Liver-resident Natural Killer Research Articles

The characteristics of liver hematopoietic progenitor cells (HEM5P.231)

Abstract During fetal life, hematopoiesis majorly occurs in the liver. After birth, the site of hematopoiesis shifts from fetal liver to the bone marrow (BM). However, some recent studies have shown that the adult liver also contains stem cell population with potent hematopoietic-reconstitution potential. To investigate the properties of adult liver hematopoiesis, we compared the phenotype of liver lineage (Lin)-negative progenitor cells with that of BM counterparts. We found that Lin- cells in the adult liver are phenotypically distinct from BM Lin- cells. Moreover, transfer of fetal liver cells, but not adult BM cells, can repopulate these liver progenitor cells with unique phenotype. In addition, hematopoietic progenitor cells in the liver and BM exhibit differential transcription factor-expression. Liver Lin- progenitor cells express much higher levels of promyelocytic leukemia zinc finger (PLZF), than their BM counterparts. Liver Lin-PLZF+ progenitors express high levels of Sca-1 and, in contrast to BM Lin-PLZF+ cells, lack c-kit and α4β7. Besides PLZF, another transcription factor, namely T-bet, is also expressed at higher levels by liver progenitors cells than their BM counterparts. Therefore, given the essential roles of PLZF and T-bet in the development of liver-resident natural killer (NK) cells, our data suggest that the unique characteristics of liver hematopoietic progenitor cells may contribute to liver-resident NK cell development.

Intrahepatic development of liver-resident natural killer cells (LYM2P.739)

Abstract Natural killer (NK) cells are an important component of the innate immune system and play critical roles in host defense against infections and tumors. Accumulating studies have shown that the NK cell pool is highly heterogeneous, consisting of different cell subsets with distinct phenotypic and functional features. Recently, in collaboration with Professor Wayne Yokoyama, we showed that a novel NK cell subset expressing CD49a and lacking DX5 rarely recirculates and selectively resides in the liver, that is, liver-resident NK cells. While bone marrow (BM) cells are efficient in generating conventional (cNK) cells, the frequency of liver-resident NK cells is significantly reduced after BM transplantation. Conversely, lethally irradiated mice adoptively transferred with fetal liver cells exhibit normal frequency of liver-resident NK cells. Further analyses reveal that the adult liver retains hematopoietic potential and contains a few hematopoietic progenitor cells (HPCs), which are phenotypically distinct from BM lineage (Lin)-negative progenitor cells but resemble fetal liver counterparts. Moreover, adoptive transfer of these adult liver HPCs have the capacity to generate liver-resident NK cells, but not cNK cells. Finally, Liver HPCs express higher levels of the transcription factors that are essential for liver-resident NK cell development than their BM counterparts. Thus, our studies suggest an alternative development pathway for liver-resident NK cells.

Fasting enhances TRAIL-mediated liver natural killer cell activity via HSP70 upregulation.

Acute starvation, which is frequently observed in clinical practice, sometimes augments the cytolytic activity of natural killer cells against neoplastic cells. In this study, we investigated the molecular mechanisms underlying the enhancement of natural killer cell function by fasting in mice. The total number of liver resident natural killer cells in a unit weight of liver tissue obtained from C57BL/6J mice did not change after a 3-day fast, while the proportions of tumor necrosis factor–related apoptosis-inducing ligand (TRAIL)+ and CD69+ natural killer cells were significantly elevated (n = 7, p <0.01), as determined by flow cytometric analysis. Furthermore, we found that TRAIL− natural killer cells that were adoptively transferred into Rag-2−/− γ chain−/− mice could convert into TRAIL+ natural killer cells in fasted mice at a higher proportion than in fed mice. Liver natural killer cells also showed high TRAIL-mediated antitumor function in response to 3-day fasting. Since these fasted mice highly expressed heat shock protein 70 (n = 7, p <0.05) in liver tissues, as determined by western blot, the role of this protein in natural killer cell activation was investigated. Treatment of liver lymphocytes with 50 µg/mL of recombinant heat shock protein 70 led to the upregulation of both TRAIL and CD69 in liver natural killer cells (n = 6, p <0.05). In addition, HSP70 neutralization by intraperitoneally injecting an anti- heat shock protein 70 monoclonal antibody into mice prior to fasting led to the downregulation of TRAIL expression (n = 6, p <0.05). These findings indicate that acute fasting enhances TRAIL-mediated liver natural killer cell activity against neoplastic cells through upregulation of heat shock protein 70.

