Liver Natural Killer Cells Research Articles

Neutralization of IL-10 Restores the Downregulation of IL-18 Receptor on Natural Killer Cells and Interferon-γ Production in Septic Mice, Thus Leading to an Improved Survival

The objective of the study was to investigate the mechanisms of insufficient interferon-γ (IFN-γ) response to interleukin 18 (IL-18) and the treatment for the insufficient response in septic mice. Interleukin 18 stimulation does not restore IFN-γ production by blood mononuclear cells in septic patients but does restore its production in postoperative patients. Although sepsis impairs the IFN-γ response to IL-18, little is known about why the IL-18/IFN-γ-mediated immune response is ineffective in patients with sepsis. A cecal ligation and puncture was made in C57BL/6 mice following a sublethal lipopolysaccharide challenge to examine their IFN-γ response to IL-18, focusing on natural killer (NK) cells and cytokines. We next examined the effect of neutralization of IL-10 on the NK cell and survival in septic mice. Interleukin 18 injection did not restore IFN-γ production in septic (cecal ligation and puncture) mice. Despite an increase in the numbers of liver NK cells, the IL-18 receptor (IL-18R) expression was decreased in the septic mice compared with sham mice. Serum IL-10 levels were positively correlated with the percentage of liver NK cells, but negatively with their IL-18R expression. Neutralization of IL-10 restored the IL-18R expression on liver NK cells and restored the IFN-γ response in the septic mice, improving their survival. Sepsis might impair IL-18R expression on liver and spleen NK cells and impair the IL-18-mediated IFN-γ response. Neutralization of IL-10 may restore this response in septic hosts, thereby improving survival.

Mechanistic analysis of the antitumor efficacy of human natural killer cells against breast cancer cells

We investigated the role of human natural killer (NK) cells in the peripheral blood (PB) and liver in controlling breast cancer. The proportion of NK cells among liver mononuclear cells was significantly higher than among PB mononuclear cells. Liver NK cells inductively expressed higher levels of tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) than PB NK cells in response to interleukin-2 (IL-2). Liver NK cells displayed higher cytotoxicity against various breast cancer cell lines (MDA-MB231, MDA-MB453, MDA-MB468, and MCF-7) after IL-2 stimulation than did PB NK cells. Anti-HER2 monoclonal antibody (mAb) promoted the cytotoxicity of both the types of NK cells toward HER2-expressing cell lines. All breast cancer cell lines highly expressed death-inducing TRAIL receptors, death receptor 4, but did not express death-inhibitory receptors (DcR1 and DcR2). Both PB and liver NK cell-induced cytotoxicity was inhibited partially by anti-TRAIL mAb and more profoundly by the combination of anti-TRAIL mAb and concanamycin A, indicating that TRAIL and perforin are involved. IL-2-stimulated liver and PB NK cells exhibited upregulated expression of CXCR3, which bind to the chemokines CXCL9, CXCL10, and CXCL11 secreted by breast cancer cells. We also found that IFN-γ promoted the production of CXCL10 from breast cancer cells. The results of this study show that IFN-γ secreted from NK cells likely promotes the production of CXCL10 from breast cancer cells, which in turn accelerates the migration of CXCR3-expressing NK cells into the tumor site. These findings suggest the possibility of a therapeutic approach by either activation of endogenous PB and liver NK cells or adoptive transfer of in vitro-activated autologous NK cells.

Hepatic macrophage migration and differentiation critical for liver fibrosis is mediated by the chemokine receptor C-C motif chemokine receptor 8 in mice

