Oral tolerance is the process by which feeding of soluble proteins induces antigen-specific systemic immune unresponsiveness. Oral tolerance is thought to have a central role in suppressing immune responses to 'harmless' food antigens, and its failure can lead to development of pathologies such as food allergies or coeliac disease. However, on the basis of long-standing experimental observations, the relevance of oral tolerance in human health has achieved new prominence recently following the discovery that oral administration of peanut proteins prevents the development of peanut allergy in at-risk human infants. In this Review, we summarize the new mechanistic insights into three key processes necessary for the induction of tolerance to oral antigens: antigen uptake and transport across the small intestinal epithelial barrier to the underlying immune cells; the processing, transport and presentation of fed antigen by different populations of antigen-presenting cells; and the development of immunosuppressive T cell populations that mediate antigen-specific tolerance. In addition, we consider how related but distinct processes maintain tolerance to bacterial antigens in the large intestine. Finally, we outline the molecular mechanisms and functional consequences of failure of oral tolerance and how these may be modulated to enhance clinical outcomes and prevent disease.

Deciphering the mechanisms by which Plasmodium parasites develop inside hepatocytes is an important step toward the understanding of malaria pathogenesis. We propose that the nature and the magnitude of the inflammatory response in the liver are key for the establishment of the infection. Here, we used mice deficient in the multidrug resistance-2 gene (Mdr2-/-)-encoded phospholipid flippase leading to the development of liver inflammation. Infection of Mdr2-/- mice with Plasmodium berghei ANKA (PbANKA) sporozoites (SPZ) resulted in the blockade of hepatic exo-erythrocytic forms (EEFs) with no further development into blood stage parasites. Interestingly, cultured primary hepatocytes from mutant and wild-type mice are equally effective in supporting EEF development. The abortive infection resulted in a long-lasting immunity in Mdr2-/- mice against infectious SPZ where neutrophils and IL-6 appear as key effector components along with CD8+ and CD4+ effector and central memory T cells. Inflammation-induced breakdown of liver tolerance promotes anti-parasite immunity and provides new approaches for the design of effective vaccines against malaria disease.

Liver transplantation is the ideal treatment approach for a variety of end-stage liver diseases. However, life-long, systemic immunosuppressive treatment after transplantation is required to prevent rejection and graft loss, which is associated with severe side effects, although liver allograft is considered more tolerogenic. Therefore, understanding the mechanism underlying the unique immunologically privileged liver organ is valuable for transplantation management and autoimmune disease treatment. The unique hepatic acinus anatomy and a complex cellular network constitute the immunosuppressive hepatic microenvironment, which are responsible for the tolerogenic properties of the liver. The hepatic microenvironment contains a variety of hepatic-resident immobile non-professional antigen-presenting cells, including hepatocytes, liver sinusoidal endothelial cells, Kupffer cells, and hepatic stellate cells, that are insufficient to optimally prime T cells locally and lead to the removal of alloreactive T cells due to the low expression of major histocompatibility complex (MHC) molecules, costimulatory molecules and proinflammatory cytokines but a rather high expression of coinhibitory molecules and anti-inflammatory cytokines. Hepatic dendritic cells (DCs) are generally immature and less immunogenic than splenic DCs and are also ineffective in priming naïve allogeneic T cells via the direct recognition pathway in recipient secondary lymphoid organs. Although natural killer cells and natural killer T cells are reportedly associated with liver tolerance, their roles in liver transplantation are multifaceted and need to be further clarified. Under these circumstances, T cells are prone to clonal deletion, clonal anergy and exhaustion, eventually leading to tolerance. Other proposed liver tolerance mechanisms, such as soluble donor MHC class I molecules, passenger leukocytes theory and a high-load antigen effect, have also been addressed. We herein comprehensively review the current evidence implicating the tolerogenic properties of diverse liver cells in liver transplantation tolerance.

Complex cell culture models such as microphysiological models (MPS) mimicking human liver functionality in vitro are in the spotlight as alternative to conventional cell culture and animal models. Promising techniques like microfluidic cell culture or micropatterning by 3D bioprinting are gaining increasing importance for the development of MPS to address the needs for more predictivity and cost efficiency. In this context, human induced pluripotent stem cells (hiPSCs) offer new perspectives for the development of advanced liver-on-chip systems by recreating an in vivo like microenvironment that supports the reliable differentiation of hiPSCs to hepatocyte-like cells (HLC). In this review we will summarize current protocols of HLC generation and highlight recently established MPS suitable to resemble physiological hepatocyte function in vitro. In addition, we are discussing potential applications of liver MPS for disease modeling related to systemic or direct liver infections and the use of MPS in testing of new drug candidates.

The liver is a major target organ for metastasis of both gastrointestinal and extra-gastrointestinal cancers. Due to its frequently inoperable nature, liver metastasis represents a leading cause of cancer-associated death worldwide. In the past years, the pivotal role of the immune system in this process is being increasingly recognised. In particular, the role of the hepatic macrophages, both recruited monocyte-derived macrophages (Mo-Mfs) and tissue-resident Kupffer cells (KCs), has been shown to be more versatile than initially imagined. However, the lack of tools to easily distinguish between these two macrophage populations has hampered the assignment of particular functionalities to specific hepatic macrophage subsets. In this Review, we highlight the most remarkable findings regarding the origin and functions of hepatic macrophage populations, and we provide a detailed description of their distinct roles in the different phases of the liver metastatic process.

