Impairment of liver regeneration correlates with activated hepatic NKT cells in HBV transgenic mice

A fraction of HBV carriers have a risk to develop liver cancer. Because liver possesses a strong regeneration capability, surgical resection of cancerous liver or transplantation with healthy liver is an alternate choice for HBV-caused hepatocarcinoma therapy. How HBV infection affects the regeneration of hepatectomized or transplanted liver remains elusive. We report that partial hepatectomy (PHx)-induced liver regeneration was reduced in HBV transgenic (HBV-tg) mice, a model of human HBV infection. PHx markedly triggered natural killer T (NKT) cell accumulation in the hepatectomized livers of HBV-tg mice, simultaneously with enhanced interferon gamma (IFN-gamma) production and CD69 expression on hepatic NKT cells at the early stage of liver regeneration. The impairment of liver regeneration in HBV-tg mice was largely ameliorated by NKT cell depletion, but not by natural killer (NK) cell depletion. Blockage of CD1d-NKT cell interaction considerably alleviated NKT cell activation and their inhibitory effect on regenerating hepatocytes. Neutralization of IFN-gamma enhanced bromodeoxyuridine incorporation in HBV-tg mice after PHx, and IFN-gamma mainly induced hepatocyte cell cycle arrest. Adoptive transfer of NKT cells from regenerating HBV-tg liver, but not from normal mice, could inhibit liver regeneration in recipient mice. CONCLUSION: Activated NKT cells negatively regulate liver regeneration of HBV-tg mice in the PHx model.     

Activation of mouse natural killer T cells accelerates liver regeneration after partial hepatectomy

BACKGROUND & AIMS: Activation of natural killer T cells with the synthetic ligand alpha-galactosylceramide (alpha-GalCer) induced hepatotoxicity through the tumor necrosis factor (TNF) and Fas-ligand-mediated pathway in aged mice. The aim of this study was to elucidate how alpha-GalCer-activated natural killer T cells function in hepatocyte proliferation and liver regeneration in partially hepatectomized (PHx) mice. METHODS: Mice were injected with alpha-GalCer at 36 hours after 70% PHx. Hepatocyte mitosis was evaluated by either mitotic figures or proliferating cell nuclear antigen staining. The role of TNF and Fas-ligand in hepatocyte mitosis also was assessed. RESULTS: In PHx mice injected with alpha-GalCer, hepatocyte mitosis was greatly enhanced at 44 hours after surgery and the increase was more obvious in aged mice than in young mice. The expression of both TNF receptor 1 and Fas-ligand in liver natural killer T cells tended to increase after alpha-GalCer injection in PHx mice. Treatment of mice with anti-NK1.1 Ab 3 days before and just after hepatectomy greatly inhibited the effect of alpha-GalCer on hepatocyte mitosis and liver regeneration. Furthermore, pretreatment of PHx mice with either anti-TNF Ab or anti-FasL Ab 1 hour before alpha-GalCer injection mostly abrogated the increase in hepatocyte proliferation. alpha-GalCer injection did not accelerate hepatocyte proliferation in Fas-mutated lpr mice after PHx. CD1d-/- mice without alpha-GalCer injection showed decreased hepatocyte mitosis after PHx. CONCLUSIONS: Activated natural killer T cells help hepatocyte proliferation and liver regeneration after PHx via the TNF and Fas/Fas-ligand-mediated pathway.     

Negative regulation of liver regeneration by innate immunity (natural killer cells/interferon-gamma)