Chemokines critically control the infiltration of immune cells upon liver injury, thereby promoting hepatic inflammation and fibrosis. The chemokine receptor CCR8 can affect trafficking of monocytes/macrophages, monocyte-derived dendritic cells (DCs) and T-helper cell (Th) subsets, but its role in liver diseases is currently unknown. To investigate the functional role of CCR8 in liver diseases, ccr8(-/-) and wild-type (WT) mice were subjected to chronic experimental injury models of carbon tetrachloride (CCl(4) ) administration and surgical bile duct ligation (BDL). CCR8 was strongly up-regulated in the injured liver. Ccr8(-/-) mice displayed attenuated liver damage (e.g., ALT, histology, and TUNEL) compared to WT mice and were also protected from liver fibrosis in two independent injury models. Flow cytometry revealed reduced infiltrates of liver macrophages, neutrophils and natural killer cells, whereas hepatic CD4(+) T cells increased. The main CCR8-expressing cells in the liver were hepatic macrophages, and CCR8 was functionally necessary for CCL1-directed migration of inflammatory but not for nonclassical monocytes into the liver. Moreover, the phenotype of liver macrophages from injured ccr8(-/-) animals was altered with increased expression of DC markers and enhanced expression of T-cell-attracting chemokine macrophage inflammatory protein 1-alpha (MIP-1α/CCL3). Correspondingly, hepatic CD4(+) T cells showed increased Th1 polarization and reduced Th2 cells in CCR8-deficient animals. Liver fibrosis progression, but also subsequent T-cell alterations, could be restored by adoptively transferring CCR8-expressing monocytes/macrophages into ccr8(-/-) mice during experimental injury. CCR8 critically mediates hepatic macrophage recruitment upon injury, which subsequently shapes the inflammatory response in the injured liver, affecting macrophage/DC and Th differentiation. CCR8 deficiency protects the liver against injury, ameliorating initial inflammatory responses and hepatic fibrogenesis. Inhibition of CCR8 or its ligand, CCL1, might represent a successful therapeutic target to limit liver inflammation and fibrosis progression.

Fate mapping analysis of lymphoid cells expressing the NKp46 cell surface receptor

NKp46 is a cell surface receptor expressed on natural killer (NK) cells, on a minute subset of T cells, and on a population of innate lymphoid cells that produce IL-22 and express the transcription factor retinoid-related orphan receptor (ROR)-γt, referred to as NK cell receptor (NKR)(+)ROR-γt(+) cells. Here we describe Nkp46(iCre) knock-in mice in which the gene encoding the improved Cre (iCre) recombinase was inserted into the Nkp46 locus. This mouse was used to noninvasively trace cells expressing NKp46 in vivo. Fate mapping experiments demonstrated the stable expression of NKp46 on NK cells and allowed a reappraisal of the sequential steps of NK cell maturation. NKp46 genetic tracing also showed that gut NKR(+)ROR-γt(+) and NK cells represent two distinct lineages. In addition, the genetic heterogeneity of liver NK cells was evidenced. Finally, Nkp46(iCre) mice also represent a unique mouse model of conditional mutagenesis specifically in NKp46(+) cells, paving the way for further developments in the biology of NKp46(+) NK, T, and NKR(+)ROR-γt(+) cells.

Donor liver natural killer cells alleviate liver allograft acute rejection in rats

Liver enriched natural killer (NK) cells are of high immune activity. However, the function of donor liver NK cells in allogeneic liver transplantation (LTx) remains unclear. Ten Gy of whole body gamma-irradiation (WBI) from a 60Co source at 0.6 Gy/min was used for depleting donor-derived leukocytes, and transfusion of purified liver NK cells isolated from the same type rat as donor (donor type liver NK cells, dtlNKs) through portal vein was performed immediately after grafting the irradiated liver. Post-transplant survival observation on recipients and histopathological detection of liver grafts were adoptive to evaluate the biological impact of donor liver NK cells on recipients' survival in rat LTx. Transfusion of dtlNKs did not shorten the survival time among the recipients of spontaneous tolerance model (BN to LEW rat) after rat LTx, but prolonged the liver graft survival among the recipients depleted of donor-derived leukocytes in the acute rejection model (LEW to BN rat). Compared to the recipients in the groups which received the graft depleted of donor-derived leukocytes, better survival and less damage in the allografts were also found among the recipients in the two different strain combinations of liver allograft due to transfusion of dtlNKs. Donor liver NK cells alone do not exacerbate liver allograft acute rejection. Conversely, they can alleviate it, and improve the recipients' survival.