The liver participates in a multitude of metabolic functions that are critical for sustaining human life. Despite constant encounters with antigenic-rich intestinal blood, oxidative stress, and metabolic intermediates, there is no appreciable immune response. Interestingly, patients undergoing orthotopic liver transplantation benefit from a high rate of graft acceptance in comparison to other solid organ transplant recipients. In fact, cotransplantation of a donor liver in tandem with a rejection-prone graft increases the likelihood of graft acceptance. A variety of players may account for this phenomenon including the interaction of intrahepatic antigen-presenting cells with CD4⁺ T cells and the preferential induction of forkhead box P3 (Foxp3) expression on CD4⁺ T cells following injurious stimuli. Ineffective insult management can cause chronic liver disease, which manifests systemically as the following: antibody-mediated disorders, ineffective antiviral and antibacterial immunity, and gastrointestinal disorders. These sequelae sharing the requirement of CD4⁺ T cell help to coordinate aberrant immune responses. In this review, we will focus on CD4⁺ T cell help due to the shared requirements in hepatic tolerance and coordination of extrahepatic immune responses. Overall, intrahepatic deviations from steady state can have deleterious systemic immune outcomes and highlight the liver's remarkable capacity to maintain a balance between tolerance and inflammatory response while simultaneously being inundated with a panoply of antigenic stimuli. Liver Transplantation 24 89-97 2018 AASLD.

Oral tolerance can prevent unnecessary immune responses against dietary antigens. Members of the B7 protein family play critical roles in the positive and/or negative regulation of T cell responses to interactions between APCs and T cells. V-set and Ig domain-containing 4 (VSIG4), a B7-related co-signaling molecule, has been known to act as a co-inhibitory ligand and may be critical in establishing immune tolerance. Therefore, we investigated the regulation of VSIG4 signaling in a food allergy and experimental oral tolerance murine models. We analyzed the contributions of the two main sites involved in oral tolerance, the mesenteric lymph node (MLN) and the liver, in VSIG4-mediated oral tolerance induction. Through the comparative analysis of major APCs, dendritic cells (DCs) and macrophages, we found that Kupffer cells play a critical role in inducing regulatory T cells (Tregs) and establishing immune tolerance against oral antigens via VSIG4 signaling. Taken together, these results suggest the possibility of VSIG4 signaling-based regulation of orally administered antigens.

Macrophages are found throughout the body, where they have crucial roles in tissue development, homeostasis and remodeling, as well as being sentinels of the innate immune system that can contribute to protective immunity and inflammation. Barrier tissues, such as the intestine, lung, skin and liver, are exposed constantly to the outside world, which places special demands on resident cell populations such as macrophages. Here we review the mounting evidence that although macrophages in different barrier tissues may be derived from distinct progenitors, their highly specific properties are shaped by the local environment, which allows them to adapt precisely to the needs of their anatomical niche. We discuss the properties of macrophages in steady-state barrier tissues, outline the factors that shape their differentiation and behavior and describe how macrophages change during protective immunity and inflammation.

The metabolic intermediate of acetaminophen (APAP) can cause severe hepatocyte necrosis, which triggers aberrant immune activation of liver non-parenchymal cells (NPC). Overzealous hepatic inflammation determines the morbidity and mortality of APAP-induced liver injury (AILI). Interleukin-1 receptor (IL-1R) signaling has been shown to play a critical role in various inflammatory conditions, but its precise role and underlying mechanism in AILI remain debatable. Herein, we show that NLRP3 inflammasome activation of IL-1β is dispensable to AILI, whereas IL-1α, the other ligand of IL-1R1, accounts for hepatic injury by a lethal dose of APAP. Furthermore, Kupffer cells function as a major source of activated IL-1α in the liver, which is activated by damaged hepatocytes through TLR4/MyD88 signaling. Finally, IL-1α is able to chemoattract and activate CD11b⁺Gr-1⁺ myeloid cells, mostly neutrophils and inflammatory monocytes, to amplify deteriorated inflammation in the lesion. Therefore, this work identifies that MyD88-dependent activation of IL-1α in Kupffer cells plays a central role in the immunopathogenesis of AILI and implicates that IL-1α is a promising therapeutic target for AILI treatment.

Kupffer cells, the largest tissue resident macrophage population, are key for the maintenance of liver integrity and its restoration after injury and infections, as well as the local initiation and resolution of innate and adaptive immunity. These important roles of Kupffer cells were recently identified in healthy and diseased liver revealing diverse functions and phenotypes of hepatic macrophages. High-level phenotypic and genomic analysis revealed that Kupffer cells are not a homogenous population and that the hepatic microenvironment actively shapes both phenotype and function of liver macrophages. Compared to macrophages from other organs, hepatic macrophages bear unique properties that are instrumental for their diverse roles in local immunity as well as liver regeneration. The diverse and, in part, contradictory roles of hepatic macrophages in anti-tumor and inflammatory immune responses as well as regulatory and regenerative processes have been obscured by the lack of appropriate technologies to specifically target or ablate Kupffer cells or monocyte-derived hepatic macrophages. Future studies will need to dissect the exact role of the hepatic macrophages with distinct functional properties linked to their differentiation status and thereby provide insight into the functional plasticity of hepatic macrophages.

The liver is known as organ with unique immune competence. Besides its unique microenvironment that is determined by gut-derived portal venous blood constituents and the presence of enzymes with immune regulatory properties, liver antigen presenting cell populations regulate antigen-specific immunity in a local fashion. In addition to bone marrow-derived dendritic cells and myeloid cells such as macrophages and monocytes, also truly liver-resident cell populations function as antigen presenting cells such as liver sinusoidal endothelial cells and hepatocytes. The functional outcome of antigen-presentation by these cell populations is diverse and ranges from generation of regulatory CD4 cells, to induction of memory CD8 T cells or deletional tolerance, which generates a complex network of antigen-presenting cells that determines hepatic immune regulation and local immune surveillance against viral infection.

