyte®-M (mouse) (Cedarlane, Ontario, Canada) and cultured in GIT medium (Nippon Seiyaku Ltd, Tokyo), stimulated with adenovirus vector at an MOI of 1–5 in the presence of 1 mug/ml ConA for 4 days and used as cytotoxic effector cells. Hepatocyte isolation was previously described.24 Hepatocytes isolated from mouse Groups 1 and 2 were plated on collagen-coated six-well plates in William's Medium E (1 times 105 cell/well). Stimulated effector cells were harvested, counted, and added to the primary hepatocyte cultures at ratios of 1:1, 10:1, and 50:1, then incubated at 37°C for 24 h. Hepatocyte lysis was evaluated by ALT and AST concentrations in the medium.

Assay for cytokine serum levels by enzyme-linked immunosorbent assay

We measured IL-18 and IFN-gamma with enzyme-linked immunosorbent assay (ELISA) kits, which we purchased from MBL (Nagoya, Japan). The assays were performed exactly as described by the manufacturer. Each sample was determined in triplicate.

Measurement of serum transaminase activities

We determined ALT and AST serum activities using a Vision® kit (Abbott, Abbott Park, IL, USA) according to the manufacturer's protocol.

Statistical analysis

We assessed the significance of mouse survival results using the Gehan's generalized Wilcoxon test. The significance of the differences in the ALT and AST concentrations between the different groups was assessed with the Student's unpaired t-test. A probability value of P<0.05 was considered to be significant in all studies. Error bars in figures represent standard deviations.

Ethics

We conducted all our experimental protocols in accordance with the policies of the Animal Ethics Committee of the National Research Institute for Child Health and Development.

