I. Nicholas Crispe

TLR-dependent cross talk between human Kupffer cells and NK cells

The liver protects the host from gut-derived pathogens yet is tolerant of antigenic challenge from food and commensal sources. Innate responses involving liver macrophages (Kupffer cells) and effector liver natural killer (NK) cells form the first line in this defense. We address the impact of Toll-like receptor (TLR) signaling on the cross talk between these two cells, and reveal how the liver displays a down-regulated inflammatory response to constitutive bacterial elements through the secretion of interleukin (IL) 10 yet retains a vigorous response to viral challenge. The data support the model that (a) human liver Kupffer cells respond to TLR ligands and indirectly activate NK cells; (b) the activation depends on cell–cell contact; (c) the Kupffer cells synthesize NK cell activating signals, among which IL-18 is critical, and NK cell inhibitory factors, including IL-10; (d) ligands that signal via myeloid differentiation factor 88 induce IL-10, giving a blunted response in the NK cells; and (e) ligands that signal via the Toll–IL-1 receptor domain–containing adaptor inducing interferon (IFN) β–IFN regulatory factor 3 pathway induce less IL-10, and also directly potentiate the stimulatory effect of IL-18 on NK cells, resulting in enhanced activation. Subversion of cellular mechanisms of innate immune response against viruses may be important for hepatotropic viruses (e.g., hepatitis B and C) to develop persistence.

Hepatitis C virus core protein subverts the antiviral activities of human Kupffer cells.

BACKGROUND & AIMS Kupffer cells (KC) are important innate immune cells of the liver, functioning as scavenging sinusoidal phagocytes and transducers of pattern recognition signals, including those of toll-like receptors (TLRs). The hepatitis C virus core protein (HCVc) engages TLR2 on peripheral blood monocytes and induces production of multiple inflammatory cytokines. We examined the effects of HCVc on human primary KC functions. METHODS KC were isolated from living donor allografts and stimulated with HCVc and/or ligands for TLRs. KC were examined for production of cytokines, expression of programmed death-ligand 1 (PD-L1), secretion of type 1 interferons (IFNs), and expression of the apoptosis-inducing protein tumor necrosis factor (TNF)-related apoptosis-inducing ligand (TRAIL). RESULTS HCVc acts as a ligand for TLR2 on human KC, inducing them to secrete interleukin (IL)-1beta, TNF-alpha, and IL-10 and up-regulate cell surface PD-L1. HCVc blocked TLR3-mediated secretion of IFN-alpha, IFN-beta, and cell surface expression of the cytotoxic molecule TRAIL. Inhibition of phosphoinositide 3 kinase with LY294002 blocked the up-regulation of PD-L1 by TLR ligands and the TLR3-specific induction of TRAIL and type 1 IFNs. CONCLUSIONS KC are intravascular macrophages that are continuously exposed to, and tolerant of, bacterial TLR ligands, which are delivered via the portal circulation. By mimicking a bacterial TLR2 ligand and effectively blocking the TLR3-mediated, double-stranded RNA-induced antiviral response, HCVc might appear to exploit this unique aspect of immunity in the liver.

Biology and significance of T-cell apoptosis in the liver

The liver has emerged as an organ with distinct immunological properties. In this review, we summarize evidence that shows that the liver can remove apoptotic, or non‐apoptotic but activated, CD8+ T cells from the circulation and induce apoptosis in these activated T cells by either active or passive mechanisms. Hepatitis viruses, particularly hepatitis C virus, often establish persistent infection. We review evidence that suggests that these viruses exploit intrahepatic tolerance mechanisms to protect themselves from immune attack.

Galactosylated LDL nanoparticles: a novel targeting delivery system to deliver antigen to macrophages and enhance antigen specific T cell responses.

We aim to define the role of Kupffer cells in intrahepatic antigen presentation, using the selective delivery of antigen to Kupffer cells rather than other populations of liver antigen-presenting cells. To achieve this we developed a novel antigen delivery system that can target antigens to macrophages, based on a galactosylated low-density lipoprotein nanoscale platform. Antigen was delivered via the galactose particle receptor (GPr), internalized, degraded and presented to T cells. The conjugation of fluoresceinated ovalbumin (FLUO-OVA) and lactobionic acid with LDL resulted in a substantially increased uptake of FLUO-OVA by murine macrophage-like ANA1 cells in preference to NIH3T3 cells, and by primary peritoneal macrophages in preference to primary hepatic stellate cells. Such preferential uptake led to enhanced proliferation of OVA specific T cells, showing that the galactosylated LDL nanoscale platform is a successful antigen carrier, targeting antigen to macrophages but not to all categories of antigen presenting cells. This system will allow targeted delivery of antigen to macrophages in the liver and elsewhere, addressing the question of the role of Kupffer cells in liver immunology. It may also be an effective way of delivering drugs or vaccines directly at macrophages.