BACKGROUND AND AIMS: Hepatic lymphocytes are composed mainly of natural killer (NK) cells and NKT cells, which play key roles in innate immune responses against pathogens and tumors in the liver. This report analyzes the effects of activation of innate immunity by viral infection or the toll-like receptor 3 (TLR3) ligand on liver regeneration. METHODS: The partial hepatectomy (PHx) method was used as a model of liver regeneration. Murine cytomegalovirus (MCMV) infection and the TLR3 ligand polyinosinic-polycytidylic acid [poly(I:C)] were used to activate innate immunity. RESULTS: NK cells are activated after PHx, as evidenced by producing interferon (IFN)-gamma. Infection with MCMV or injection of poly(I:C) further activates NK cells to produce IFN-gamma and attenuates liver regeneration in the PHx model. Depletion of NK cells or disruption of either the IFN-gamma gene or the IFN-gamma receptor gene enhances liver regeneration and partially abolishes the negative effects of MCMV and polyI:C on liver regeneration, whereas NKT cells may only play a minor role in suppression of liver regeneration. Adoptive transfer of IFN-gamma +/+ NK cells, but not IFN-gamma -/- NK cells, restores the ability of polyI:C to attenuate liver regeneration in NK-depleted mice. Finally, administration of polyI:C or IFN-gamma enhances expression of several antiproliferative proteins, including STAT1, IRF-1, and p21cip1/waf1 in the livers of partially hepatectomized mice. CONCLUSIONS: Our findings suggest that viral infection and the TLR3 ligand negatively regulate liver regeneration via activation of innate immunity (NK/IFN-gamma), which may play an important role in the pathogenesis of viral hepatitis.     

The variations during the circadian cycle of liver CD1d-unrestricted NK1.1+TCR gamma/delta+ cells lead to successful ageing. Role of metallothionein/IL-6/gp130/PARP-1 interplay in very old mice

NKT cells derive from the thymus and home to the liver. Liver NKT cells can be divided in two groups: 'classical' and 'non-classical'. The first is CD1d-restricted, the second is CD1d-unrestricted. NKT cells (classical and non-classical) co-express T-cell receptor (TCR) and NK-cell marker (NK1.1), display cytotoxicity and produce IFN-gamma under IL-12 stimulation affecting, thereby, Th1 response and innate immunity. NK1.1(+)TCR alpha/beta(+) cells belong to both groups. NK1.1(+)TCR gamma/delta(+) cells belong to the second group. Anyway, both NKT cell subtypes, via IFN-gamma production, protect against viruses and bacteria from early in life. Immune variations as well as zinc rhythmicity during the circadian cycle confer the immune plasticity, which is essential for successful ageing. Liver NK1.1(+)TCR gamma/delta(+) cells, rather than TCR alpha/beta(+), from young and very old mice display 'in vitro' (under IL-12 stimulation) nocturnal peaks in cytotoxicity and IFN-gamma production. The acrophase of liver NK1.1(+)TCR gamma/delta(+) cells is present in young and very old mice, not in old. The interplay among zinc-bound metallothionein (MT)/IL-6/gp130/poly(ADP-ribose) polymerase-1 (PARP-1) may be involved in conferring plasticity to liver NK1.1(+)TCR gamma/delta(+) cells. IL-6, via sub-unit receptor gp130, induces MTmRNA. At night, gene expressions of MT, IL-6, gp130 are lower in very old mice than old and young MT-I transgenic mice (MT-I*). In very old mice, this phenomenon allows limited sequester of intracellular zinc from MT leading to good free zinc ion bioavailability for immune efficiency and zinc-dependent PARP-1 activity. Indeed (1) in vitro, high IL-6 provokes strong accumulation of MT, impaired cytotoxicity and low zinc ion bioavailability in liver NK1.1(+)TCR gamma/delta(+) cells exclusively from old and MT-I* mice. (2) The ratio total/endogen PARP-1 activity is higher in very old than in old and MT-I* mice, suggesting a higher capacity of PARP-1 in base excision DNA-repair in very old age thanks to low zinc-bound MT. Cytotoxicity and IFN-gamma production from liver NK1.1(+)TCR gamma/delta(+) cells are thus preserved leading to successful ageing. In conclusion, MT/IL-6/gp130/PARP-1 interplay may confer plasticity to liver CD1d-unrestricted NK1.1(+)TCR gamma/delta(+) cells, where MT, IL-6, gp130 are the main upstream protagonists, and PARP-1 is the main downstream protagonist in immunosenescence.     

Selective elimination of hepatic natural killer T cells with concanavalin A improves liver regeneration in mice.

