29 Apoptosis and Cell Survival in the Immune System

Apoptosis is essential in the generation and function of the immune system. Many of the millions of T and B cells that are produced daily in the primary lymphoid organs are destined to die after a sorting process that keeps only cells that meet strict selection criteria. Successful cells receive a survival signal and emigrate to the periphery, where they will become the many soldiers that protect the organism against viruses, bacteria, and other unfriendly agents. In response to infection or immunization, antigen-specific cells become activated and proliferate, and some differentiate into effector cells. When the infection battle is won, most of these cells are eliminated by apoptosis to prevent their accumulation and the potential problems that it would cause. Defects in the apoptotic process in the hematopoietic system can promote autoimmunity, whereas too much apoptosis promotes lymphopenia and immunodeficiency. This review focuses on the two main pathways of apoptosis and their specific roles during the development and the function of the main cell populations of the immune system.

1. TWO APOPTOTIC PATHWAYS CONVERGE

IN CASPASE ACTIVATION

Execution of the apoptotic program is the result of the activation of a family of aspartate-specific cysteine proteases, called caspases. Caspases pre-exist in healthy cells as inactive zymogens. Depending on their structure and their mode of activation, they are classified as initiator (i.e., caspases-8, -9, -10) or effector (i.e., caspases- 3, -6, -7) caspases. Initiator caspase zymogens contain long pro-domains that allow their recruitment to activation platforms where they autoactivate. They then cleave and activate effector caspases that are responsible for the destruction of essential cellular proteins and the ultimate demise of the cell.

Two different pathways lead to caspase activation (Figure 29-1). The extrinsic pathway, also called death receptor pathway, is triggered by the oligomerization of death receptors of the tumor necrosis factor receptor (TNF-R) family (e.g., Fas, TNF-R1, DR4, DR5) after ligation by their specific ligands (including FasL, TNF, TNF-related apoptosis-inducing ligand [TRAIL]).1 Death receptors contain an intracellular death domain (DD) responsible for the recruitment of the adaptor protein Fas-associated death domain (FADD) by DD homophilic interaction. FADD in turn recruits the initiator caspase- 8 (and -10 in humans) through death effector domain (DED) interaction in a complex named death-inducing signaling complex (DISC), in which caspase-8 is activated by cleavage. Death receptor signaling can be inhibited by cellular FADD-like interleukin-1 beta-converting enzyme (FLICE) inhibitory proteins (cFLIP), which can be recruited to the DISC and block the activation of caspase-8.

Activation of caspase-8 triggers the caspase cascade leading to apoptosis. In type I cells, such as lymphocytes, the death-receptor pathway does not require the Bcl-2-regulated pathway, and initiator caspases-8 and -10 activate the effector caspases directly.2 Genetic alteration of FADD has shown that these proteins are essential for death receptor–mediated apoptosis, but loss of either of these proteins does not affect sensitivity of T cells to a variety of death stimuli, including cytokine deprivation and cytotoxic stress.3,4,5 By contrast, in type II cells, such as hepatocytes, the death-receptor pathway and the Bcl-2–regulated pathway appear to cooperate to kill cells in response to death receptor activation. In this instance, the Bcl-2 family member Bid is activated by cleavage by caspase-8 and triggers mitochondrial events that amplify the initial signal through death receptors.6

Extrinsic pathway

Ligand

Pro-caspases-3, -6, -7

Intrinsic pathway

Cytokine withdrawal, chemotherapeutic drugs, radiations…

BH3-only

proteins

Anti-apoptotic Bcl-2 family members

Bid

Bax/Bak

tBid

Members of the Bcl-2 family are crucial regulators of apoptosis. The Bcl-2 regulated pathway (also called intrinsic, or mitochondrial pathway) requires the involvement of mitochondria (Figure 29-1).7,8 Proteins of the Bcl-2 family all contain 1–4 regions of homology, called Bcl-2 homology (BH) domains. According to the function and the structure of its members, the Bcl- 2 family can be subdivided into three groups. The prosurvival members (Bcl-2, Bcl-xL, Bcl-w, A1, Mcl-1, and BOO) contain three or four BH domains. When overexpressed, they confer to the cells a resistance to various stimuli, such as cytokine withdrawal or cytotoxic stress,

including γ-radiations or chemotherapeutic drugs. The proapoptotic BH3-only proteins (Bad, Bik, Bid, Hrk, Bim, Noxa, Puma, and Bmf) act as sensors of cellular stress. They bind with high affinity to (at least some) prosurvival members and trigger apoptosis when over-expressed. Bim and Puma are potent cell death activators, capable of binding all of the prosurvival proteins. BH3-only proteins with a more limited binding repertoire such as Bad (which binds Bcl-2, Bcl-xL, and Bcl-w, but not Mcl-1 or A1) or Noxa (which binds only A1 and Mcl-1) appear to be weaker apoptotic inducers than Bim or Puma.7 The multidomain proapoptotic proteins (Bax, Bak, and

maybe Bok) contain two or three BH domains and trigger apoptosis when over-expressed. They act downstream of BH3 only proteins, because Bax−/− Bak−/− cells are resistant to BH3-only protein over-expression. In healthy cells, Bax is found in the cytoplasm, but it gets activated and moves to mitochondria on induction of apoptosis. Bak always localizes to the mitochondrial outer membrane, but it also becomes activated on apoptosis induction.9 Activation of Bax and/or Bak results in their oligomerization and mitochondrial outer membrane permeabilization (MOMP). This allows the release of apoptogenic proteins (in particular, cytochrome c) from the mitochondrial intermembrane space. In the cytosol, cytochrome c associates with apoptosis protease activating factor 1 (APAF-1) and the initiator caspase- 9 to form the apoptosome, which triggers the autoactivation of caspase-9 and starts the cascade of effector caspases.