The liver has unique immune regulatory functions that promote the induction of tolerance rather than responses to antigens encountered locally. These functions are mediated by local expression of coinhibitory receptors and immunosuppressive mediators that help prevent overwhelming tissue damage. Over the years, we have gained more insight into the local regulatory cues that determine the functional complexity of immune responses regulated locally in the liver. Both the unique hepatic microenvironment and the particular liver sinusoidal cell populations, in addition to hepatocytes, actively modulate immune responses locally in the liver and thereby determine the outcome of hepatic immune responses. This is of high biological and clinical relevance in hepatitis B virus and hepatitis C virus infections, which can cause acute and persistent infections associated with chronic inflammation in humans that eventually progress to cirrhosis and hepatocellular carcinoma. Here, we review current knowledge about the balance between immunity and tolerance in the liver and how this may affect our understanding of the determinants of hepatitis B virus and hepatitis C virus clearance, persistence, and virus-induced liver disease.

The liver is the largest organ in the body and is generally regarded by nonimmunologists as having little or no lymphoid function. However, such is far from accurate. This review highlights the importance of the liver as a lymphoid organ. Firstly, we discuss experimental data surrounding the role of liver as a lymphoid organ. The liver facilitates tolerance rather than immunoreactivity, which protects the host from antigenic overload of dietary components and drugs derived from the gut and it is instrumental to fetal immune tolerance. Loss of liver tolerance leads to autoaggressive phenomena, which if not controlled by regulatory lymphoid populations, may lead to the induction of autoimmune liver diseases. Liver-related lymphoid subpopulations also act as critical antigen-presenting cells. The study of the immunological properties of liver and delineation of the microenvironment of the intrahepatic milieu in normal and diseased livers provides a platform to understand the hierarchy of a series of detrimental events that lead to immune-mediated destruction of the liver and the rejection of liver allografts. The majority of emphasis within this review will be on the normal mononuclear cell composition of the liver. However, within this context, we will discuss selected, but not all, immune-mediated liver disease and attempt to place these data in the context of human autoimmunity.

Because of its unique blood supply, the liver maintains a special local immune tolerogenic microenvironment. Moreover, the liver can impart this immune tolerogenic effect on other organs, thus inducing systemic immune tolerance. The network of hepatic regulatory cells is an important mechanism underlying liver tolerance. Many types of liver-resident antigen-presenting cells (APCs) have immune regulatory function, and more importantly, they can also induce the differentiation of circulating immune cells into regulatory cells to further extend systemic tolerance. Thus, the liver can be seen as a type of 'school', where liver APCs function as 'teachers' and circulating immune cells function as 'students.'

The liver has a role in T cell tolerance induction, which is mainly achieved through the functions of tolerogenic hepatic antigen-presenting cells (APCs) and regulatory T cells. Hepatic stellate cells (HSCs) are known to have various immune functions, which range from immunogenic antigen presentation to the induction of T cell apoptosis. Here we report a novel role for stellate cells in vetoing the priming of naive CD8 T cells. Murine and human HSCs and stromal cells (but not hepatocytes) prevented the activation of naive T cells by dendritic cells, artificial APCs, and phorbol 12-myristate 13-acetate/ionomycin by a cell contact-dependent mechanism. The veto function for inhibiting T cell activation was directly correlated with the activation state of HSCs and was most pronounced in HSCs from fibrotic livers. Mechanistically, high expression levels of CD54 simultaneously restricted the expression of interleukin-2 (IL-2) receptor and IL-2 in T cells, and this was responsible for the inhibitory effect because exogenous IL-2 overcame the HSC veto function.

Our results demonstrate a novel function of HSCs in the local skewing of immune responses in the liver through the prevention of local stimulation of naive T cells. These results not only indicate a beneficial role in hepatic fibrosis, for which increased CD54 expression on HSCs could attenuate further T cell activation, but also identify IL-2 as a key cytokine in mediating local T cell immunity to overcome hepatic tolerance.

The demands that are imposed on the liver as a result of its function as a metabolic organ that extracts nutrients and clears gut-derived microbial products from the blood are met by a unique microanatomical and immunological environment. The inherent tolerogenicity of the liver and its role in the regulation of innate and adaptive immunity are mediated by parenchymal and non-parenchymal antigen-presenting cells (APCs), cell-autonomous molecular pathways and locally produced factors. Here, we review the central role of liver APCs in the regulation of hepatic immune function and also consider how recent insights may be applied in strategies to target liver tolerance for disease therapy.

Dendritic cell activation through ligation of pattern recognition receptors leading to full functional maturation causes induction of CD8(+) T-cell immunity through increased delivery of costimulatory signals instead of tolerance. Here we investigate whether organ-resident antigen-presenting cells, such as liver sinusoidal endothelial cells (LSECs), also switch from tolerogenic to immunogenic CD8(+) T-cell activation upon such stimulation.

Murine LSECs were isolated by immunomagnetic separation and analyzed for functional maturation upon triggering pattern recognition receptors or viral infection employing gene expression analysis and T cell coculture assays. In vivo relevance of the findings was confirmed with bone-marrow chimeric animals.

LSECs expressed numerous pattern recognition receptors that allowed for sentinel function, but ligand-induced activation of these receptors was not sufficient to overcome tolerance induction of CD8(+) T cells. Importantly, viral infection with murine cytomegalovirus caused functional maturation of antigen-presenting LSECs and was sufficient to promote antigen-specific differentiation into effector CD8(+) T cells in the absence of dendritic cells and independent of CD80/86.

