Liver immunology

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Chapter 9 Liver immunology

Fundamentals of Immunology

The immune system is composed of two major components: innate and adaptive immunity (Fig. 9.1; Janeway, 2001). Innate immune cells are armed with hard-wired detection systems that recognize common structures found on pathogens or changes in surface molecules of host cells that signal danger. Such pattern recognition receptors (PRRs) account for the rapid response of innate immune cells to infection or host injury. A variety of PRRs exist, of which the best characterized is the Toll-like receptor (TLR) family. In humans, 11 TLRs are currently known that recognize various structurally conserved components of bacteria and viruses in addition to endogenous intracellular ligands. Engagement of PRRs by their respective ligands causes activation of innate immune cells, such as neutrophils, monocytes, macrophages, natural killer (NK) cells, and dendritic cells (DCs). The ensuing response results in destruction of the invading pathogen or tumor via phagocytosis or release of various cytotoxic or inflammatory agents.

In contrast, adaptive immunity refers to the arm of the immune system that is involved in antigen-specific responses, which occur either later in a particular immune response or rapidly after repeat exposure to a particular pathogen. The adaptive immune system is essentially comprised of T and B cells that circulate within the blood, lymphatic tissues, and organs. These lymphocytes interact with various antigen-presenting cells (APCs) of the innate immune system, such as DCs and macrophages. Activation occurs in the presence of specific signals between APCs and T or B cells. These signals include 1) stable antigen presentation within the context of major histocompatibility complex (MHC) molecules MHC-I or MHC-II, 2) appropriate costimulation by APCs and the corresponding receptor on T or B cells, and 3) cytokine-mediated signals that can modulate the overall response (Fig. 9.2). Innate and adaptive immune responses provide protection against a vast array of pathogens, preserving normal cells and tissues from attack. Failure of the immune system’s ability to correctly recognize foreign or abnormal cells may facilitate the development or spread of infection or malignancy. In contrast, inability to distinguish self from foreign antigens may result in autoimmune disease.

Modern clinical and experimental work has shown that the liver is particularly important in immunity (Fig. 9.3). First, the fact that the liver is one of the most common sites for metastatic disease suggests that it may have an increased propensity toward immunologic acceptance. Second, allogeneic liver transplantation can be accomplished in certain strains of mice without the need for immunosuppression, and in humans it often requires less immunosuppression compared with kidney or other solid organ transplants. Third, the liver is home to chronic viral infection in the form of hepatitis B and C. Additionally, oral ingestion or portal vein injection of foreign proteins can lead to tolerance in animal models. Conversely, the liver is the site of several autoimmune processes, including primary sclerosing cholangitis and primary biliary sclerosis. Despite the significant role that the liver plays in altering the balance between tolerance and immunity, the study of liver immunology remains in its infancy. This chapter discusses our current understanding of the function of liver immune cells and their role in disease.

Tolerance Versus Immunity

One of the most intriguing aspects of liver immunology is the propensity of antigens passing through the liver to produce tolerance rather than immunity. In experimental animal models and clinical studies of humans undergoing liver transplantation, a greater propensity for graft acceptance has been noted compared with transplantation of other solid organs. A liver transplant protects a kidney allograft transplanted simultaneously from the same donor (Creput et al, 2003). Trying to decipher the reasons for the liver’s inherent propensity for tolerance has been a particularly active area of research in the field of transplantation and cancer immunobiology.

Multiple competing theories to explain the cause of liver tolerance have been advanced. One postulate is that, as in central thymic tolerance, clonal deletion of antigen-specific T cells occurs in the liver. Another theory is that liver allografts release large amounts of soluble MHC class I molecules that potentially could function to neutralize donor-specific antibodies or cytolytic T cells. Kupffer cells (KCs) and natural killer (NK) T cells have been implicated as being important in the development of hepatic tolerance, because the depletion of either has been associated with a loss of oral tolerance. Liver DCs also have been suggested to play a role in liver tolerance, because as a group they have been shown to be less immunogenic than DCs from lymphoid organs such as the spleen (Bamboat et al, 2009). In addition, the numbers of several unusual cell types are elevated in the liver, and more recent studies have shown that multiple lineages of intrahepatic leukocytes are unique in function and phenotype compared with their counterparts from lymphoid organs. Controversy exists, however, as to whether these differences are due to de novo development, conditioning within the liver, or chemotactic signals in the liver that attract a unique population of cells. Regardless, the differences in composition and function are likely to play an important role in defining the nature of liver immunology. We devote a portion of this chapter to the major populations of liver immune cells and their role in various liver diseases.

Liver Immune Cells

Although the liver is primarily a nonlymphoid organ, it contains numerous nonparenchymal cells (NPCs; see Chapter 6), one quarter of which are leukocytes. The composition of the intrahepatic leukocyte population is markedly different from that seen in other organs (Fig. 9.5). As in the rest of the body, however, the liver contains most of the cellular components of innate and adaptive immunity. Advances in technology have allowed for the precise delineation and isolation of cell types based on the expression of a distinct set of surface markers.