Early Changes in Natural Killer Cell Function Indicate Virologic Response to Interferon Therapy for Hepatitis C

Mathematical modeling of hepatitis C virus (HCV) kinetics indicated that cellular immune responses contribute to interferon (IFN)-induced clearance of HCV. We investigated a potential role of natural killer (NK) cells in this process. Phenotype and function of blood and liver NK cells were studied during the first 12 weeks of treatment with pegylated IFN-alfa and ribavirin, the time period used to define the early virological response. Within hours of treatment initiation, NK cells of patients that had an early virological response increased expression of activating receptors NKG2D, NKp30, and CD16 and decreased expression of NKG2C and 2B4, along with inhibitory receptors SIGLEC7 and NKG2A, resulting in NK cell activation. NK cell cytotoxicity, measured by degranulation and tumor necrosis factor-related apoptosis-inducing ligand production, peaked after 24 hours (P<.01), concomitant with an increase in alanine aminotransferase levels (P<.05), whereas IFN-γ production decreased within 6 hours and did not recover for more than 4 weeks (P<.05). NK cells from liver biopsies taken 6 hours after treatment initiation had increased numbers of cytotoxic CD16+NK cells (P<.05) and a trend toward increased production of tumor necrosis factor-related apoptosis-inducing ligand. Degranulation of peripheral blood NK cells correlated with treatment-induced, first-phase decreases in viral load (P<.05) and remained higher in early virological responders than in nonresponders for weeks. IFN activates NK cells early after treatment is initiated. Their cytotoxic function, in particular, is strongly induced, which correlates to virologic response. Therefore, NK cell activation indicates responsiveness to IFN-α-based treatment and suggests the involvement of the innate immune cells in viral clearance.

A novel concept of adoptive immunotherapy using interleukin-2-stimulated liver natural killer cells for treatment of liver transplantation with hepatocellular carcinoma.

e13006 Background: Tumor recurrence is the main limitation of liver transplantation (LT) in patients with hepatocellular carcinoma (HCC) and can be promoted by immunosuppressants. However, there is no prevention or treatment for HCC recurrence after LT. Here, we describe a novel concept of adoptive immunotherapeutic approach that uses natural killer (NK) cells derived from deceased donor liver graft perfusate to prevent tumor recurrence after LT. Methods: To determine whether NK cells extracted from deceased donor liver graft perfusate acquire anti-tumor effect, we demonstrated the phenotypical and functional properties of activated liver NK cells under current Good Manufacturing Practice (cGMP) conditions. Results: Liver mononuclear cells (LMNC) that were extracted from deceased donor liver graft perfusate contained a large percentage of NK cells (45.0 ± 4.0 %) compared with peripheral blood mononuclear cells (PBMC) (21.8 ± 5.2 %) from the same donor. The CD69 activation marker and the natural cytotoxicity receptors, NKp44 and NKp46, were expressed at high levels in freshly isolated liver NK cells. Furthermore, interleukin (IL)-2-stimulated NK cells showed greater upregulation of activation markers and the tumor necrosis factor-related apoptosis-inducing ligand (TRAIL), which is critical for NK cell-mediated anti-tumor cell death and increased production of interferon. Moreover, IL-2 stimulation induced LMNC to exhibit a stronger cytotoxicity against NK-susceptible K562 target cells compared with PBMC (p < 0.01). Finally, we also showed that the final product contained a very low T-cell contamination (0.02 × 106 cells×kg-1), which could be acceptable in allogeneic cell transplantation. Conclusions: It was strongly suggested that the adoptive transfer of IL-2 stimulated NK cells from deceased donor liver graft perfusate could be safe and effective in LT patients with HCC.

Liver Natural Killer Cells Play a Role in the Destruction of Islets After Intraportal Transplantation

A significant loss of islet function observed in type 1 diabetic recipients after intraportal islet transplantation raises a question about the suitability of the liver as a transplant site. We hypothesize that natural killer (NK) cells in the liver play a role in the islet graft destruction. Phenotypical and functional differences between liver and splenic NK cells isolated from mice were examined by flow cytometry and in vitro cytotoxicity assays. In vivo, the role of liver NK cells was determined by examining the function of intraportally administered islet iso- and allografts by treating recipients with anti-asialo GM1 to deplete NK cells. NK cell-depleted diabetic C57BL/6 mice receiving 400 syngeneic islets into the liver rapidly ameliorated hyperglycemia, whereas control recipients did not. The same number of BALB/c islets grafted in the liver of diabetic nonimmunosuppressed C57BL/6 mice failed to function, whereas NK cell-depleted recipients reversed hyperglycemia for up to 10 days. NK cells from the liver of naive C57BL/6 mice showed significantly higher cytotoxicity than splenic NK cells as tested with a β-cell line and allogeneic islets. The cell proportion and the expression level of activation markers on liver NK cells significantly increased after intraportal islet transplantation as compared with the control. Liver NK cells also increased anti-islet cytotoxicity, but not splenic NK cells, after islet transplantation. Our results clearly show the destructive activity of liver NK cells toward islets, suggesting that NK cells play a role in early islet graft loss after intraportal islet transplantation.