The E2F-1 Transcription Factor Promotes Caspase-8 and Bid Expression, and Enhances Fas Signaling in T Cells

The immune system depends on the extensive proliferation of rare Ag-specific precursor T lymphocytes, followed by their differentiation, the delivery of effector function, and finally death by apoptosis. T cells that lack the E2F-1 transcription factor, which is activated as cells pass the restriction point and enter S phase, show defects in activation-induced cell death. We now report that E2F-1 increases the activity of an apoptotic pathway that is important in murine primary T cells. Thus, E2F-1 promotes the transcription of Bid, a molecule that links death receptor signaling to the activation of apoptotic mechanisms in mitochondria. It also promotes the transcription of caspase-8, the enzyme that cleaves and activates Bid. Enforced expression of Bid can partially restore apoptosis in E2F-1-deficient T cells. Thus, E2F-1 integrates cell cycle progression with apoptosis.

TNF-α Controls Intrahepatic T Cell Apoptosis and Peripheral T Cell Numbers

At the end of an immune response, activated lymphocyte populations contract, leaving only a small memory population. The deletion of CD8+ T cells from the periphery is associated with an accumulation of CD8+ T cells in the liver, resulting in both CD8+ T cell apoptosis and liver damage. After adoptive transfer and in vivo activation of TCR transgenic CD8+ T cells, an increased number of activated CD8+ T cells was observed in the lymph nodes, spleen, and liver of mice treated with anti-TNF-α. However, caspase activity was decreased only in CD8+ T cells in the liver, not in those in the lymphoid organs. These results indicate that TNF-α is responsible for inducing apoptosis in the liver and suggest that CD8+ T cells escaping this mechanism of deletion can recirculate into the periphery.

Immune role of hepatic TLR‐4 revealed by orthotopic mouse liver transplantation

Activated CD8+ T cells migrate to the liver at the end of an immune response and go through apoptosis there, but this mechanism is impaired in mice lacking Toll‐like receptor‐4. This allowed us to test the importance of liver trapping in an ongoing immune response. In the absence of Toll‐like receptor‐4, reduced liver accumulation was associated with an increase in the circulating CD8+ T cell pool, more long‐lived memory T cells and increased CD8+ T cell memory responses. Using experimental orthotopic liver transplantation, we showed that the effect of Toll‐like receptor‐4 on the formation of the CD8+ T cell memory resides in the liver. Conclusion: These studies reveal a new function for the liver, which is to regulate the magnitude of T cell memory responses through a Toll‐like receptor‐4–dependent mechanism. (HEPATOLOGY 2007;45:178–186.)

P27kip1 regulates the cell cycle arrest and survival of activated T lymphocytes in response to interleukin-2 withdrawal

The majority of activated T lymphocytes undergo cell death at the end of a primary immune response, while a minority survive as memory cells. The mechanisms that control the decision between these two fates are unknown. In the present study we examined the response of activated T cells to interleukin‐2 (IL‐2) withdrawal. Within hours, the percentage of T lymphocytes in cell cycle showed a steady decrease, while the percentage arrested in G1 increased proportionally. Deprivation of IL‐2 resulted in upregulation of the cell cycle inhibitor p27kip1. Comparison with resting T‐cell populations revealed that the highest expression of p27kip1 occurs in activated T cells undergoing cell cycle arrest following IL‐2 withdrawal. T cells deficient in p27kip1 expression showed an impaired ability to undergo cell cycle arrest in response to IL‐2 deprivation. Moreover, T cells deficient in p27kip1 showed significantly more apoptosis after IL‐2 withdrawal. Collectively, this study demonstrates that p27kip1 regulates both the cell cycle arrest and the apoptosis of antigen‐specific T lymphocytes.

Cleavage of E2F-1-regulating proteins and activation of E2F-1 during CD95-induced death of thymocytes

The CD95 death receptor activates caspases that cleave a variety of intracellular substrates, including cell cycle control proteins. However, the significance of this cleavage for the induction of apoptosis is unclear. In this study, CD95‐induced cleavage of the G1/S checkpoint regulator proteins, retinoblastoma protein (pRb) and murine‐double‐minute‐2 (mdm‐2), was associated with an increased protein concentration of a key transcription factor, E2F‐1, which is regulated by both of them. Furthermore, DNA‐binding activity to E2F sites is increased. In thymocytes, CD95‐induced apoptosis was associated with increased E2F‐1 DNA‐binding activity, while thymocytes that lacked E2F‐1 were less susceptible to CD95‐induced apoptosis. We conclude that the G1/S checkpoint is an important target of CD95 signalling. CD95‐activated caspases cleave regulator proteins to increase E2F‐1 activity, and inappropriate activation of E2F‐1 is part of the mechanism of CD95‐induced apoptosis.

Contrasting Urban and Rural Lifestyles of Memory CD8+ T Cells

Memory CD8(+) T cells in lymphoid tissue exhibit an unexpectedly high apoptosis rate, while memory cells located in several nonlymphoid tissues do not. This may be because the lymphoid CD8(+) memory T cell repertoire is being continuously updated, while the tissue repertoire is more stable.