Antigen-Presenting Cells

The experimental observation that antigens passing through the liver can lead to tolerance makes the understanding of antigen presentation in the liver particularly germane (Li et al, 2004). Antigen-presenting cells (APCs) play a crucial role in determining the nature of adaptive immune responses. The manner in which an APC presents any particular antigen can alter dramatically the response of antigen-specific T cells. Specifically, when antigen presentation occurs in conjunction with the appropriate costimulatory molecules, T cells proliferate and develop an immunogenic phenotypic and functional profile (Fig. 9.6). In contrast, antigens presented in the absence of costimulation lead to anergy or activation-induced T-cell death, two of the mechanisms of peripheral tolerance induction and maintenance.

Dendritic Cells

Dendritic cells (DCs) are rare, heterogeneous leukocytes that are primarily responsible for the capture of antigens in the periphery and subsequent presentation to immune effector cells. DCs are now recognized as the most potent APCs of the immune system and seem to arise from multiple hematopoietic cell lineages. Murine DCs are typically isolated based on their expression of the integrin CD11c. Immature DCs are specialized to capture antigens and then migrate to lymph nodes, where they can interact with T cells. After an encounter with pathogenic stimulus, such as bacterial lipopolysaccharide, DCs undergo phenotypic and functional changes, whereby their ability to capture antigens is diminished, but they increase their expression of MHC class II and T cell costimulatory molecules. Costimulatory molecule expression is essential in facilitating antigen presentation and efficient T cell activation. The ability of DCs to capture antigens in the periphery, translocate to areas rich in T cells, and then present antigens places DCs in a central role in immune regulation, bridging innate and adaptive immunity.

The body of literature studying the function of DCs grown in vitro from murine bone marrow progenitors or human peripheral blood mononuclear cells is vast. DCs isolated directly from the spleen, lymph nodes, or thymus also have been well studied. In contrast, because of the rarity of DCs in the liver, few studies have shown the phenotype or function of liver DCs. Initial studies focused on DC progenitors grown from liver nonparenchymal cells cultured with various cytokines, including granulocyte-macrophage colony-stimulating factor (GM-CSF). These early studies showed that liver DC progenitors are relatively immature cells with poor immunostimulatory ability that can promote immunologic tolerance and prolong solid organ transplants in animal models (Lu et al, 1994, 2001; Lau & Thomson, 2003).

To gain a better understanding of liver DCs as they function in situ, recent focus has been on studying freshly isolated liver DCs. In our initial studies, we wished to increase the number of liver DCs to facilitate their isolation. Because GM-CSF had been used to grow DC progenitors in culture from liver nonparenchymal cells, and because we had previously shown that in vivo GM-CSF overexpression increases the number of myeloid DCs in the spleen (Miller et al, 2002a), we suspected that it might expand liver DCs as well. We found that overexpression of GM-CSF using an adenoviral vector led to a dramatic increase in the number of highly immunostimulatory liver DCs (Pillarisetty et al, 2003). We also showed that a large proportion of the cells recruited to the liver by GM-CSF were DC precursor cells that develop into CD11c+ DCs in culture, which suggests that DC development can occur in the liver. We used immunohistochemistry to locate both types of recruited cells. The liver DCs were concentrated around the central veins, whereas the DC precursors were distributed diffusely throughout the liver parenchyma.

Our experience with expanding liver DCs sheds light on in situ liver DC development, but we wished to understand the phenotype and function of liver DCs in normal, untreated mice. When our ability to isolate liver DCs from normal mice improved, we began to study them in depth. We found that compared with the relatively well-studied DCs from the spleen, CD11c+ liver DCs were immature and only weakly immunostimulatory (Pillarisetty et al, 2004). We also noticed that, in contrast to spleen DCs, liver DCs are heterogeneous in their expression of MHC class II and costimulatory molecules. We further separated CD11c+ liver DCs based on their expression of the myeloid marker CD11b and the lymphoid marker CD8α+, which have been commonly used to define murine DC subtypes. Myeloid (CD11b+) and lymphoid (CD8α+) liver DCs, which each comprise approximately 10% of the total population of DCs in the liver, were as able to activate T cells as were their splenic counterparts. The bulk of the remaining cells, which had low to no expression of CD11b and CD8α, were only poorly immunostimulatory. This study showed that the presence of these atypical DCs was primarily responsible for the weakly activating nature of liver DCs on the whole. More recently, we discovered that despite having multiple APCs, the CD11chi subset of DCs within the liver are required for effective presentation of soluble protein (Plitas et al, 2008). Using a transgenic mouse in which CD11chi DCs can be depleted selectively, we found that activation of antigen-specific CD8+ T cells in the liver only occurred in the presence of CD11chi DCs.

Our investigation into liver DCs has now been extended into humans. We found that freshly isolated DCs from human liver exhibit tolerogenic properties when compared with autologous blood DCs. Liver DCs are weaker stimulators of T cells and favor the production of antiinflammatory IL-10, which induces the differentiation of naïve CD4+ T cells into regulatory T cells with suppressive function (Fig. 9.7; Bamboat et al, 2009a).