Adaptive immunity mediated by Natural Killer cells. (45.20)

Abstract Adaptive immunity is thought to be a unique feature of T and B lymphocytes. However, there is mounting evidence that adaptive immunity can be mediated by a discrete subset of liver-resident natural killer (NK) cells. To date, Rag-independent NK memory has been documented for four distinct haptens and six viral antigens (Ag). We have developed a sorting strategy to identify and isolate Ag-specific NK cells from livers of Rag-/- mice sensitized with the hapten DNFB. By transplanting nuclei from sorted Rag-/- NK cells into enucleated oocytes, embryonic stem (ES) cell lines were generated that were used to clone mice. NK cells from the cloned animals instantly responded to DNFB without requiring prior sensitization, but they could not be sensitized to other Ags. Thus, the nuclear 'information' for Ag specificity of memory NK cells persists even in animals generated from a donor nucleus whose epigenetic state was reset by nuclear reprogramming. This apparent genetic fixation would be expected if Ag receptor specificity were encoded at the level of genomic DNA, rather than by epigenetic regulation of conventional 'hardwired' NK receptors, and suggest an entirely novel mechanism to generate Ag receptor diversity. Thus, current views of the immune system as a duality of adaptive (T and B cells) and innate (everything else) arms, need to be re-imagined as a trinity that includes memory NK cells as a third branch.

Preactivation of NKT cells with α-GalCer protects against hepatic ischemia-reperfusion injury in mouse by a mechanism involving IL-13 and adenosine A2Areceptor

Hepatic preconditioning has emerged as a promising strategy of activating natural pathways to augment tolerance to liver ischemia-reperfusion (IR) injury. Liver-resident natural killer T (NKT) cells play an important role in modulating the local immune and inflammatory responses. This work was aimed to investigate whether preactivation of NKT cells could provide a beneficial "preconditioning" effect to ameliorate the subsequent hepatic IR injury. To selectively activate NKT cells, C57BL/6 mice were treated intraperitoneally with the glycolipid antigen alpha-galactosylceramide (alpha-GalCer) 1 h prior to hepatic ischemia. Significantly reduced liver IR injury was observed in mice pretreated with alpha- GalCer, and this protective effect was specifically abrogated by a CD1d blocking antibody. Serum TNF-alpha, IFN-gamma, and IL-13 levels were markedly increased shortly after alpha-GalCer injection. Pretreatment with a neutralizing antibody against TNF-alpha or IFN-gamma did not influence the protective effect of alpha-GalCer preconditioning, whereas preadministration of an IL-13 neutralizing antibody completely abolished the effect. Treatment with alpha-GalCer also led to an increased expression of adenosine A2A receptor (A2AR) in the liver, and blockade of A2AR by SH58261 diminished alpha-GalCer pretreatment-mediated attenuation of liver IR injury. In contrast, administration of the selective A2AR agonist CGS21680 reversed the counteracting effect of the IL-13 neutralizing antibody on alpha-GalCer preconditioning. Additionally, alpha-GalCer pretreatment was associated with a decreased neutrophil accumulation in the ischemic liver. These findings provide the first evidence that hepatic preconditioning by preactivation of NKT cells with alpha-GalCer protects the liver from IR injury via an IL-13 and adenosine A2AR-dependent mechanism.

Mammalian augmenter of liver regeneration protein is a sulfhydryl oxidase.