BACKGROUND: Although concanavalin A (Con A) as a T cell stimulant can cause natural killer T (NKT) cell-mediated liver injury in mice and a nonhepatotoxic dose of Con A can trigger innate immune cells including NKT cells to prevent tumor metastasis in the liver, little is known about the role of Con A-primed NKT cells in liver repair. In this study, we aimed to investigate the effect of pretreatment with a nontoxic dose of Con A on subsequent liver regeneration in mice. METHODS: A nontoxic dose of Con A was injected intravenously 24 h before partial hepatectomy (PHx), which was used as a model of liver regeneration. Ratios of remnant liver mass to body weight, bromodeoxyuridine (BrdU) incorporation and proliferating cell nuclear antigen (PCNA) labeling were used to assess liver regeneration. RESULTS: Hepatic mononuclear cells were isolated and analyzed by flow cytometry. After PHx, the ratios of liver weight to body weight, PCNA-positive hepatocytes and BrdU-positive hepatocytes in Con A-pretreated mice were significantly higher than that of phosphate-buffered saline-treated mice, indicating that Con A pretreatment can accelerate liver regeneration. Flow cytometric analysis showed that NKT cells were significantly activated and selectively eliminated after the Con A administration. Moreover, NKT cells expressed more apoptosis-related molecules, Fas and Annexin V. CONCLUSIONS: Taken together, Con A accelerates liver regeneration in mice by eliminating hepatic NKT cells via activation-induced cell death.     

Defective development of NK1.1+ T-cell antigen receptor alphabeta+ cells in zeta-associated protein 70 null mice with an accumulation of NK1.1+ CD3- NK-like cells in the thymus

Development of natural killer 1.1+ (NK1.1+) CD3+ (NK1.1+ T) cells was analyzed in zeta-associated protein 70 (ZAP-70) null ((-/-)) mice. Both NK1.1+ TCRalphabeta+ and NK1.1+ TCRgammadelta+ cell populations were absent in the thymus and spleen. By contrast, the number of NK1.1+ CD3- cells was increased in these tissues. The NK1.1+ CD3- thymocytes in ZAP-70(-/-) mice had surface phenotypes in common with NK or NK1.1+ T cells. However, some of them were discordant either with NK cells or with NK1.1+ T cells. The NK1.1+ CD3- cells produced interferon-gamma upon stimulation with NK1.1 cross-linking in the presence of interleukin-2 and exhibited a substantial cytotoxicity against YAC-1 cells. Moreover, the generation of NK1.1+ T cells with invariant Valpha14Jalpha281 chains was induced from the NK1.1+ CD3- thymocytes following stimulation with phorbol myristate acetate and ionomycin in a neonatal thymic organ culture. An introduction of TCRalpha and beta transgenes to the ZAP-70(-/-) mice resulted in generation of an NK1.1+ TCRalphabeta(dim) population, whereas no substantial CD4+ CD8- or CD4- CD8+ population that expressed the introduced TCRalphabeta was generated in the mainstream T lineage. These findings demonstrate that ZAP-70 kinase is indispensable for the development of NK1.1+ T cells and that the unique NK1.1+ CD3- thymocytes in ZAP-70(-/-) mice contain immediate precursors of NK1.1+ T cells.     

Concanavalin a injection activates intrahepatic innate immune cells to provoke an antitumor effect in murine liver

Concanavalin A (ConA), directly injected into mice, induces T cell-mediated liver injury. However, it remains unclear whether ConA injection can activate innate immune cells, including natural killer (NK) cells and natural killer T (NKT) cells, both of which exist abundantly in the liver. Here we report that ConA injection stimulated interferon (IFN)-gamma production from liver NKT cells as early as 2 hours after injection and augmented YAC-1 cytotoxicity of liver NK cells. ConA-induced NK cell activation required other types of immune cells and critically depended on IFN-gamma. Because a nonhepatotoxic low dose of ConA was capable of fully activating both NKT cells and NK cells, we next addressed the possibility of ConA injection displaying an antitumor effect in the liver without liver injury. A nonhepatotoxic low-dose ConA injection augmented the cytotoxicity of liver NK cells against Colon-26 colon cancer cells and suppressed hepatic metastasis of Colon-26 cells in a NK cell- and IFN-gamma-dependent manner. In conclusion, a nonhepatotoxic low dose of ConA might serve as an immunomodulator that can preferentially activate the innate immune cells to induce an antitumor effect against metastatic liver tumor.     