These results shed new light on the generation of organ-specific immunity and may contribute to overcoming tolerance in relevant situations, such as cancer.

Tumor-induced immunosuppression plays a key role in tumor evasion of the immune system. A key cell population recognized as myeloid-derived suppressor cells (MDSC) contributes and helps orchestrate this immunosuppression. MDSC can interact with T cells, macrophages, and natural killer cells to create an environment favorable for tumor progression. In various tumor models, their presence at high levels has been reported in the bone marrow, blood, spleen, and tumor. We report for the first time that MDSC accumulate and home to the liver in addition to the other organs. Liver MDSC suppress T cells and accumulate to levels comparable with splenic MDSC. Additionally, hematopoiesis in the liver contributes to the dramatic expansion of MDSC in this organ. Furthermore, MDSC in the liver interact with macrophages, also known as Kupffer cells, and cause their up-regulation of PD-L1, a negative T-cell costimulatory molecule. The liver is thus an organ in which MDSC accumulate and can contribute to immunosuppression directly and indirectly. MDSC play a role in various pathologic states in addition to cancer, and these results contribute to our understanding of their biology and interactions with immune-related cells.

Peripheral CD8 T-cell tolerance can be generated outside lymphatic tissue in the liver, but the course of events leading to tolerogenic interaction of hepatic cell populations with circulating T-cells remain largely undefined. Here we demonstrate that preferential uptake of systemically circulating antigen by murine liver sinusoidal endothelial cells (LSECs), and not by other antigen-presenting cells in the liver or spleen, leads to cross-presentation on major histocompatibility complex (MHC) I molecules, which causes rapid antigen-specific naïve CD8 T-cell retention in the liver but not in other organs. Using bone-marrow chimeras and a novel transgenic mouse model (Tie2-H-2K(b) mice) with endothelial cell-specific MHC I expression, we provide evidence that cross-presentation by organ-resident and radiation-resistant LSECs in vivo was both essential and sufficient to cause antigen-specific retention of naïve CD8 T-cells under noninflammatory conditions. This was followed by sustained CD8 T-cell proliferation and expansion in vivo, but ultimately led to the development of T-cell tolerance.

Our results show that cross-presentation of circulating antigens by LSECs caused antigen-specific retention of naïve CD8 T-cells and identify antigen-specific T-cell adhesion as the first step in the induction of T-cell tolerance.

This review aims to give an update of the field of the hepatic sinusoid, supported by references to presentations given at the 14th International Symposium on Cells of the Hepatic Sinusoid (ISCHS2008), which was held in Tromsø, Norway, August 31-September 4, 2008. The subtitle of the symposium, 'Integrating basic and clinical hepatology', signified the inclusion of both basal and applied clinical results of importance in the field of liver sinusoidal physiology and pathophysiology. Of nearly 50 oral presentations, nine were invited tutorial lectures. The authors of the review have avoided writing a 'flat summary' of the presentations given at ISCHS2008, and instead focused on important novel information. The tutorial presentations have served as a particularly important basis in the preparation of this update. In this review, we have also included references to recent literature that may not have been covered by the ISCHS2008 programme. The sections of this review reflect the scientific programme of the symposium (http://www.ub.uit.no/munin/bitstream/10037/1654/1/book.pdf): 1. Liver sinusoidal endothelial cells. 2. Kupffer cells. 3. Hepatic stellate cells. 4. Immunology. 5. Tumor/metastasis. Symposium abstracts are referred to by a number preceded by the letter A.

The liver is known to favor the induction of immunological tolerance rather than immunity. Although Kupffer cells (KC) have been indicated to play a role in liver tolerance to allografts and soluble antigens, the mechanisms involved remain unclear. We hypothesized that KCs could promote immune tolerance by acting as incompetent antigen-presenting cells (APC), as well as actively suppressing T cell activation induced by other potent APCs. The expression of antigen presentation-related molecules by KCs was phenotyped by flow cytometry. The abilities of KCs to act as APCs and to suppress T cell activation induced by splenic dendritic cells (DC) were examined by in vitro proliferation assays using CD4(+) OVA-TCR (ovalbumin T cell receptor) transgenic T cells. We found that, compared with DCs, KCs expressed significantly lower levels of major histocompatibility complex (MHC) II, B7-1, B7-2, and CD40. This result is consistent with our observation that KCs were not as potent as DCs in eliciting OVA-specific T cell proliferation. However, KCs isolated from polyinosinic:polycytidylic acid-treated mice expressed significantly higher levels of MHC II and costimulatory molecules than did naïve KCs and could stimulate stronger T cell responses. More importantly, we found that KCs could inhibit DC-induced OVA-specific T cell activation. Further investigation of the underlying mechanism revealed that prostaglandins produced by KCs played an important role. The results ruled out the possible involvement of interleukin-10, nitric oxide, 2,3-dioxygenase, and transforming growth factor beta in KC-mediated T cell suppression.

Our data indicate that KCs are a tolerogenic APC population within the liver. These findings suggest that KCs may play a critical role in regulating immune reactions within the liver and contributing to liver-mediated systemic immune tolerance. (HEPATOLOGY 2008.).