Synergy between TLR3 and IL-18 promotes IFN-y dependent TRAIL expression in human liver NK cells (108.6)

Abstract Toll-like receptor 3 (TLR3), which recognizes double-stranded RNA (dsRNA), was the first identified antiviral TLR, and because dsRNA is a universal viral molecular pattern, TLR3 has been assumed to have a central role in the host response to viruses. Natural killer (NK) cells are a component of innate immunity against viral infections through their rapid cytotoxic activity and cytokine production. The synthetic dsRNA polyinosinic-polycytidylic acid (poly I:C), a mimic of a common product of viral infections, is known to rapidly up-regulate their in vivo functions; however, intrahepatic NK cells’ ability to respond to dsRNA is still mostly unknown. Our results show that poly I:C activates liver NK cells directly in the context of cytokines found in the liver, i.e: poly I:C plus inflammatory cytokines (IL-18, IL-12, and IL-2) induced NK cell IFN-g production and TRAIL expression, and anti-inflammatory cytokines (TGF-β and IL-10) inhibit NK cell IFN-γ production. Neutralization of IFN-g blocks poly I:C plus inflammatory cytokines-induced NK cell TRAIL expression, suggesting that IFN-g is an autocrine differentiation factor for these cells. A better understanding of the intrahepatic NK cell activation against viral infection may help in the design of therapies and vaccines for the control of viral hepatitis.

CD27‐deficient mice show normal NK‐cell differentiation but impaired function upon stimulation

Natural killer (NK) cells are part of the first line defense against tumors, parasites and virus-infected cells. Therefore, factors that control NK-cell numbers and their function are important. CD27 is constitutively expressed on NK cells and its expression correlates with sequential phases in NK-cell development, discriminating phenotypically and functionally different subsets within the NK-cell population. Although CD27 has been described to have an important regulatory role in effector and memory T and B lymphocytes, its role in NK-cell biology remains to be addressed. In this study, we used CD27(-/-) mice to investigate the role of CD27 in NK-cell development and function, both during the resting state and upon stimulation. The results show that NK-cell numbers are not impaired in CD27(-/-) mice. Moreover, CD27(-/-) NK cells reach full phenotypic maturity, evidenced by normal expression of CD49b, CD43 and CD11b. Expression of activating receptors is unaltered, whereas expression of several inhibitory receptors is increased. Cytotoxicity and interferon-γ production by NK cells from CD27(-/-) mice in the resting state are normal. However, upon in vivo anti-CD40- or poly-I:C-mediated activation, or in vitro interleukin-15 priming plus anti-NKp46 stimulation, the absence of CD27 results in decreased cytolytic activity and cytokine production by spleen and liver NK cells. In conclusion, this study demonstrates that CD27 is dispensable for the development of functional NK cells. However, upon stimulation of NK cells, CD27 displays an important role in their activation and functionality.

Synergy between TLR3 and IL-18 promotes IFN-γ dependent TRAIL expression in human liver NK cells

Natural killer (NK) cells are a component of innate immunity against viral infections through their rapid cytotoxic activity and cytokine production. However, intra-hepatic NK cells’ ability to respond to virus is still mostly unknown. Our results show that the synthetic dsRNA polyinosinic–polycytidylic acid (poly I:C), a mimic of a common product of viral infections, activates NK cells directly in the context of cytokines found in the liver, i.e.: poly I:C plus inflammatory cytokines (IL-18, IL-12, and IL-2) induced NK cell IFN-γ production and TRAIL expression, and anti-inflammatory cytokines (TGF-β and IL-10) inhibit NK cell IFN-γ production. Neutralization of IFN-γ blocks poly I:C plus inflammatory cytokines-induced NK cell TRAIL expression, suggesting that IFN-γ is an autocrine differentiation factor for these cells. A better understanding of the intra-hepatic NK cell activation against viral infection may help in the design of therapies and vaccines for the control of viral hepatitis.