NK1.1+ cell depletion in vivo fails to prevent protection against infection with the murine nematode parasite Trichuris muris

Protection against the murine nematode parasite Trichuris muris has been shown to involve interleukin 4 (IL-4). NK1.1+ T cell receptor alphabeta+ cells, designated Natural Killer T (NKT) cells, produce a large amount of IL-4 in response to anti-CD3 stimulation and numerous pieces of evidence suggest that NKT cells provide the initial source of IL-4 for T helper 2 (Th2) priming. These observations allow the hypothesis that NKT cells produce a large amount of IL-4 in response to T. muris infection and augment Th2 responses and IL-4 production, thus achieving protection against T. muris. To investigate the involvement of NKT cells in protection against T. muris infection, NK1.1+ cell-depleted B10.BR mice were prepared by anti-NK1.1 monoclonal antibody injection. Efficient expulsion of T. muris worms occurred in NK1.1+ cell-depleted infected mice, and the expulsion kinetics of T. muris worms, the levels of IL-4 production by mesenteric lymph node cells, and the kinetics of the specific IgG1 and IgG2a responses to T. muris were similar to those in mouse IgG-treated or non-treated control B10.BR mice. These observations suggest that NK1.1+ cells and NKT cells are not involved in the induction of Th2 responses and protective immunity to T. muris infection.     

Interleukin-12 regulates natural killer cell-dependent Propionibacterium acnes-primed, lipopolysaccharide-induced liver injury.

Aim: Interleukin (IL)-12, produced primarily by macrophage/monocytes, modulates mature T and natural killer (NK) cell functions, including cytotoxicity and cytokine production. Methods: To determine the role of IL-12 in Propionibacterium acnes (P. acnes)-primed, lipopolysaccharide (LPS)-induced liver injury, mice were injected with an anti-IL-12 monoclonal antibody (mAb) 1 and 2 days before P. acnes injection (day 0) or 5 and 6 days before LPS challenge (day 7). The survival rates, plasma cytokine levels, and liver mononuclear cell phenotypes were evaluated for the mice treated with and without anti-IL-12 mAb. Results: The observed mortality with P. acnes-primed, LPS-induced liver injury in C57BL/6 (B6) mice was 100%, but was reduced to 0% in interferon (IFN)-gamma receptor-deficient mice and B6 mice treated with anti-IL-12 mAb on 1 and 2 days before P. acnes exposure (day 0). The plasma IFN-gamma levels weresignificantly lower (P < 0.05), and significantly less ( approximately 90% reduction) hepatic infiltrating mononuclear and NK1.1 cells were also found in the IL-12 mAb-treated, P. acnes-primed mice. The plasma cytokine levels after LPS challenge and in vitro cytokine release by liver mononuclear cells were significantly lower (P < 0.05) in the mice treated with anti-IL-12 mAb prior to P. acnes exposure. The in vivo administration of anti-NK1.1 mAb also improved survival in this liver injury model. Conclusion: IL-12-regulated IFN-gamma production is crucial during the priming phase by P. acnes, but not at the time of the subsequent LPS challenge. NK1.1(+)CD3(-)CD4(-) NK or NK1.1(+)CD3(+)CD4(-) NKT cells are important in this model of liver injury.     