How exactly the proteins of the Bcl-2 family induce MOMP is the subject of an intense debate (for review see references 7, 8, 9).

Genetic experiments involving gene ablation or transgenic expression of many Bcl-2 family members, as well as proteins of the death receptor pathway, have helped define the role of these two major apoptotic pathways in the homeostasis and function of the immune system. Their role in the multiple maturation steps of T and B cells in particular is discussed.

2. APOPTOSIS AND SURVIVAL IN THE DEVELOPMENT AND

HOMEOSTASIS OF THE IMMUNE SYSTEM

Apoptosis is essential for the development of all cell lineages but plays a very particular role in the immune system. Throughout most of adult life, lymphocyte numbers remain constant, as a result of a fine balance between proliferation and apoptosis.10,11 Cells of the immune system are generated from progenitors residing in the bone marrow. Most of these cells have to undergo several developmental stages to become functional immune cells. For instance, the survival of lymphocyte precursors is mediated by cytokines, which regulate the number of progenitor cells and initiate the rearrangement of the antigen receptor genes. B- and T-cell maturation involves the production by these cells of a functional antigen receptor (BCR or TCR, respectively). Cells that fail this process do not receive a survival signal from the receptor and die by apoptosis. The resulting pre-TCRs or pre-BCRs signal survival of progenitors and initiate their further differentiation. The processes of positive and negative selection then ensure that the interaction between the functional receptor and the

major histocompatibility complex (MHC) is adequate. All the lymphocytes whose receptors do not fit the selection criteria are eliminated by apoptosis.

The last important role of programmed cell death in the immune system is the elimination of responding lymphocytes at the end of an immune response. This mechanism of immune homeostasis is essential to prevent the nonspecific tissue damage that prolonged immune responses could cause and limit the risk of autoimmunity.

2.1. Survival of early hematopoietic progenitors

The hematopoietic stem cells (HSCs) give rise to multipotent progenitors (MPPs), which in turn give rise to the common lymphoid progenitors (CLPs) and the common myeloid progenitors (CMPs). CMPs give rise to the erythroid, megakaryocytic, granulocytic and monocytic lineages (Figure 29-2). In the present chapter, we focus on immune system development, without considering the erythroid and megakaryocytic lineage.

Because none of the differentiated lineages have selfrenewal capacity and most have a limited life span (e.g., neutrophils have a life span of 6–18 hours), there is a constant requirement for the production of new cells from the bone marrow to ensure the turnover of the differentiated cells. The role of the Bcl-2 family members in the different developmental stages of B and T cells has been defined more precisely in the last 5 years.

Ectopic expression of Bcl-2 increases the number of hematopoietic progenitor populations and increases their ability to repopulate irradiated hosts.12,13,14 These cells are also protected from several death stimuli.15 These observations suggest that some proapoptotic BH3-only proteins are involved in the killing of progenitors, but do not prove that Bcl-2 is the main prosurvival member of the family involved in this process. Indeed, loss-of-function studies have demonstrated that mice deficient for Bcl-2 could still generate all blood lineages,16,17,18,19 showing that other prosurvival proteins are involved in the protection of HSC.

Similarly, Bcl-xL–deficient embryonic stem (ES) cells could also give rise to mature T cells, but not mature B cells.20,21 Bcl-xL expression increases dramatically when T cells differentiate from CD4–CD8– (double negative, DN) thymocytes to CD4+ CD8+ (double positive, DP) thymocytes. In contrast, single-positive (SP) thymocytes express negligible amounts of Bcl-xL protein. This expression pattern differs from that of Bcl-2, which is present in DN thymocytes, downregulated in DP thymocytes, and re-induced upon maturation to SP thymocytes.21

Bcl-w RNA is detectable in most myeloid and some lymphoid cell lines, but loss of Bcl-w had no effect on the numbers of bone marrow HSCs.22

The role of A1 in the immune system has been poorly characterized, mainly because of the fact that three A1 genes exist in the genome and that inactivation of the three genes at the same time has not been possible. Downregulation of the expression of the three genes may now be possible through the use of transgenic RNA interference.

By contrast, Mcl-1 has been described as an essential prosurvival protein during early hematopoiesis. The high level of Mcl-1 expression in long-term HSC and its subsequent decline in MPP, CLP, and CMP suggest that Mcl-1 may play a pivotal role in the earliest stages of hematopoiesis.23 Interestingly, deletion of Mcl-1 in early hematopoietic development results in a rapid, fatal, and multilineage hematopoietic failure.24 Later in myeloid development, Mcl-1 exhibits a selective role

being required for the terminal stages of granulocyte development but is dispensable for monocytic differentiation.25 BH3-only proteins Bim, Puma, and Noxa have been shown to interact with Mcl-1 and thus are potential candidates for the downsizing of early progenitor populations.26 Bim-, Puma-, and Noxa-deficient mice do not exhibit abnormalities in the resting hematopoietic progenitor populations,27,28,29 suggesting that none of them has an exclusive role in the determination of HSC numbers.

Consistent with the observation that Bcl-2 overexpression protects cells from cytokine withdrawal or ionomycin-induced apoptosis, Bcl-2 and Bim proteins display critical opposing roles in controlling lymphocyte homeostasis. Importantly, it has been described that the removal of Bim is able to rescue defects observed in Bcl-2-/- mice.30 Whereas a deficiency in Bcl-2 reduced the number of lymphoid and myeloid cells, the loss of one allele of Bim ameliorated this deficit and loss of both