Harvard-MIT Division of Health Sciences and Technology HST.176: Cellular and Molecular Immunology Course Director: Dr. Shiv Pillai Memory and death in the immune system Apoptosis Apoptosis refers to a form of death in which a cell initiates a suicide program resulting in characteristic morphological changes that accompany death. These changes include chromatin condensation, nucleolar disruption and cytoplasmic contraction..Growth factor induced serine/threonine kinase DNA damage activity Fas Bax / Bad / Bak / Bcl-XS Caspase 3/CPP32/Ced-3 Bcl-2/ Bcl-XL Ced-4 mitochondrial permeability Cytochrome c release p53 g -irradiation JNK/SAPK ? Ceramide Glucocorticoids GR Nur 77 Atypical PKCs Antigen receptors Daxx FADD/Mort-1 FLICE/Caspase-8 Caspase cascade XIAP Granzyme B Channel formation DFF Ca++/Mg++ dependent endonuclease PARP, LaminB, DNA-PK, SREBP etc. Figure 1. An overview of pathways leading to apoptosis. The “common death pathway” is highlighted in the box. GR refers to the glucocorticoid receptor. PKC refers to protein kinase C. Some atypical PKCs may prevent apoptosis while others may contribute to it. Similarly the JNK pathway may actually protect lymphocytes from apoptosis. Apoptosis is often readily recognized by the cleavage of DNA into a “ladder” of oligonucleosome length fragments. This form of death is also referred to as “programmed cell death” or “physiological cell death”. Apoptosis is of critical importance in the development of virtually every multicellular organism. This form of death is initiated many times during lymphoid ontogeny - during the process of negative selection and whenever lymphocytes fail to be positively selected. The termination or down-regulation of immune responses is also in part achieved by the induction of the apoptotic death of activated T and B cells. Cytotoxic CD8+T, Natural killer cells, as well as CD4+ T lymphocytes express specificmolecules that are designed to assist in the execution of targets by apoptosis. The expression or activation of genes whose products (such as the Fas ligand, Perforin, and Granzyme B) are involved in the apoptotic elimination of other target cells is an important aspect of lymphocyte development. The induction of DNA damage, the activation of a stress response, the withdrawal of growth factors, or the triggering of specific signaling receptors - all of the above may lead to the initiation of apoptosis. The cell type or state of differentiation of a cell can influence whether a particular receptor induces proliferation or apoptosis. Apoptosis may be kept at bay in certain cell types by the induction of anti-apoptotic signals. Death may occur by default in other cell types or stages of differentiation when proliferative signals are withdrawn, presumably because in these cells protective pathways are not turned on. Some of the signals involved in the initiation of apoptosis in lymphocytes are outlined in Figure 1. The death of large pre-B and double negative T cells that fail to be selected by the pre-B and pre-T receptors may reflect the failure of these cells to receive cell survival or growth promoting signals. Signals from antigen receptors induce apoptotic death primarily in immature B and CD4+/CD8+ T cells as part of negative selection. The death by default of CD4+/CD8+ T cells may be mediated by endogenous glucocorticoids and the glucocorticoid receptor (GR). Apoptosis of activated T and B cells as well as of B cells that have been activated as bystanders (and not by specific antigen) depends on triggering of the Fas receptor. The Fas pathway may also be used by CD4+ T cells to kill infected targets. Cytotoxic T lymphocytes (CTLs) and Natural Killer (NK) cells both secrete proteins such as perforin and Granzyme B to induce apoptosis of their targets. The common death pathway Over the past few years a molecular understanding of a common apoptotic death pathway has emerged from a range of studies in a number of organisms and cell types. This pathway is summarized in the box in Figure16. The induction of death is generally linked to the activation of a set of cysteine proteases that cleave proteins immediately after aspartic acid residues. The first such enzyme to be identified was the mammalian homolog of the C. elegans cell death gene, Ced-3. This protein was identical to an enzyme previously described as the Interleukin-1b Converting Enzyme (ICE) which was known to beresponsible for proteolytically converting pro-Interleukin-1b to mature IL-1. It soon became apparent that the ICE protease was a member of a large family of related proteases most of which appear to be involved in cell death. These enzymes are now referred to as caspases. Apart from ICE itself (caspase 1) a critical mediator of cell death appears to be caspase-3 (also known as CPP32/ Yama/Apopain). Activation of the caspase cascade may depend on the induction in the cell of a reducing state which would favor the catalytic activation of these cysteine proteases (which contain a QACRG motif in which the cysteine forms a part of the active site). A number of substrates of caspase-3 have been identified. These include Poly-ADP ribose polymerase (PARP), huntingtin (the protein product encoded by the gene that is mutated in Huntington’s disease), lamin B, the sterol regulatory element binding proteins (SREBPs), D4-GDI, and the U1 associated 70 kD protein. The relevance of these substrates to the initiation or progression of apoptotic death is unclear. A particularly interesting substrate of caspase-3 is the DNA Fragmentation Factor or DFF. DFF is a cytosolic heterodimeric protein that consists of a 45kD subunit and a 40 KD subunit. DFF is activated by caspase-3 mediated cleavage of the 45 kd subunit at least two cleavage sites. The activated DFF in turn, by an unknown process, contributes to the activation of a Ca++/Mg++ dependent endonuclease which is responsible for the fragmentation of DNA that is characteristic of apoptosis. Caspases are activated by proteolytic cleavage after specific aspartate residues. Cleavage of a given caspase may be mediated by another member of the family or by autoproteolysis. In the case of Fas signaling, a caspase molecule, caspase-8 is activated and targets caspase-3. Cytotoxic or NK cell mediated killing (also discussed later in the chapter) involves the introduction of a caspase-3 activating