Isolation of murine hepatic lymphocytes using mechanical dissection for phenotypic and functional analysis of NK1.1^＋ cells

AIM: To choose an appropriate methods for the isolation of hepatic lymphocytes between the mechanical dissection and the enzymatic digestion and investigate the effects of two methods on phenotype and function of hepaticlymphocytes.METHODS: Hepatic lymphocytes were isolated from untreated, poly (I:C)-stimulated or ConA-stimulated mice using the two methods, respectively. The cell yield per liver was evaluated by direct counting under microscope.Effects of digestive enzymes on the surface markers involved in hepatic lymphocytes were represented by relative change rate [(percentage of post-digestionpercentage of pre-digestion)/percentage of pre-digestion].Phenotypic analyses of the subpopulations of hepatic lymphocytes and intracellular cytokines were detected by flow cytometry. The cytotoxicity of NK cells from wild C57BL/6 or poly (I:C)-stimulated C57BL/6 mice was analyzed with a 4-h ^51Cr release assay.RESULTS: NK1.1^+ cell markers, NK1.1 and DX5, were significantly down-expressed after enzymatic digestion and their relative change rates were about 28% and 32%,respectively. Compared with the enzymatic digestion, the cell yield isolated from unstimulated, poly (I:C)-treated or ConA-treated mice by mechanical dissection was not significantly decreased. Hepatic lymphocytes isolated by the mechanical dissection comprised more innate immune cells like NK, NKT and γδ cells in normal C57BL/6 mice.After poly (I:C) stimulation, hepatic NK cells rose to about 35%, while NKT cells simultaneously decreased. Following ConA injection, the number of hepatic NKT cells was remarkably reduced to 3.67%. Higher ratio of intracellular IFN-γ^+(68%) or TNF-α^+(15%) NK1.1^+ cells from poly (I:C)treated mice was obtained using mechanical dissection method than control mice. There was no difference in viability between the mechanical dissection and the enzymatic digestion, and hepatic lymphocytes obtained with the two methods had similar cytotoxicity against YAC-1 cells.CONCLUSION: There is no difference in the cell yield and viability of the hepatic lymphocyte isolated with the two methods. The mechanical dissection, but not the enzymatic digestion, may be suitable for the phenotypic analysis of hepatic NK1.1^+ cell.     

Role of Valpha 14 NKT cells in the development of impaired liver regeneration in vivo

Although we have previously demonstrated that IL-12 stimulation increases the number of hepatic natural killer (NK) T (NKT) cells and enhances liver injury during the early phase of liver regeneration, the role of NKT cells has remained unknown. We therefore evaluated the influence of NKT cells activated by IL-12 or by alpha-galactosylceramide (alpha-GalCer) on murine liver regeneration using Valpha 14 NKT knockout (Jalpha 281(-/-)) mice. Levels of serum alanine aminotransferase (sALT) 24 hours after partial hepatectomy were enhanced in Jalpha 281(+/+) but not in Jalpha 281(-/-) mice by both procedures. Hepatic NKT cells expressed considerably more interferon (IFN) gamma and tumor necrosis factor alpha (TNF-alpha) messenger RNA (mRNA) after stimulation with both factors in Jalpha 281(+/+) mice. Either anti-IFN-gamma or TNF-alpha antibody inhibited the enhancement of liver injury. Furthermore, recombinant TNF-alpha injection similarly caused injury in hepatectomized livers of both Jalpha 281(+/+) and Jalpha 281(-/-) mice; indeed, adoptively transferred TNF-alpha(+/+) NKT cells enhanced liver injury after hepatectomy in TNF-alpha knockout mice. TNF receptor expressions on hepatocytes increased and peaked 24 hours after partial hepatectomy. In conclusion, simultaneous TNF-alpha synthesis and high levels of TNF receptor expression on hepatocytes cause severe liver damage by activated NKT cells during liver regeneration.     

