Increased activated natural killer T cells in the liver of patients with advanced stage primary biliary cirrhosis.

Although growing evidence suggests a major role for T cells in the pathogenesis of primary biliary cirrhosis (PBC), the roles of natural killer (NK) and natural killer T (NKT) cells, which predominate in the liver, in the pathogenesis of PBC remain unclear. We investigated the status of NK and NKT cells in the liver and peripheral blood samples obtained from 11 patients with asymptomatic PBC diagnosed as stage I or II (early PBC) and 7 patients with symptomatic PBC who underwent liver transplantation (advanced PBC) using flow cytometry and immunohistochemical staining. The proportions of NK and NKT cells were significantly decreased in the liver of patients with early PBC compared with normal donors. However, the proportion of CD56+ NKT cells was increased in the liver of patients with advanced PBC. Moreover, the proportion of activated Fas ligand (FasL)-positive NKT cells was significantly increased in the liver of patients with advanced PBC compared with early PBC (P=0.013). We also found increased expression of FasL on lymphocytes infiltrating around the injured bile duct in advanced PBC using immunohistochemical staining. Our results suggest that activated NKT cells may contribute to the biliary epithelial cell death resulting in the progression of PBC.

Hepatic natural killer and natural killer T cells markedly decreased in two cases of drug-induced fulminant hepatic failure rescued by living donor liver transplantation.

The human liver contains significant numbers of innate immune cells, such as natural killer (NK) cells and natural killer T (NKT) cells, which express both T-cell receptors and NK-cell receptors simultaneously. It has been suggested that the innate immune system plays a crucial role in the liver. In this report, the distribution of NK and NKT cells in the liver and peripheral blood of two patients with drug-induced fulminant hepatic failure (FHF) who had undergone living donor liver transplantation was examined. In both the liver and peripheral blood, the proportions of NK and NKT cells markedly decreased compared with those in healthy donors. It was also revealed that, unlike murine NKT cells, human CD56(+) T cells and CD57(+) T cells did not constitutively express CD28, which is one of the important costimulatory molecules on T cells. Additionally, the residual CD56(+) T cells and CD57(+) T cells in the patients expressed more CD28 than in controls. This result suggests that NKT cells might be more activated in FHF. Although the accumulation of further cases is required, it is suggested that both NK and NKT cells might be involved in hepatic injury in FHF.

Characterization of extrathymic CD8 alpha beta T cells in the liver and intestine in TAP-1 deficient mice.

TAP-1 deficient (-/-) mice cannot transport MHC class I antigens onto the cell surface, which results in failure of the generation of CD8+ T cells in the thymus. In a series of recent studies, it has been proposed that extrathymic T cells are generated in the liver and at other extrathymic sites (e.g. the intestine). It was therefore investigated whether CD8+ extrathymic T cells require an interaction with MHC class I antigens for their differentiation in TAP-1(-/-) mice. Although CD8+ thymically derived T cells were confirmed to be absent in the spleen as well as in the thymus, CD8 alpha beta+ T cells were abundant in the livers and intestines of TAP-1(-/-) mice. These CD8+ T cells expanded in the liver as a function of age and were mainly confined to a NK1.1-CD3int population which is known to be truly of extrathymic origin. Hepatic lymphocytes, which contained CD8+ T cells and which were isolated from TAP-1(-/-) mice (H-2b), responded to neither mutated MHC class I antigens (bm1) nor allogeneic MHC class I antigens (H-2d) in in vitro mixed lymphocyte cultures. However, the results from repeated in vivo stimulations with alloantigens (H-2d) were interesting. Allogeneic cytotoxicity was induced in liver lymphocytes in TAP-1(-/-) mice, although the magnitude of cytotoxicity was lower than that of liver lymphocytes in immunized B6 mice. All allogeneic cytotoxicity disappeared with the elimination of CD8+ cells in TAP-1(-/-) mice. These results suggest that the generation and function of CD8+ extrathymic T cells are independent of the existence of the MHC class I antigens of the mouse but have a limited allorecognition ability.