Induction of hematopoietic chimerism and subsequent donor-specific immune tolerance via bone marrow transplantation is an ideal approach for islet transplantation to treat type-1 diabetes. We examined the potential of mesenchymal stem cells (MSCs) in the induction of chimerism and islet allograft tolerance without the incidence of graft-versus-host disease (GVHD). Streptozotocin-diabetic rats received a conditioning regimen consisting of antilymphocyte serum and 5 Gy total body irradiation, followed by an intraportal co-infusion of allogeneic MSCs, bone marrow cells (BMCs) and islets. Although all the recipients rejected the islets initially, half of them developed stable mixed chimerism and donor-specific immune tolerance, shown by the engraftment of donor skin and second-set islet transplants and acute rejection of a third-party skin. The engraftment of the primary islet allografts with stable chimerism was achieved by the addition of a 2-week peritransplant administration of 15-deoxyspergualin (DSG). Without MSCs, none of the recipients treated with DSG developed chimerism or reversal of diabetes. GVHD was not observed in any of the recipients infused with MSCs (0/15), whereas it occurred in 4/11 recipients without MSCs. These results indicate a potential use of MSCs for induction of hematopoietic chimerism and subsequent immune tolerance in clinical islet transplantation.

A portal venous injection of allogeneic donor cells is known to prolong the survival of subsequently transplanted allografts. In this study, we investigated the role of liver sinusoidal endothelial cells (LSECs) in immunosuppressive effects induced by a portal injection of allogeneic cells on T cells with indirect allospecificity. To eliminate the direct CD4+ T cell response, C57BL/6 (B6) MHC class II-deficient C2tatm1Ccum (C2D) mice were used as donors. After portal injection of irradiated B6 C2D splenocytes into BALB/c mice, the host LSECs that endocytosed the irradiated allogeneic splenocytes showed enhanced expression of MHC class II molecules, CD80, and Fas ligand (FasL). Due to transmigration across the LSECs from BALB/c mice treated with a portal injection of B6 C2D splenocytes, the naive BALB/c CD4+ T cells lost their responsiveness to stimulus of BALB/c splenic APCs that endocytose donor-type B6 C2D alloantigens, while maintaining a normal response to stimulus of BALB/c splenic APCs that endocytose third-party C3H alloantigens. Similar results were not observed for naive BALB/c CD4+ T cells that transmigrated across the LSECs from BALB/c FasL-deficient mice treated with a portal injection of B6 C2D splenocytes. Adaptive transfer of BALB/c LSECs that had endocytosed B6 C2D splenocytes into BALB/c mice via the portal vein prolonged the survival of subsequently transplanted B6 C2D hearts; however, a similar effect was not observed for BALB/c FasL-deficient LSECs. These findings indicate that LSECs that had endocytosed allogeneic splenocytes have immunosuppressive effects on T cells with indirect allospecificity, at least partially via the Fas/FasL pathway.

The initial site of replication for Plasmodium parasites in mammalian hosts are hepatocytes, cells that offer unique advantages for the extensive parasite replication occurring prior to the erythrocytic phase of the life cycle. The liver is the metabolic centre of the body and has an unusual relationship to the immune system. However, to reach hepatocytes, sporozoites must cross the sinusoidal barrier, composed of specialized endothelia and Kupffer cells, the resident macrophages of the liver. Mounting evidence suggests that, instead of taking what would seem a safer route through endothelia, the parasites traverse Kupffer cells yet suffer no harm. Kupffer cells have a broad range of responses towards incoming microorganisms, toxins and antigens which depend on the nature of the intruder, the experimental conditions and the environmental circumstances. Kupffer cells may become activated or remain anergic, produce pro- or anti-inflammatory mediators. Consequently, outcomes are diverse and include development of immunity or tolerance, parenchymal necrosis or regeneration, chronic cirrhotic transformation or acute liver failure. Here we review data concerning the unique structural and functional characteristics of Kupffer cells and their interactions with Plasmodium sporozoites in the context of a model in which these hepatic macrophages function as the sporozoite gate to the liver.

Endothelial cells lining the blood vessels form a barrier between circulating immune cells and parenchymal tissue. While the molecular mechanisms involved in antigen-independent recruitment of leukocytes into infected tissue have been extensively studied, the mechanisms involving antigen-specific recruitment of T cells into tissue have remained largely elusive. Here I shall review the experimental evidence that endothelial cells function as antigen-presenting cells and in this function contribute first to regulation of immune responses and second, to antigen-specific recruitment of T cells.

The idiosyncratic nature and poor prognosis of drug-induced liver injury (DILI) make this type of reaction a major safety issue during drug development, as well as the most common cause for the withdrawal of drugs from the pharmaceutical market. The key to predicting and preventing DILI is understanding the underlying mechanisms. DILI is initiated by direct hepatotoxic effects of a drug, or a reactive metabolite of a drug. Parenchymal cell injury induces activation of innate and/or adaptive immune cells, which, in turn, produce proinflammatory and tissue hepatotoxic mediators, and/or mount immune reactions against drug-associated antigens. Understanding the molecular and cellular elements associated with these pathways can help identify risk factors and may ultimately facilitate the development of strategies to predict and prevent DILI.

After ingestion, oral antigens distribute systemically and provoke T cell stimulation outside the gastrointestinal tract. Within the liver, scavenger liver sinusoidal endothelial cells (LSEC) eliminate blood-borne antigens and induce T cell tolerance. Here we investigated whether LSEC contribute to oral tolerance. Oral antigens were efficiently cross-presented on H-2K(b) by LSEC to naive CD8 T cells. Cross-presentation efficiency in LSEC but not dendritic cells was increased by antigen-exposure to heat or low pH. Mechanistically, cross-presentation in LSEC requires endosomal maturation, involves hsc73 and proteasomal degradation. H-2K(b)-restricted cross-presentation of oral antigens by LSEC in vivo induced CD8 T cell priming and led to development of CD8 T cell tolerance in two independent experimental systems. Adoptive transfer of LSEC from mice fed with antigen (ovalbumin) into RAG2-/- knockout mice, previously reconstituted with naive ovalbumin-specific CD8 T cells, prevented development of specific cytotoxicity and expression of IFN-gamma in CD8 T cells. Using a new transgenic mouse line expressing H-2K(b) only on endothelial cells, we have demonstrated that oral antigen administration leads to tolerance in H-2K(b)-restricted CD8 T cells. Collectively, our data demonstrate a participation of the liver, in particular scavenger LSEC, in development of CD8 T cell tolerance towards oral antigens.