Alterations in natural killer cells and natural killer T cells during acute viral hepatitis E

The mechanism of liver damage in acute hepatitis E is poorly understood. In this study, we assessed the frequency and activation status of natural killer (NK) and natural killer T (NKT) cells and cytotoxic activity of NK cells in the peripheral blood mononuclear cells (PBMCs) obtained from patients with hepatitis E (n = 41) and healthy controls (n = 61). Flow cytometry was used to assess NK (CD3(-)/CD56(+)) and NKT cell (CD3(+)/CD56(+)) fractions (% of PBMCs) and activation status (CD69(+); % of NK, NKT cells). NK cell cytotoxicity was assessed using major histocompatibilities complex-deficient K562 cells as target cells. In 14 patients, the studies were repeated during the convalescence period. Patients had fewer median (range) NK cells [8.9% (2.4-47.0) vs 11.2% (2.6-35.4)] and NKT cells [8.7% (2.8-34.1) vs 13.6% (2.3-36.9)] than controls (P < 0.05 each). Activation markers were present on large proportion of NK cells [43.5% (11.2-58.6) vs 15.5% (3.0-55.8)] and NKT cells [41.5% (17.4-71.1) vs 12.8% (3.3-63.2); P < 0.05 each] from patients. NK cell cytotoxicity was similar in patients and controls. During convalescence, all the parameters normalized. In conclusion, reversible alterations in NK and NKT cell number and activation status during acute hepatitis E suggest a role of these cells in the pathogenesis of this disease.     

Functional alterations of liver innate immunity of mice with aging in response to CpG-oligodeoxynucleotide

Immune functions of liver natural killer T (NKT) cells induced by the synthetic ligand alpha-galactosylceramide enhanced age-dependently; hepatic injury and multiorgan dysfunction syndrome (MODS) induced by ligand-activated NKT cells were also enhanced. This study investigated how aging affects liver innate immunity after common bacteria DNA stimulation. Young (6 weeks) and old (50-60 weeks) C57BL/6 mice were injected with CpG oligodeoxynucleotides (CpG-ODN), and the functions of liver leukocytes were assessed. A CpG-ODN injection into the old mice remarkably increased tumor necrosis factor (TNF) production in Kupffer cells, and MODS and lethal shock were induced, both of which are rarely seen in young mice. Old Kupffer cells showed increased Toll-like receptor-9 expression, and CpG-ODN challenge augmented TNF receptor and Fas-L expression in liver NKT cells. Experiments using mice depleted of natural killer (NK) cells by anti-asialoGM1 antibody (Ab), perforin knockout mice, and mice pretreated with neutralizing interferon (IFN)-gamma Ab demonstrated the important role of liver NK cells in antitumor immunity. The production capacities of old mice for IFN-gamma, IFN-alpha, and perforin were much lower than those of young mice, and the CpG-induced antitumor cytotoxicity of liver NK cells lessened. Lethal shock and MODS greatly decreased in old mice depleted/deficient in TNF, FasL, or NKT cells. However, depletion of NK cells also decreased serum TNF levels and FasL expression of NKT cells, which resulted in improved hepatic injury and survival, suggesting that NK cells are indirectly involved in MODS/lethal shock induced by NKT cells. Neutralization of TNF did not reduce the CpG-induced antitumor effect in the liver. CONCLUSION: Hepatic injury and MODS mediated by NKT cells via the TNF and FasL-mediated pathway after CpG injection increased, but the antitumor activity of liver NK cells decreased with aging.     

Lack of endothelial autophagy does not impair liver regeneration after partial hepatectomy in mice

Liver sinusoidal endothelial cells (LSEC) are key elements in regulating the liver response to injury and regeneration. While endothelial autophagy is essential to protect endothelial cells from injury-induced oxidative stress and fibrosis, its role in liver regeneration has not been elucidated. This study was intended to investigate the role of endothelial autophagy in liver regeneration in the context of partial hepatectomy (PHx). Analysis of autophagy levels in rat LSEC after PHx indicated a tendency to decrease activity the first 2 days after surgery. PHx performed in mice with impaired endothelial autophagy (Atg7^flox/flox;VE-Cadherin-Cre⁺) and their littermate controls showed no differences neither in liver-to-body weight ratio, histological analysis, hepatocyte proliferation nor vascular integrity during the first 7 days after PH and liver regeneration was completely achieved. Our results indicate that endothelial autophagy does not play an essential role in the coordination of the liver regeneration process after PHx.     

Sustained response to interferon-alpha plus ribavirin therapy for chronic hepatitis C is closely associated with increased dynamism of intrahepatic natural killer and natural killer T cells.