Identification of effector cells for TNFalpha-mediated cytotoxicity against WEHI164S cells.

WEHI164S cells were found to be very sensitive targets for in vitro killing in a 6-h culture when liver or splenic lymphocytes were used as effector cells in mice. Of particular interest, a limiting cell-dilution analysis showed that effector cells were present in the liver with a high frequency (1/4,300). In contrast to YAC-1 cells as NK targets, perforin-based cytotoxicity was not highly associated with WEHI164S killing. The major killer mechanism for WEHI164S targets was TNFalpha-mediated cytotoxicity. By cell sorting experiments, both NK cells and intermediate T cells (i.e., TCR(int) cells) were found to contain effector cells against WEHI164S cells. However, the killer mechanisms underlying these effector cells were different. Namely, NK cells killed WEHI164S cells by perforin-based cytotoxicity, TNFalpha-mediated cytotoxicity, Fas ligand cytotoxicity, and other mechanisms, whereas intermediate T cells did so mainly by TNFalpha-mediated cytotoxicity. These results suggest that TNFalpha-mediated cytotoxicity mediated by so-called natural cytotoxic (NC) cells comprised events which were performed by both NK and intermediate T cells using somewhat different killer mechanisms. Intermediate T cells which were present in the liver were able to produce TNFalpha if there was appropriate stimulation.

Unconventional NK1.1(-) intermediate TCR cells as major T lymphocytes expanding in chronic graft-versus-host disease.

Chronic graft-versus-host disease (GVHD) accompanying autoimmune disease was induced in (C57BL/6xDBA/2) F(1) mice (H-2(b/d)) by an injection of splenic T cells of parental DBA/2 origin (H-2(d)). In parallel with the onset of proteinuria, an expansion of lymphocytes was induced in the liver and kidney, showing a peak at 2 weeks after the onset of disease. The majority of lymphocytes were of recipient origin (H-2(b/d)). The main lymphocyte subset among T cells at the pre-onset stage and after the onset of disease was CD8(+) NK1.1(-) CD3(int) cells (of extrathymic, hepatic origin) in both the liver and kidney. NK1.1(-) CD3(int) cells confer primarily neither NK-like nor NKT-like cytotoxicity. No induction of these types of cytotoxicity was observed in these mice with the expansion of NK1.1(-) CD3(int) cells. This raised the possibility that granulocytes induced in the liver and kidney might be associated with tissue damage. The present results suggest that, similarly to the case of autoimmune-prone mice with genetic background (e.g. MRL-lpr/lpr mice and BXSB mice), NK1.1(-) CD3(int) cells of extrathymic, hepatic origin might be crucial lymphocytes involved in the induction of the autoimmune-like disease in mice with chronic GVHD, in conjunction with Bcells (e.g. B-1 cells).

Mechanisms underlying the activation of cytotoxic function mediated by hepatic lymphocytes following the administration of glycyrrhizin.

Stronger neo-minophagen C (SNMC), a glycyrrhizin (GL) preparation, has been used for the treatment of chronic viral hepatitis. It has been reported that a single administration of SNMC induced the activation of hepatic lymphocytes in number and function in animal studies. However, it is still unknown how SNMC augments the cytotoxic function and why such augmentation of cytotoxicity occurs in the liver and other organs. In this study, SNMC was daily injected into mice (2 mg GL/day/mouse) for 2 weeks. A significant augmentation of cytotoxicity mediated by NK cells, NKT cells and TNFalpha was demonstrated mainly in the liver. The presence of TNFalpha-mediated cytotoxicity in the liver was demonstrated for the first time. In contrast to CD8+ cytotoxic T cells (CD8+ CTL), all these cytotoxicities were preexistent in lymphocytes without the immunization of a specific antigen or alloantigens. NK cytotoxicity was mediated by a perforin system, while NKT cytotoxicity was mediated by a Fas ligand system. The present results suggest that the entire cytotoxic function mediated by hepatic lymphocytes was simultaneously augmented by SNMC.