Antigen injected subcutaneously (SQ) results in a strong systemic immune response, whereas antigen infused orally or by the portal vein tends to induce tolerance. Nontransgenic C57BL/6J (H-2(b)), or B6, mice and transgenic 2C mice (that express a cytotoxic T cell receptor against major histocompatibility complex Class I L(d)) were used to investigate the regional and systemic responses to oral antigen.

B6 (H-2(b)) and 2C (H-2(b)) mice were given either saline or BALB/cByJ (H-2(d), L(d+)) (BALB/c) spleen cells (SCs) (25 x 10(6)) by oral gavage (PO) on day 0. Injection of 10 x 10(6) BALB/c SCs SQ was performed after 7 days, followed by a footpad injection of 10 x 10(6) BALB/c SC on day 14. Specific footpad swelling was measured 24 hours later by means of a micrometer. Mixed lymphocyte culture was performed on splenic and mesenteric lymph node (MLN) lymphocytes. The percentage of splenic and MLN CD4+ and CD8+ T cells was quantitated by fluorescence activated cell sorter. Cytokine messenger RNA (mRNA) and protein was measured by reverse transcriptase-polymerase chain reaction and enzyme-linked immunosorbent assay.

Whereas B6 mice were shown to have specifically decreased responsiveness to Balb/c cells in vivo and in vitro after oral Balb/c, 2C mice did not demonstrate any downregulation in delayed-type hypersensitivity responsiveness or splenic lymphocyte proliferation. However, MLN lymphocytes from 2C mice did demonstrate decreased proliferation against Balb/c after oral Balb/c gavage (P < .01). Subset percentages of naïve B6 (n=8) spleen and MLN lymphocytes were 50% to 52% for CD4+ and 46% to 47% for CD8+ T cells. These percentages were unchanged in B6 mice after PO Balb/c. Subset percentages of naïve 2C (n=6) spleen and MLN lymphocytes were 2% to 5% for CD4+ and 61% to 64% for CD8+ T cells. After PO Balb/c, MLN CD4+ and CD8+ T cells were unchanged, whereas splenic CD8+ T cells decreased to 44% to 48% and CD4+ T cells increased to 32% to 33% (P < .05, P < .05 vs naïve 2C spleen). After PO Balb/c, inflammatory interleukin (IL)-2 and interferon gamma mRNA and protein were increased in 2C spleen cells, whereas they were decreased in 2C MLN. Anti-inflammatory cytokine IL-4 and IL-10 mRNA and protein were increased in 2C MLN after PO Balb/c, whereas they were not detectable in 2C spleen cells.

Systemic oral tolerance can be induced in B6 mice, whereas only regional mesenteric lymph node tolerance develops in transgenic 2C mice. The inability to induce systemic oral tolerance in 2C mice appears to be due to a significant increase in peripheral (splenic) CD4+ T cells.

Intraportal injection of donor antigens delays rejection of allografts (portal venous tolerance). The study aimed to investigate the possible influence of prior gadolinium chloride (Gd)-induced Kupffer cell blockade on tolerance to non-vascularized skin allografts induced by means of donor-specific intraportal blood transfusion.

Wistar rats (n = 10) were used as donors and Sprague-Dawley rats (n = 70) as recipients of a non-vascularized skin graft. Recipients were divided into groups according to the manipulations prior to transplantation, as follows: (1) no manipulation; (2) donor-specific intrajugular blood transfusion; (3) donor-specific intraportal blood transfusion; (4) Gd administration and donor-specific intrajugular blood transfusion; (5) Gd administration and donor-specific intraportal blood transfusion; (6) Gd administration, and (7) intraportal saline infusion. In a first set of experiments, these manipulations were performed once. In a second set of experiments, the same manipulations were performed twice. Skin allograft was performed 7 days after the last manipulation in all groups.

Group 3 showed the highest skin graft survival, particularly after repeated blood transfusion. Graft survival in this group was significantly higher than in any other group. Conversely, group 5 showed the lowest graft survival, particularly after repeated blood transfusion. Graft survival in this group was significantly lower than that of groups 1, 2, 3 and 7.

In this model of skin allograft transplantation, Gd administration abrogates and can even reverse the tolerogenic effect of repeated donor-specific intraportal blood transfusion.

The ability of the immune system to distinguish between harmful and harmless antigens is essential for mounting protective immune responses and preventing the induction of pathology. Tolerance is a mechanism that prevents or suppresses potentially injurious immune responses. Natural killer T (NKT) lymphocytes, a subset of regulatory T lymphocytes, can induce pro-inflammatory or anti-inflammatory immune responses. This subset of cells appears to be crucial for induction of tolerance by several immune-modulatory interventions; these include immune manipulations in the setting of transplantation, induction of tolerance by introduction of antigen into immune-privileged sites, and oral administration of disease-associated-antigen. The ability to predict whether tolerance or immunity will be generated in a given situation is essential for development of NKT lymphocyte-based immune-modulatory treatments. The role of NKT lymphocytes in these settings, and the requirements for development of tolerance, rather than immunity, are discussed.