A novel concept of adoptive immunotherapy with tumor necrosis factor-related, apoptosis-inducing, ligand-expressing natural killer cells for eliciting an anti-breast-cancer response.

e13048 Background: Previous studies have demonstrated that natural killer (NK) cells can destroy breast cancer cells. In the present study, we have reported that liver NK cells exhibit greater cytotoxic activity against tumor cells than do peripheral blood (PB) NK cells in humans. Methods: We investigated the phenotypic and functional properties of NK cells extracted from liver perfusates of living donors in clinical liver transplantation. Next, the susceptibility of various breast cancer cell lines to the activity of the liver and PB NK cells was tested. Results: Stimulation with IL-2 increased the expression of the tumor necrosis factor (TNF)-related apoptosis-inducing ligand (TRAIL) on both the PB and liver NK cells. Accordingly, both the liver and PB NK cells exhibited strong cytotoxicity against various breast cancer cell lines (MDA-MB231, MDA-MB453, MDA-MB468, and MCF-7) after in vitro stimulation with IL-2. Further treatment with anti-HER2 monoclonal antibodies (mAbs) remarkably promoted the NK cell-mediated cytotoxicity toward the HER2-expressing breast cancer cell line MDA-MB453. All the breast cancer cell lines expressed remarkable levels of a death-inducing TRAIL receptor—death receptor (DR)4 and 5—which contains cytoplasmic death domains and mediates apoptosis, but they did not express the death-inhibitory receptors (DcRs) 1 and 2. Treatment with anti-TRAIL neutralizing mAbs partially reduced the liver and PB NK cell-mediated cytotoxicity toward breast cancer cells (32% ± 9%, 64% ± 15% reduction respectively at E:T = 5:1); this finding indicated the involvement of TRAIL-induced NK cell-mediated cytotoxicity. The expression of CXC chemokine receptor 3 (CXCR3), which binds to chemokines CXCL9, CXCL10, and CXCL11 secreted by breast cancer cells, was upregulated in the IL-stimulated NK cells. Conclusions: TRAIL+ NK cells kill breast cancer cells via death receptor/ligand interactions. This finding provides the basis for a novel concept, i.e., adoptive transfer of IL-2-stimulated NK cells to breast cancer patients for eliciting an anti-breast-cancer response. No significant financial relationships to disclose.

Liver grafts contain a unique subset of natural killer cells that are transferred into the recipient after liver transplantation

In contrast to other solid organ transplantations, liver grafts have tolerogenic properties. Animal models indicate that donor leukocytes transferred into the recipient after liver transplantation (LTX) play a relevant role in this tolerogenic phenomenon. However, the specific donor cell types involved in modulation of the recipient alloresponse are not yet defined. We hypothesized that this unique property of liver grafts may be related to their high content of organ-specific natural killer (NK) and CD56(+) T cells. Here, we show that a high proportion of hepatic NK cells that detach from human liver grafts during pretransplant perfusion belong to the CD56bright subset, and are in an activated state (CD69(+)). Liver NK cells contained perforin and granzymes, exerted stronger cytotoxicity against K562 target cells when compared with blood NK cells, and secreted interferon-gamma, but no interleukin-10 or T helper 2 cytokines, upon stimulation with monokines. Interestingly, whereas the CD56bright subset is classically considered as noncytolytic, liver CD56bright NK cells showed a high content of cytolytic molecules and degranulated in response to K562 cells. After LTX, but not after renal transplantation, significant numbers of donor CD56dim NK and CD56(+) T cells were detected in the recipient circulation for approximately 2 weeks. In conclusion, during clinical LTX, activated and highly cytotoxic NK cells of donor origin are transferred into the recipient, and a subset of them mixes with the recirculating recipient NK cell pool. The unique properties of the transferred hepatic NK cells may enable them to play a role in regulating the immunological response of the recipient against the graft and therefore contribute to liver tolerogenicity.