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References

  1. Tiegs G, Hentschel J, Wendel A. A T cell-dependent experimental liver injury in mice inducible by concanavalin A. J Clin Invest 1992; 90: 196–203. | PubMed | ISI | ChemPort |
  2. Tiegs G, Gantner F. Immunotoxicology of T cell-dependent experimental liver injury. Exp Toxicol Pathol 1996; 48: 471–476.
  3. Watanabe Y, Morita M, Akaike T. Concanavalin A induces perforin-mediated but not Fas-mediated hepatic injury. Hepatology 1996; 24: 702–710. | Article | PubMed | ISI | ChemPort |
  4. Tagawa Y, Kakuta S, Iwakura Y. Involvement of Fas/Fas ligand system-mediated apoptosis in the development of concanavalin A-induced hepatitis. Eur J Immunol 1998; 28: 4105–4113.
  5. Ksontini R et al. Disparate roles for TNF-alpha and Fas ligand in concanavalin A-induced hepatitis. J Immunol 1998; 160: 4082–4089. | PubMed | ISI | ChemPort |
  6. Nicoletti F et al. Essential pathogenetic role for interferon (IFN-) gamma in concanavalin A-induced T cell-dependent hepatitis: exacerbation by exogenous IFN-gamma and prevention by IFN-gamma receptor-immunoglobulin fusion protein. Cytokine 2000; 12: 315–323.
  7. Tagawa Y, Sekikawa K, Iwakura Y. Suppression of concanavalin A-induced hepatitis in IFN-gamma(-/-) mice, but not in TNF-alpha(-/-) mice: role for IFN-gamma in activating apoptosis of hepatocytes. J Immunol 1997; 159: 1418–1428. | PubMed | ISI | ChemPort |
  8. Farrar MA, Schreiber RD. The molecular cell biology of interferon-gamma and its receptor. Annu Rev Immunol 1993; 11: 571–611. | Article | PubMed | ISI | ChemPort |
  9. Dinarello CA et al. Overview of interleukin-18: more than an interferon-gamma inducing factor. J Leukoc Biol 1998; 63: 658–664. | PubMed | ISI | ChemPort |
  10. Okamura H et al. A novel costimulatory factor for gamma interferon induction found in the livers of mice causes endotoxic shock. Infect Immun 1995; 63: 3966–3972. | PubMed | ISI | ChemPort |
  11. Okamura H et al. Cloning of a new cytokine that induces IFN-gamma production by T cells. Nature 1995; 378: 88–91. | Article | PubMed | ISI | ChemPort |
  12. Ushio S et al. Cloning of the cDNA for human IFN-gamma-inducing factor, expression in Escherichia coli, and studies on the biologic activities of the protein. J Immunol 1996; 156: 4274–4279. | PubMed | ISI | ChemPort |
  13. Matsui K et al. Propionibacterium acnes treatment diminishes CD4+ NK1.1+ T cells but induces type I T cells in the liver by induction of IL-12 and IL-18 production from Kupffer cells. J Immunol 1997; 159: 97–106. | PubMed | ISI | ChemPort |
  14. Takeda K et al. Defective NK cell activity and Th1 response in IL-18-deficient mice. Immunity 1998; 8: 383–390. | Article | PubMed | ISI | ChemPort |
  15. Puren AJ et al. Interleukin-18 (IFNgamma-inducing factor) induces IL-8 and IL-1beta via TNFalpha production from non-CD14+ human blood mononuclear cells. J Clin Invest 1998; 101: 711–721. | PubMed | ISI | ChemPort |
  16. Tsutsui H et al. IFN-gamma-inducing factor up-regulates Fas ligand-mediated cytotoxic activity of murine natural killer cell clones. J Immunol 1996; 157: 3967–3973. | PubMed | ISI | ChemPort |
  17. Ray CA et al. Viral inhibition of inflammation: cowpox virus encodes an inhibitor of the interleukin-1 beta converting enzyme. Cell 1992; 69: 597–604. | Article | PubMed | ISI | ChemPort |
  18. Vaux DL, Weissman IL, Kim SK. Prevention of programmed cell death in Caenorhabditis elegans by human bcl-2. Science 1992; 258: 1955–1957. | Article | PubMed | ISI | ChemPort |
  19. Nicholson DW et al. Identification and inhibition of the ICE/CED-3 protease necessary for mammalian apoptosis. Nature 1995; 376: 37–43. | Article | PubMed | ISI | ChemPort |
  20. Tewari M et al. Yama/CPP32 beta, a mammalian homolog of CED-3, is a CrmA-inhibitable protease that cleaves the death substrate poly(ADP-ribose) polymerase. Cell 1995; 81: 801–809. | Article | PubMed | ISI | ChemPort |