Plasmodium sporozoite invasion of liver cells has been an extremely elusive event to study. In the prevailing model, sporozoites enter the liver by passing through Kupffer cells, but this model was based solely on incidental observations in fixed specimens and on biochemical and physiological data. To obtain direct information on the dynamics of sporozoite infection of the liver, we infected live mice with red or green fluorescent Plasmodium berghei sporozoites and monitored their behavior using intravital microscopy. Digital recordings show that sporozoites entering a liver lobule abruptly adhere to the sinusoidal cell layer, suggesting a high-affinity interaction. They glide along the sinusoid, with or against the bloodstream, to a Kupffer cell, and, by slowly pushing through a constriction, traverse across the space of Disse. Once inside the liver parenchyma, sporozoites move rapidly for many minutes, traversing several hepatocytes, until ultimately settling within a final one. Migration damage to hepatocytes was confirmed in liver sections, revealing clusters of necrotic hepatocytes adjacent to structurally intact, sporozoite-infected hepatocytes, and by elevated serum alanine aminotransferase activity. In summary, malaria sporozoites bind tightly to the sinusoidal cell layer, cross Kupffer cells, and leave behind a trail of dead hepatocytes when migrating to their final destination in the liver.

Nerve allotransplantation has been used successfully in human subjects to restore function after traumatic nerve injury and avoid subsequent limb amputation. However, due to the morbidity associated with nonspecific immunosuppression, this reconstructive approach has been limited to patients with particularly severe nerve injuries. It would be desirable to broaden the indications for such procedures through development of less toxic antirejection therapies. A miniature swine model of nerve transplantation was used to investigate the effects of preoperative ultraviolet-B (UV-B)-irradiated donor alloantigen portal venous infusion and injection of cultured major histocompatibility complex (MHC)-matched Schwann cells into the nerve graft. The transplanted ulnar nerves were harvested at 20 weeks. Histomorphometry showed marked enhancement in nerve regeneration through allografts injected with Schwann cells. Serial mixed lymphocyte assays demonstrated suppression of the recipient immune response to the donor antigen after pretreatment, but no additional neuroregenerative effect of donor alloantigen pretreatment.

Malaria infection is caused by sporozoites, the life cycle stage of Plasmodium that is transmitted by female anopheline mosquitoes. The inoculated sporozoites migrate in the skin, enter a capillary and use the bloodstream for the long haul to the liver. Here, the parasites invade hepatocytes and differentiate to thousands of merozoites that specifically infect red blood cells. Hepatocytes, however, are not directly accessible to sporozoites entering the liver sinusoid. The liver phase of the malaria life cycle can occur only if the parasites first cross the layer of sinusoidal cells that line the liver capillaries. Experimental observations show that sporozoite entry into the liver parenchyma involves a complex cascade of events, from binding to extracellular matrix proteoglycans via passage through Kupffer cells and transmigration through several hepatocytes, until the final host cell is found. By choosing the liver as their initial site of replication, Plasmodium sporozoites can exploit the tolerogenic properties of this unique immune organ to evade the host's immune response.

Cell death through apoptosis plays a critical role in regulating cellular homeostasis. Whether the disposal of apoptotic cells through phagocytosis can actively induce immune tolerance in vivo, however, remains controversial. Here, we report in a rat model that without using immunosuppressants, transfusion of apoptotic splenocytes from the donor strain prior to transplant dramatically prolonged survival of heart allografts. Histological analysis verified that rejection signs were significantly ameliorated. Splenocytes from rats transfused with donor apoptotic cells showed a dramatically decreased response to donor lymphocyte stimulation. Most importantly, blockade of phagocytosis in vivo, either with gadolinium chloride to disrupt phagocyte function or with annexin V to block binding of exposed phosphotidylserine to its receptor on phagocytes, abolished the beneficial effect of transfused apoptotic cells on heart allograft survival. Our results demonstrate that donor apoptotic cells promote specific allograft acceptance and that phagocytosis of apoptotic cells in vivo plays a crucial role in maintaining immune tolerance.

Liver sinusoidal endothelial cells (LSEC) have been reported to express MHC class II, CD80, CD86, and CD11c and effectively stimulate naive T cells. Because dendritic cells (DC) are known to possess these characteristics, we sought to directly compare the phenotype and function of murine LSEC and DC. Nonparenchymal cells from C57BL/6 mice were obtained by collagenase digestion of the liver followed by density gradient centrifugation. From the enriched nonparenchymal cell fraction, LSEC (CD45(-)) were then isolated to 99% purity using immunomagnetic beads. Flow cytometric analysis of LSEC demonstrated high expression of CD31, von Willebrand factor, and FcgammaRs. However, unlike DC, LSEC had low or absent expression of MHC class II, CD86, and CD11c. LSEC demonstrated a high capacity for Ag uptake in vitro and in vivo. Although acetylated low-density lipoprotein uptake has been purported to be a specific function of LSEC, we found DC captured acetylated low-density lipoprotein to a similar extent in vivo. Consistent with their phenotype, LSEC were poor stimulators of allogeneic T cells. Furthermore, in the absence of exogenous costimulation, LSEC induced negligible proliferation of CD4(+) or CD8(+) TCR-transgenic T cells. Thus, contrary to previous reports, our data indicate that LSEC alone are insufficient to activate naive T cells.

Previous studies have shown that portal venous transfusion (PVT) induces a state of immunosuppression, and Kupffer cells may be involved in the mechanism.