Regulatory natural killer cells in murine liver and their immunosuppressive capacity

Abundant amounts of natural killer (NK) cells are present in the liver, most of which are endowed with direct cytotoxic and inflammatory cytokine production capacities. However, the control of compromised immunity in the liver may be accomplished by a population of regulatory NK cells possessing suppressive or tolerogenic functions. To identify and characterize regulatory NK cells in murine liver. NK cells were isolated from the liver of C57BL/6 mice by magnetic-activated cells sorting (MACS). NK cells were stimulated with different agents and those cells that produced interleukin (IL)-10 were detected by flow cytometry and isolated by MACS. IL-10-producing NK cells were regarded as regulatory NK cells and the functional capacities of liver-derived regulatory NK cells were assessed in vitro. The frequencies of regulatory NK cells in the liver were 4.1 +/- 0.3% of hepatic NK cells and 0.45 +/- 0.02% of liver nonparenchymal cells. Regulatory NK cells produced abundant amounts of IL-10 in culture. These cells also suppressed the proliferative capacities of T cells and B cells in vitro. However, another population of NK cells that did not produce IL-10 (immunogenic NK cells) could not suppress lymphocyte proliferation. The presence of regulatory NK cells in the liver and their immunosuppressive capacities endowed these cells with the critical function of maintaining homeostasis under normal conditions. Exaggerated or impaired functions of these cells may also contribute to different pathological processes.

Involvement of Hepatic Innate Immunity in Alcoholic Liver Disease

Excessive alcohol consumption is one of the critical causative factors leading to alcoholic liver disease (ALD). ALD is characterized by a wide spectrum of liver damage, ranging from simple uncomplicated liver steatosis (fatty liver) to steatohepatitis and liver fibrosis/cirrhosis. It has been believed that the obvious underlying cause for ALD is due to hepatocyte death induced by alcohol itself. However, recent sparkling studies have shown that diverse immune responses contribute to ALD because liver is enriched with numerous immune cells. Especially, a line of evidence has suggested that innate immune cells such as Kupffer cells and natural killer (NK)/NKT cells are significantly involved in the pathogenesis of ALD via production of pro-inflammatory cytokines and other mediators. Indeed, more interestingly, hepatic stellate cells (HSCs), known as a major cell inducing liver steatosis and fibrosis, can be killed by liver NK cells, which could be suppressed by chronic alcohol consumption. In this review, with the view of liver as predominant innate immune organ, we describe the pathogenesis of ALD in which what roles of innate immune cells are and how they are interacting with HSCs.

A potential role for RAG‐1 in NK cell development revealed by analysis of NK cells during ontogeny

Little is known regarding natural killer (NK) cell development in hematopoietic and visceral organs during ontogeny. We sought to determine NK cell accumulation during ontogeny and whether organ-specific niches altered development. Neonatal NK cells in the bone marrow, spleen and lung exist as immature CD27(+)/CD11B(lo) cells. The first appearance of mature CD27(lo)/CD11B(+) cells occurs at 3 weeks of age whereas maturation is complete by 8 weeks. In contrast, maturation of liver NK cells is essentially finished at 2 weeks. A role for RAG-1 (recombination-activating gene-1 product) in NK cell development was suggested as RAG-1-deficient mice accumulated NK cells differently. Surprisingly, bone marrow and spleen NK cells are absent in neonatal RAG-1(-/-) mice and an overrepresentation of a precursor NK cell subset, found normally in the liver, was observed in the bone marrow of RAG-1(-/-) mice. As mice lacking specific adaptive immune elements, including T and/or B cells, have normal NK cell repertoires, a more direct role for RAG during NK cell development cannot be excluded. Liver NK cells may represent an independent pool of cells from those developing out of the bone marrow, and RAG-1 itself may have a significant role in NK cell development.