  21. Lowin B, Peitsch MC, Tschopp J. Perforin and granzymes: crucial effector molecules in cytolytic T lymphocyte and natural killer cell-mediated cytotoxicity. Curr Top Microbiol Immunol 1995; 198: 1–24. | PubMed | ChemPort |
  22. Tewari M et al. CrmA, a poxvirus-encoded serpin, inhibits cytotoxic T-lymphocyte-mediated apoptosis. J Biol Chem 1995; 270: 22705–22708. | Article | PubMed | ISI | ChemPort |
  23. Fujino M et al. In vitro prevention of cell-mediated xeno-graft rejection via the Fas/FasL-pathway in CrmA-transducted porcine kidney cells. Xenotransplantation 2001; 8: 115–124.
  24. Li XK et al. Inhibition of Fas-mediated fulminant hepatitis in CrmA gene-transfected mice. Biochem Biophys Res Commun 2000; 273: 101–109. | Article | PubMed | ChemPort |
  25. Galle PR et al. Involvement of the CD95 (APO-1/Fas) receptor and ligand in liver damage. J Exp Med 1995; 182: 1223–1230. | Article | PubMed | ISI | ChemPort |
  26. Gantner F et al. Concanavalin A-induced T-cell-mediated hepatic injury in mice: the role of tumor necrosis factor. Hepatology 1995; 21: 190–198. | Article | PubMed | ISI | ChemPort |
  27. Kusters S et al. Interferon gamma plays a critical role in T cell-dependent liver injury in mice initiated by concanavalin A. Gastroenterology 1996; 111: 462–471. | Article | PubMed | ChemPort |
  28. Mizuhara H et al. T cell activation-associated hepatic injury: mediation by tumor necrosis factors and protection by interleukin 6. J Exp Med 1994; 179: 1529–1537. | Article | PubMed | ISI | ChemPort |
  29. Mizuhara H et al. Critical involvement of interferon gamma in the pathogenesis of T-cell activation-associated hepatitis and regulatory mechanisms of interleukin-6 for the manifestations of hepatitis. Hepatology 1996; 23: 1608–1615. | PubMed | ChemPort |
  30. Seino K et al. Contribution of Fas ligand to T cell-mediated hepatic injury in mice. Gastroenterology 1997; 113: 1315–1322. | Article | PubMed | ISI | ChemPort |
  31. Ogasawara J et al. Lethal effect of the anti-Fas antibody in mice. Nature 1993; 364: 806–809. | Article | PubMed | ISI | ChemPort |
  32. Trautwein C et al. Concanavalin A-induced liver cell damage: activation of intracellular pathways triggered by tumor necrosis factor in mice. Gastroenterology 1998; 114: 1035–1045. | Article | PubMed | ChemPort |
  33. Jones RA et al. Fas-mediated apoptosis in mouse hepatocytes involves the processing and activation of caspases. Hepatology 1998; 27: 1632–1642. | Article | PubMed | ISI | ChemPort |
  34. Bradham CA et al. Mechanisms of hepatic toxicity. I. TNF-induced liver injury. Am J Physiol 1998; 275: G387–G392. | PubMed | ISI | ChemPort |
  35. Lemasters JJV. Necrapoptosis and the mitochondrial permeability transition: shared pathways to necrosis and apoptosis. Am J Physiol 1999; 276: G1–G6. | PubMed | ISI | ChemPort |
  36. Ekert PG, Silke J, Vaux DL. Inhibition of apoptosis and clonogenic survival of cells expressing crmA variants: optimal caspase substrates are not necessarily optimal inhibitors. EMBO J 1999; 18: 330–338. | Article | PubMed | ISI | ChemPort |
  37. Dbaibo GS, Hannun YA. Cytokine response modifier A (CrmA): a strategically deployed viral weapon. Clin Immunol Immunopathol 1998; 86: 134–140.
  38. Fujino M et al. Distinct pathways of apoptosis triggered by FTY720, etoposide, and anti-Fas antibody in human T-lymphoma cell line (Jurkat cells). J Pharmacol Exp Ther 2002; 300: 939–945.
  39. Ekert PG, Silke J, Vaux DL. Caspase inhibitors. Cell Death Differ 1999; 6: 1081–1086. | Article | PubMed | ISI | ChemPort |
  40. Dbaibo GS et al. Cytokine response modifier A (CrmA) inhibits ceramide formation in response to tumor necrosis factor (TNF)-alpha: CrmA and Bcl-2 target distinct components in the apoptotic pathway. J Exp Med 1997; 185: 481–490. | Article | PubMed | ISI | ChemPort |
  41. Quan LT et al. Granzyme B is inhibited by the cowpox virus serpin cytokine response modifier A. J Biol Chem 1995; 270: 10377–10379. | Article | PubMed | ISI | ChemPort |