This study was aimed to investigate the effect of PVT on Kupffer cell gene expression.

Each BALB/C mouse was subjected to laparotomy and received one of five treatments: PVT, portal venous saline injection (PVS), inferior vena caval transfusion (IVCT), inferior vena caval saline injection (IVCS) or sham operation (S). The blood for PVT and IVCT was sampled from C57BL/6J mice. Kupffer cells were then isolated 1 or 24 h after each of the 5 treatments, for a total of 10 experimental groups (1-h PVT, PVS, IVCT, IVCS and S, and 24-h PVT, PVS, IVCT, IVCS and S) from BALB/C mice. To examine the effect of PVT on Kupffer cell gene expression, RT-PCR differential display was performed.

Increase in the expression of MIP-1alpha mRNA post PVT and IVCT was identified by differential display. PVT groups revealed higher levels of serum MIP-1alpha than any other groups.

These results suggest that MIP-1alpha may be involved in a cascade of signaling events associated with the PVT-mediated immunologic modulation in Kupffer cells.

Portal blood leukocytes play an important, but still poorly defined, role in the immune processes of the liver. In our previous studies, we showed that certain leukocyte subsets are selectively halted in the liver. These cells marginate in sinusoids and, together with resident Kupffer and endothelial sinusoidal cells, participate in antiviral and antitumor processes. The molecular mechanisms of margination and cooperation with resident sinusoidal cells require clarification. However, in vivo harvesting of portal blood leukocytes is associated with cumbersome cannulation of portal and hepatic veins and manipulation of the liver, causing major disturbances in splanchnic blood flow and liver blood supply, totally distorting sinusoidal blood perfusion and leukocyte margination. To overcome these difficulties, we have developed an in situ normothermic rat liver perfusion model permitting quantitative observations of blood leukocyte extraction in sinusoids. First, liver was flushed through the portal vein and the effluent leukocytes, named liver-associated leukocytes (LAL1), were collected from hepatic veins. Then, the liver was perfused for 60 min with 50 ml of blood using a semiclosed perfusion system. Upon completion of perfusion, the liver portal vasculature was flushed again to retrieve the leukocytes extracted from the perfusing blood (LAL2). These cells were characterized with respect to their phenotype and cytotoxicity. The mean leukocyte count of the washout before perfusion was 1.04+/-0.2x10(6)/g of liver tissue and 0.9+/-0.1x10(6)/g after 60 min of perfusion, indicating retention by the perfused liver, the live leukocyte extracting capacity. To further evaluate the efficiency of perfusion, FITC-labelled leukocytes were added to the perfusing leukocyte-free blood. Around 95% of the postperfusion washout LALs were FITC(+). Heat-killed leukocytes did not marginate in sinusoids. Preincubation of leukocytes with substances able to lower adhesion capacity, such as lidocaine, trypsin and AAGM1, significantly decreased the postperfusion LAL2 washout population. The numbers of extracted postperfusion LAL2 CD5(+), CD4(+), CD8(+), CD56(+) and class II(+) subsets did not differ statistically from those of preperfusion LAL1. Moreover, the cytotoxicity of LAL2 and LAL1 against CC531 and K562 remained at a similar level. Thus, perfused liver also retained its selective leukocyte extraction capacity. This model shows that the process of selective margination of portal blood cell subsets in the liver can be studied in an artificially perfused liver subject to physiological blood flow parameters, temperature, oxygenation and minimal ischemic time before connection to the perfusion device. Furthermore, it is suitable for studies of the selective recruitment of blood cells in sinusoids in a wide large of situations including liver tumors, infections, rejection after transplantation, graft vs. host disease, as well as in the investigation of the effect of drugs on these processes.

Local antigen presentation via either the oral (PO) or the portal venous (PV) routes results in suppression of systemic delayed-type hypersensitivity (DTH). The responsible cell populations are not well defined. Because NK1.1(+) T cells express the Fas ligand and produce high levels of the immunosuppressive cytokine, IL-4, they may play a role in both activated T-cell apoptosis and a Th1 to Th2 immune shift, thus promoting tolerance induction.

C57BL/6 mice were tolerized to BALB/c alloantigen by PV or PO spleen cells (25 x 10(6)) on Day 0. Subcutaneous (SQ) challenge with 10 x 10(6) BALB/c cells on Day 7 was followed by footpad injection of 10 x 10(6) BALB/c cells on Day 14. Footpad swelling was measured 24 h later. A single injection of the NK1.1(+) cell-depleting antibody, PK-136, was given IP (10 mg/kg) 2 days prior to PV or PO antigen. Flow cytometry evaluated NK1.1(+) cell depletion. CD1 knockout (KO) mice, lacking NK1.1(+) T cells, were also challenged with PV and PO Balb/c in parallel experiments.

The DTH to BALB/c antigen was markedly suppressed in C57BL/6 mice when this alloantigen was given by either PO or PV routes (P < 0.001, P < 0.001). The maintenance of an unaltered response to third-party C3H/HeJ demonstrated alloantigenic specificity. Administration of the anti-NK1.1 T cell monoclonal antibody, PK-136, resulted in complete restoration of in vivo DTH responsiveness in PO tolerance (P < 0.01), and partial restoration in PV tolerance (P < 0.05) in C57BL/6 mice. FACS confirmed virtually complete depletion of liver, splenic, Peyer's patch, and mesenteric lymph node NK1.1(+) lymphocytes. Development of both PO and PV tolerance was prevented in CD1 KO mice.

NK1.1(+) T cells play an essential role in antigen-specific suppression of the DTH response mediated by both oral and portal venous tolerance.