Aim: Previous studies have revealed that functional impairment of innate immune cells, including natural killer (NK) and natural killer T (NKT) cells, might be associated with the persistence of hepatitis C virus (HCV) infection. However, the involvement of innate immune cells, which predominate in the liver, in therapeutic HCV clearance is still unclear. Methods: To clarify the role of intrahepatic innate immune cells in the clinical outcome of patients with chronic hepatitis C (CHC) treated with interferon-alpha plus ribavirin (IFN/RBV), we prospectively investigated the status of NK and NKT cells in paired liver biopsy and peripheral blood (PB) samples obtained from 21 CHC patients before and immediately after IFN/RBV treatment by flow cytometry. Normal liver and PB samples were obtained from 10 healthy donors for living donor liver transplantation. Results: Before treatment, intrahepatic NK and NKT cells constituted a significantly lower proportion in CHC patients than in healthy individuals (P < 0.05). After IFN/RBV treatment, the proportions and absolute numbers of CD3(-)CD161(+) NK and CD3(+)CD56(+) NKT cells in the liver, but not in PB, were significantly increased in sustained responders (SR) as compared with poor responders (P < 0.05). The proportion of CD3(+)CD161(+) NKT cells was also increased in the liver of SR after the treatment. Moreover, there was a striking increase of activated CD152(+) cells among CD3(+)CD56(+) NKT cells in the liver of SR (P = 0.041). Conclusion: These findings demonstrate that sustained response to IFN/RBV treatment for patients with CHC is closely associated with increased dynamism of NK and NKT cells in the liver.     

Role of NKT cells and alpha-galactosyl ceramide

Alfa-Galactosyl Ceramide was isolated from Ocean sponge which has antitumor effect against several tumors in in vivo animal model with no cytotoxicity. KRN7000(KRN) is the most potent alpha-Galactosyl Ceramide modified from the one isolated from Ocean sponge. KRN is also active against metastatic tumors through the activation ofanimal immune system. Research efforts in learning the mechanism of action, we found the important role of dendritic cells(DC) and NKT cells. NKT cells was first characterized in 1988 which is overlap some part with NK cells and T-Cells and majority is different from NK and T. KRN is active through the activation of DC and NKT in giving antigen specific immune stimulation in animal. This antigen specific stimulation is memorized by immune system and can reject second tumor challenge. KRN is not active in nude mice and NKT deficient animal. NKT cells level in blood is lower in patients with autoimmune disease, cancer, HIV positive or aplastic anemia. NKT rapidly releases IL-4 and IFN-gamma at high level when activated. NKT is CD1d and TCR restricted. NKT plays important role in autoimmune disease such as Type 1 Diabetes, Scleroderma and Systemic Lupus Erythematosus, infections such as Mycobacteria, Listeria and Malaria, GVHD control and tumor rejection. NKT acts as double edge sword, aggressive and suppressive ways. KRN can prevent the onset of Type 1 Diabetes, inhibit replication of hepatitis virus B in liver and suppress malaria replication in activating NKT cells. KRN can activate NKT through DC and activated NKT activates NK, T and macrophage. KRN also expands NKT cells and expanded NKT has full function. Although the exact role of DC and NKT is not clear, KRN clinical study results in conjunction with DC and NKT cell activation are expected.     

Hepatic gene induction in murine bone marrow after hepatectomy

BACKGROUND/AIMS: Bone marrow cells are highly plastic and differentiate into various cell types, including hepatocytes. To explore the mechanisms underlying these processes, we focused on the initial responses of bone marrow to hepatectomy, using a mouse model. METHODS: To evaluate hepatic differentiation in bone marrow cells we measured hepatocyte-related gene expression in mice undergoing partial hepatectomy with or without pretreatment for 1 week with 2-acetyl aminofluorene (AAF). RESULTS: Hepatectomy induced several genes related to early hepatic differentiation in bone marrow. Expression of these genes was enhanced by the administration of AAF, whereas genes specific for mature hepatocytes were not detected. We characterised the bone marrow cell population expressing hepatocyte differentiation genes. alpha-fetoprotein mRNA was induced in Lin- and either CD34+, c-kit+, Sca-1+, CD49f+ or CD45+ cells. The genes upregulated in the liver after AAF treatment and hepatectomy were identified using oligonucleotide microarrays. These included genes associated with the acute phase response. Dexamethasone inhibited the expression of early hepatic differentiation genes in the bone marrow of AAF/PHx mice. CONCLUSIONS: Early hepatic differentiation genes were induced in bone marrow in response to hepatectomy, especially when regeneration of the remnant liver was suppressed. Circulating signals generated in the AAF/PHx liver might activate this differentiation.     