Augmenter of Liver Regeneration is an important secondary hepatic growth factor. Augmenter of liver regeneration protein has been shown to control mitochondrial gene expression and the lytic activity of liver-resident Natural Killer cells through the levels of interferon-gamma, but the precise enzymatic function of this protein is unknown. To define the enzymatic activity of augmenter of liver regeneration protein. The carboxy terminus of augmenter of liver regeneration protein contains a special CXXC motif characteristic for redox proteins and with faint homologies to the redox-active site of sulfhydryl oxidases. Tests were, therefore, carried out to establish whether isolated augmenter of liver regeneration protein can also function in the formation of sulfur bridges. Purified augmenter of liver regeneration proteins from rat and human were tested in enzyme assays for the ability to introduce disulfide bonds into protein substrates. The isolated proteins were tested for the formation of dimers and the presence of bound FAD was investigated spectroscopically. The function of the conserved CXXC motif was investigated by in vitro mutagenesis experiments and subsequent enzyme assays. In this study, we demonstrate that rat and human augmenter of liver regeneration protein are flavin-linked sulfhydryl oxidases that catalyze the formation of disulfide bonds in reduced protein substrates. A flavin moiety is firmly but not covalently attached to the protein. In human cell cultures augmenter of liver regeneration protein is expressed in a long and short form that both exist as covalently linked dimers. The active site of the enzyme is associated with a conserved CXXC motif in the carboxy-terminal domain, that is present in the homologous proteins from yeast to humans and also in the human Q6 growth regulator protein. In vitro mutagenesis of one cysteine residue in the CXXC motif results in loss of enzymatic function and the mutated protein no longer binds FAD. For the first time, these data assign an enzymatic activity to the important hepatic growth factor augmenter of liver regeneration protein. The finding that augmenter of liver regeneration protein acts as a FAD-linked sulfhydryl oxidase is essential to identify the molecular targets inside liver cells and to elucidate the precise role of mammalian augmenter of liver regeneration protein in hepatic cell growth, liver disease and regeneration.

Molecular mechanisms of augmenter of liver regeneration as immunoregulator: its effect on interferon-γ expression in rat liver

Background. We have shown that the administration of exogenous Augmenter of Liver Regeneration protein in intact rats i) regulates mitochondrial gene expression by inducing the transcription and translation of the nuclear-encoded mitochondrial transcription factor A, and ii) inhibits the lytic activity of liver-resident Natural Killer cells. Aims. The present investigation was carried out to study the effect, in intact rats, of exogenous administration of Augmenter of Liver Regeneration protein on Interferon-γ, a cytokine produced by activated Natural Killer cells and known to control the expression of mitochondrial transcription factor A, a nuclear gene responsible for mitochondrial metabolism. Methods. Interferon-γ was measured as messenger RNA in liver-derived mononuclear leukocytes and as protein in liver-derived Natural Killer cells after a single injection of Augmenter of Liver Regeneration protein. Results. The data obtained demonstrate that: i) in intact rats, Augmenter of Liver Regeneration protein administration induces a reduction of Interferon-γ in the liver-resident Natural Killer cells and ii) the administration of Interferon-γ in 70% hepatectomized rats is followed by a significant reduction both of the mitochondrial transcription factor A expression and of liver regeneration. Conclusions. These data demonstrate the pivotal role of Augmenter of Liver Regeneration as Growth Factor and as immunoregulator by controlling, through Interferon-γ levels, the mitochondrial transcription factor A expression and the lytic activity of liver-resident Natural Killer cells.

FK506 promotes liver regeneration by suppressing natural killer cell activity.