  42. MacDonald G et al. Mitochondria-dependent and -independent regulation of Granzyme B-induced apoptosis. J Exp Med 1999; 189: 131–144. | Article | PubMed | ISI | ChemPort |
  43. Fiorucci S et al. NO-aspirin protects from T cell-mediated liver injury by inhibiting caspase-dependent processing of Th1-like cytokines. Gastroenterology 2000; 118: 404–421. | Article | PubMed | ISI | ChemPort |
  44. Chisari FV, Ferrari C. Hepatitis B virus immunopathogenesis. Annu Rev Immunol 1995; 13: 29–60. | Article | PubMed | ISI | ChemPort |
  45. Okamura H et al. Regulation of interferon-gamma production by IL-12 and IL-18. Curr Opin Immunol 1998; 10: 259–264. | Article | PubMed | ISI | ChemPort |
  46. Thornberry NA et al. A novel heterodimeric cysteine protease is required for interleukin-1 beta processing in monocytes. Nature 1992; 356: 768–774. | Article | PubMed | ISI | ChemPort |
  47. Salvesen GS, Dixit VM. Caspases: intracellular signaling by proteolysis. Cell 1997; 91: 443–446. | Article | PubMed | ISI | ChemPort |
  48. Thornberry NA, Lazebnik Y. Caspases: enemies within. Science 1998; 281: 1312–1316. | Article | PubMed | ISI | ChemPort |
  49. Alnemri ES et al. Human ICE/CED-3 protease nomenclature. Cell 1996; 87: 171. | Article | PubMed | ISI | ChemPort |
  50. Ghayur T et al. Caspase-1 processes IFN-gamma-inducing factor and regulates LPS-induced IFN-gamma production. Nature 1997; 386: 619–623. | Article | PubMed | ISI | ChemPort |
  51. Gu Y et al. Activation of interferon-gamma inducing factor mediated by interleukin-1beta converting enzyme. Science 1997; 275: 206–209. | Article | PubMed | ISI | ChemPort |
  52. Li P et al. Mice deficient in IL-1 beta-converting enzyme are defective in production of mature IL-1 beta and resistant to endotoxic shock. Cell 1995; 80: 401–411. | Article | PubMed | ISI | ChemPort |
  53. Fantuzzi G et al. Interleukin-18 regulation of interferon gamma production and cell proliferation as shown in interleukin-1beta-converting enzyme (caspase-1)-deficient mice. Blood 1998; 91: 2118–2125. | PubMed | ISI | ChemPort |
  54. Miwa K et al. Caspase 1-independent IL-1beta release and inflammation induced by the apoptosis inducer Fas ligand. Nat Med 1998; 4: 1287–1292. | Article | PubMed | ISI | ChemPort |
  55. Akita K et al. Involvement of caspase-1 and caspase-3 in the production and processing of mature human interleukin 18 in monocytic THP.1 cells. J Biol Chem 1997; 272: 26595–26603. | Article | PubMed | ISI | ChemPort |
  56. Ishiwata Y et al. Protection against concanavalin A-induced murine liver injury by the organic germanium compound, propagermanium. Scand J Immunol 1998; 48: 605–614.
  57. Wolf D et al. TNF-alpha-induced expression of adhesion molecules in the liver is under the control of TNFR1 – relevance for concanavalin A-induced hepatitis. J Immunol 2001; 166: 1300–1307. | PubMed | ISI | ChemPort |
  58. Kaneko Y et al. Augmentation of Valpha14 NKT cell-mediated cytotoxicity by interleukin 4 in an autocrine mechanism resulting in the development of concanavalin A-induced hepatitis. J Exp Med 2000; 191: 105–114. | Article | PubMed | ISI | ChemPort |
  59. Toyabe S et al. Requirement of IL-4 and liver NK1+ T cells for concanavalin A-induced hepatic injury in mice. J Immunol 1997; 159: 1537–1542. | PubMed | ISI | ChemPort |
  60. Takeda K et al. Critical contribution of liver natural killer T cells to a murine model of hepatitis. Proc Natl Acad Sci USA 2000; 97: 5498–5503. | Article | PubMed | ChemPort |
  61. Yamamoto S et al. Consistent infiltration of thymus-derived T cells into the parenchymal space of the liver in normal mice. Hepatology 1999; 30: 705–713. | Article | PubMed | ChemPort |
  62. Fogler WE et al. Recruitment of hepatic NK cells by IL-12 is dependent on IFN-gamma and VCAM-1 and is rapidly down-regulated by a mechanism involving T cells and expression of Fas. J Immunol 1998; 161: 6014–6021. |