Kupffer Cells

Liver macrophages, referred to as Kupffer cells (KCs), have long been believed to be the primary phagocytic cells of the liver. KCs represent the largest pool of macrophages in the body, derived from monocytic precursors in the blood. They are typically found in the hepatic sinusoids; however, they also can migrate through the space of Disse to interact with hepatocytes (see Fig. 9.4). KCs have been thought to play a major role in antigen presentation and have been implicated in portal venous tolerance. Evidence also suggests that KCs may regulate T-cell responses to antigens and induce immune tolerance in the setting of liver allografts (Sun et al, 2003).

Techniques that allow for the more precise delineation of cellular subtypes have called into question some of the concepts about KCs and many other traditionally defined immune cells. Specifically, the advent of flow cytometry has allowed us to define cells by their surface marker expression, rather than simply by size or other crude physical characteristics shared by many cell types. This point is illustrated by the fact that many liver DCs express the cell surface marker CD11b, which is typically expressed by cells of monocytic lineage and has been used to define KCs in the liver. Much of the antigen-presenting ability previously attributed to KCs may be a result of DC function. The lack of consistent definitions of cells by cell surface marker expression and the continued use of less precise methods of cell isolation have led to continued ambiguity regarding KCs. An alternative explanation for the overlap in functional definitions for DCs and KCs is that DCs represent a more differentiated state of the KC lineage.

Liver Sinusoidal Endothelial Cells

Liver sinusoidal endothelial cells (LSECs) are highly specialized cells that line the hepatic sinusoids. LSECs are distinguished by the presence of fenestrations, or windows, in their cellular membranes. These fenestrations are believed to facilitate the selective passage of antigens between the sinusoid and the hepatic parenchyma. This strategic placement puts LSECs in the ideal position to interact with antigens and immune cells passing between the liver and the portal venous system.

Several studies have shown that in addition to serving as the building blocks lining the sinusoids, LSECs are immune cells with the ability to capture and present antigen and activate T cells (Knolle & Limmer, 2001). As with KCs, considerable controversy surrounds the immunologic function of LSECs. In contrast to earlier work, we have shown more recently that although LSECs are highly capable of capturing various antigens in vivo and in vitro, they lack the ability to activate T cells in the absence of exogenous costimulation (Katz et al, 2004). The differences in results may derive from the use of more specific methods of cell isolation in the latter study. The finding that LSECs are not independently capable of triggering a T cell–mediated immune response does not, however, exclude the possibility that LSECs, in concert with DCs or KCs, play an important role in antigen presentation in the liver.

Effector Cells

T Cells

As is the case for DCs, T cells are implicated in determining the liver’s unique tolerogenicity. T cells are the primary effector cells of adaptive immunity. The nature of the interactions between APCs and T cells determines the overall character of the response to a particular antigen. It is difficult to know whether liver APCs or T cells are primarily responsible.

T cells are classified based on the types of antigen-recognition receptors present on their surface. Most of the T-cell subtypes in the body also have been identified in the liver. The broadest way to classify T cells is as either conventional or unconventional. Conventional T cells express the αβ T-cell receptor in association with either CD4 or CD8. These are the most prevalent T cells in the body and account for about one third of the murine liver T-cell population. In contrast, unconventional T cells expressing NK markers or the γδ T-cell receptor compose a greater proportion of liver T cells, about 50% and 10%, respectively. More recently, the discovery of the transcription factor FoxP3 has enabled the study of another family of T cells with immunosuppressive function. These FoxP3+ or regulatory T cells coexpress either CD4 or CD8 and are present in the liver. Their relative contribution to liver tolerance remains to be defined.

The diversity of conventional T cells is based on their recognition of specific peptide antigen motifs within the context of MHC class I or II molecules on the surface of APCs. The αβ T-cell receptor is highly variable, and numerous T cells, each recognizing a different antigen presented by APC, are present in the immune system. CD8+ T cells respond to peptides presented on MHC class I molecules, which are expressed by nearly every cell in the body, excluding erythrocytes. Activated CD8+ T cells become cytolytic T lymphocytes. CD4+ T cells also are called helper T lymphocytes; they recognize antigens presented on MHC class II molecules on the surface of professional APCs and are capable of regulating and further amplifying the immune response through the secretion of cytokines that act on nearby effector cells.

Liver T cells are divided into the same types of cells, as are the cells of lymphoid organs, however, evidence shows differences in the proportions of T-cell subtypes found within the liver. The interaction between CD4+ and CD8+ cells is clearly important, and more recent studies have suggested that the ratio between these two cell types—which is approximately 2 : 1 in the spleen, lymph nodes, and peripheral blood—is reversed in the liver (Crispe, 2003). Preliminary murine data from our laboratory, using more inclusive isolation techniques, have shown that the liver has a similar CD4+ to CD8+ T-cell ratio of approximately 1.5 : 1 as found in the spleen and node (Katz et al, 2005). Prior studies have shown that unconventional T cells are highly represented in the liver compared with the spleen or lymph nodes, and our data are in agreement with these findings.