We examined the mechanism of promotion of liver regeneration by tacrolimus hydrate (FK506), a potent immunosuppressant, after partial hepatectomy. The administration of FK506 significantly increased the bromodeoxyuridine (BrdU) labelling index at 36 and 48 h after 70% hepatectomy compared with the placebo group. Using the same model, we examined the effect of FK506 on the expression of hepatocyte growth factor (HGF) and transforming growth factor-beta 1 (TGF-beta 1) and found no changes in HGF and TGF-beta 1 mRNA expression in the liver or in the HGF protein concentration in plasma. We found that pretreatment with FK506 markedly reduced the activity and number of liver-resident natural killer (NK) cells at the time of partial hepatectomy. Our observations suggest that the promotion of liver regeneration by FK506 may be attributable to a reduction in the number of liver-resident NK cells and to inhibition of their activity.

Crystallization and preliminary crystallographic data for the augmenter of liver regeneration.

A new cellular growth factor termed augmenter of liver regeneration (ALR) has been crystallized. ALR has been shown to have a proliferative effect on liver cells while at the same time producing an immunosuppressive effect on liver-resident natural killer cells and liver-resident mononuclear leukocytes. In addition, ALR appears to play an important role in the synthesis and stabilization of mitochondrial gene transcripts in actively regenerating cells. ALR crystals diffract to beyond 2 A resolution and belong to space group P2(1)2(1)2, with a = 125.1, b = 108.1 and c = 38.5 A. Based on four molecules per asymmetric unit, the Matthews coefficient is calculated to be 2.16 A(3) Da(-1) which corresponds to a solvent content of 43%.

The in vivo effect of hepatotrophic factors augmenter of liver regeneration, hepatocyte growth factor, and insulin-like growth factor-II on liver natural killer cell functions.

Fine balanced sequential changes of the levels of circulating hepatotrophic factors are essential for normal liver regeneration. Our recent studies have indicated that liver-resident natural killer (NK) cells are important regulators of liver regeneration and have raised the possibility that hepatotrophic factors might mediate their activities through NK cells. In the present study, we assessed the effects of in vivo administration of three hepatotrophic factors (augmenter of liver regeneration [ALR], insulin-like growth factor-II [IGF-II], and hepatocyte growth factor [HGF]) on NK cells in normal rats. Each of the three, given over a 1-day period in doses known to produce hepatotrophic activity, induced inhibition of NK cell cytotoxic activities in the population of mononuclear leukocytes (MNL) in the liver, but not in MNL from the spleen or peripheral blood. In contrast to these results obtained by the whole animal treatment, the three molecules had no effect on NK cell functions when added to cultures of MNL from the livers, spleens, or blood of untreated rats. These data support and extend our previously advanced hypothesis that ALR and other hepatotrophic factors play an important role in liver regeneration by regional regulation of NK cells through some as-yet-unknown intermediary mechanism.

The in vivo effect of hepatotrophic factors augmenter of liver regeneration, hepatocyte growth factor, and insulin-like growth factor-II on liver natural killer cell functions

Fine balanced sequential changes of the levels of circulating hepatotrophic factors are essential for normal liver regeneration. Our recent studies have indicated that liver-resident natural killer (NK) cells are important regulators of liver regeneration and have raised the possibility that hepatotrophic factors might mediate their activities through NK cells. In the present study, we assessed the effects of in vivo administration of three hepatotrophic factors (augmenter of liver regeneration [ALR], insulin-like growth factor-II [IGF-II], and hepatocyte growth factor [HGF]) on NK cells in normal rats. Each of the three, given over a 1-day period in doses known to produce hepatotrophic activity, induced inhibition of NK cell cytotoxic activities in the population of mononuclear leukocytes (MNL) in the liver, but not in MNL from the spleen or peripheral blood. In contrast to these results obtained by the whole animal treatment, the three molecules had no effect on NK cell functions when added to cultures of MNL from the livers, spleens, or blood of untreated rats. These data support and extend our previously advanced hypothesis that ALR and other hepatotrophic factors play an important role in liver regeneration by regional regulation of NK cells through some as-yet-unknown intermediary mechanism.(Hepatology 1997 Feb;25(2):411-5)