Role of hepatic stellate cell/hepatocyte interaction and activation of hepatic stellate cells in the early phase of liver regeneration in the rat

BACKGROUND/AIMS: Hepatic stellate cells (HSCs) are known to play a role in hepatic regeneration. We investigated hepatocyte/HSC interaction and HSC activation at various times after 70% partial hepatectomy (PHx) in the rat. METHODS: The hepatic microcirculation was studied using intravital fluorescence microscopy (IVFM). Desmin and alpha-SMA within liver tissue were detected by immunohistochemistry. In isolated parenchymal liver cells (PLCs) and HSCs, double immunostaining was used to identify activated HSC. RESULTS: Using IVFM, hepatocyte-clusters were often seen in vivo at 3 days after PHx (PHx3). Distance between HSC fell from 61.7+/-2.1 microm in controls to 36.1+/-1.4 microm (P<0.001) while the HSC/hepatocyte ratio rose (0.71+/-0.01 to 1.08+/-0.03; P<0.001). In >80% of in vivo microscopic fields in the PHx3 group, clusters of HSCs were observed especially near hepatocyte-clusters. At PHx1 and PHx3, >20% of cells in the PLC-fraction were HSCs which adhered to hepatocytes. At PHx3, in addition to desmin staining, isolated HSCs were also positive for BrdU and alpha-SMA, and formed clusters. HSCs in the HSC-fraction were only positive for desmin which indicated that adherence to hepatocytes is required for HSC activation. CONCLUSIONS: Our data suggest that HSCs are activated by adhering to hepatocytes in the early phase of liver regeneration.     

Liver NK cells expressing TRAIL are toxic against self hepatocytes in mice

Although it is known that activation of natural killer (NK) cells causes liver injury, the mechanisms underlying NK cell-induced killing of self-hepatocytes are not clear. We demonstrated that liver NK cells have cytotoxicity against normal syngeneic hepatocytes in mice. Polyinosinic-polycytidylic acid (poly I:C) treatment enhanced hepatocyte toxicity of liver NK cells but not that of spleen NK cells. Unlike NK cells in other tissues, approximately 30%-40% of liver NK cells constitutively express tumor necrosis factor-related apoptosis-inducing ligand (TRAIL). An in vitro NK cell cytotoxic assay revealed that hepatocyte toxicity of liver NK cells from both na?ve and poly I:C-treated mice was inhibited partially by an anti-TRAIL monoclonal antibody (mAb) alone and completely by the combination with anti-Fas ligand (FasL) mAb and a perforin inhibitor, concanamycin A, indicating contribution of TRAIL to NK cell-mediated hepatocyte toxicity. The majority of TRAIL(+) NK cells lacked expression of Ly-49 inhibitory receptors recognizing self-major histocompatibility complex class I, indicating a propensity to targeting self-hepatocytes. Poly I:C treatment significantly upregulated the expression of Ly-49 receptors on TRAIL(-) NK cells. This might be a compensatory mechanism to protect self-class I-expressing cells from activated NK cell-mediated killing. However, such compensatory alteration was not seen at all in the TRAIL(+) NK cell fraction. Thus, liver TRAIL(+) NK cells have less capacity for self-recognition, and this might be involved in NK cell-dependent self-hepatocyte toxicity. In conclusion, our findings are consistent with a model in which TRAIL-expressing NK cells play a critical role in self-hepatocyte killing through poor recognition of MHC.