214. The Lytic Potential of Human Liver Natural Killer Cells is Tempered by Their Repertoire of Licensed Cells

214. The Lytic Potential of Human Liver Natural Killer Cells is Tempered by Their Repertoire of Licensed Cells

Systemic Delivery of Nanoliposomal Ceramide Displays Anti-Leukemic Activity in a Rat Model of Large Granular Lymphocytic Leukemia

We have previously demonstrated that enhanced survival rather than increased proliferation accounts for the accumulation of natural killer (NK) cells in large granular lymphocyte (LGL) leukemia patients. To further elucidate the mechanism by which NK survival is enhanced, we analyzed leukemic NK cells isolated either from patient peripheral blood or rat splenic cells for altered expression of members of the inhibitor of apoptosis protein (IAP) family, which act as suppressors of apoptosis in a variety of human solid tumors and hematological malignancies. We now report that the IAP, survivin, was highly expressed in NK cells. In contrast, survivin was barely detectable in NK cells from the blood of normal human donors or the splenic cells from normal rats. We next asked if the lipid-derived second messenger, ceramide, which selectively induces apoptosis in cancer cells would diminish survivin protein expression. Treatment of NKL, a human NK-LGL leukemia cell line, or RNK-16, a rat NK-LGL leukemia cell line, with the cell-permeable, short-chain C6-ceramide (C6) in a pegylated liposomal formulation, led to cell apoptosis and diminished survivin protein expression, in a time and dose dependent manner. We next extended these in vitro studies to an in vivo rat model of NK-LGL leukemia. Systemic i.v. delivery of liposomal ceramide displayed significant anti-leukemia activity in a syngeneic Fischer F344 rat NK-LGL leukemia model that exhibits clonal expansion of CD3-CD8a+ lymphocytes. Over a 6-week treatment period, a well-tolerated dose of 40 mg/kg liposomal-C6, three times a week, elicited a 3 to 10- fold reduction in the weight of various lymphoid and non-lymphoid organs, compared with liposomal formulations that did not contain ceramide (ghost). Untreated or ghost-treated leukemic rats presented with lymphocytosis (LGL counts between 2 × 1011 and 3.5 × 1011/L), anemia and thrombocytopenia. Furthermore, the percentage of NK LGL cells, defined as CD3-CD8a+ by flow cytometry, was drastically elevated in the spleen, lymph node, thymus, bone marrow, blood, liver and lung in these leukemic rats, compared with their normal counterparts. In contrast, leukemic rats treated with liposomal ceramide had undetectable LGL cells in the blood and normal counts of red blood cells and platelets. Additionally, the CD3-CD8a+ NK cells in spleen, thymus and liver were found to be remarkably decreased, whereas the NK cells in bone marrow, blood and lung were within normal range. Collectively, these results indicate that bioactive ceramide analogues can be incorporated into pegylated liposomal vehicles for anti-leukemic efficacy in a rat model of NK LGL leukemia, possibly via decreased survivin expression or signaling.

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. 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.

NKG2A and the functional competence of liver natural killer cells

The liver is a unique environment that maintains tolerance to a heavy load of foreign antigens derived from the intestine. The liver is also the site of several chronic viral infections, including hepatitis B &amp; C viruses. Defining the mechanism(s) underlying the tolerogenic environment of the liver will broaden our understanding of normal liver function and contribute to better therapeutic strategies to persistent infection. Natural killer cells (NKs) make up a large percentage of the liver lymphocyte population. NKs play a critical role in innate immunity and generating adaptive immunity via crosstalk with dendritic cells (DCs). To determine the role of liver NK cells in modulating host immunity, we examined cytokine production upon interaction of NKs with DCs. When liver or spleen NKs were co‐cultured with splenic DC in the presence of IL‐12 or IL‐15, IFN‐γ was significantly decreased in the liver NK‐DC coculture compared to the spleen NK‐DC coculture, in the presence of either IL‐12 or IL‐15. We next determined the status of NK cell differentiation and the expression of both inhibitory and activating NK receptors in the liver and spleen on specific NK subsets defined by the expression of CD27 and CD11b. The inhibitory receptor NKG2A was expressed at higher levels on liver NKs compared to spleen NKs. This higher expression was observed in all NK subsets, but most pronounced in the CD27highCD11blow subset. In contrast, expression within all subsets of the activating receptor NKG2D, as well as all Ly49 receptors, was lower on liver NKs compared to spleen NKs. These results suggest that the liver NK cell may be less functionally competent than spleen NK cells, and that the inhibitory receptor NKG2A may play a prominent role in the effector status of NKs within the liver.