Apoptosis

Illustrations of cells undergoing apoptosis were made almost as soon as stains were used to examine the appearance of cells in different tissues. Drawings of ovarian follicles undergoing cell death made over 100 years ago demonstrate cell shrinkage and nuclear condensation. Subsequent descriptions of “pyknosis,” chromatin margination, and other terms used to convey the appearance of subcellular particles during cell death included many features now recognized as apoptosis. The history of this subject is reviewed elsewhere.¹

Our modern understanding of apoptosis began with electron microscopic descriptions of morphologic changes characterized by shrinkage of hepatocytes (i.e., shrinkage necrosis) after ischemic or toxic injury to the liver. The name apoptosis was coined by Kerr, Wyllie, and Currie in 1972 to describe the form of death that was “consistent with an active, inherently controlled phenomenon” characterized by cell shrinkage, nuclear condensation, and cell blebbing (Figure 27-1).² This term also conveyed the concept of cell death that was similar to leaves falling from a tree (apo means “from,” and ptosis, “a fall,” in Greek), implying a regulated “mechanism of cell deletion, which is complementary to mitosis.”²

Figure 27-1 Electron microscopic morphology of cell death. A, A cytotoxic T cell (lower left) conjugated to its target, P815 (a murine mast cell), before initiation of cell death. B, Induction of apoptotic changes in P815. Note the reduction in target cell size, the nuclear condensation, and the vacuoles with relative preservation of organelles. C, Osmotic lysis and necrosis in P815 induced by antibody and complement. Note the increased size of the nucleus and the apparently random fragmentation of the chromatin. Organelles are severely disrupted. C, dense chromatin; M, mitochondria; P, nuclear pore; V, vacuoles. D, Electron microscopic appearance of autophagy. An autophagosome (AP) is observed in a normal rat kidney cell. The autophagosome is surrounded by two cisterns of rough endoplasmic reticulum (ER). A Golgi stack (Go) is visible next to the autophagosome.

(C, Adapted from Russell JH, Masakowski V, Rucinsky T, et al: Mechanisms of immune lysis III: characterization of the nature and kinetics of the cytotoxic T lymphocyte induced nuclear lesion in the target, J Immunol 128:2087, 1982, with permission. D, Figure kindly provided by Eeva-Liisa Eskelinen, Department of Biosciences, Helsinki, Finland.)

Further developments in apoptosis paralleled advances in molecular biology, genetics, and biochemistry. The detection of a nucleosomal ladder³ was of considerable importance, because it defined a biochemical event (i.e., nucleosomal cleavage) and provided a simple electrophoretic test for detection of apoptotic cell death that remains a standard in the field (see Laboratory Detection of Apoptosis later in this chapter). Another landmark was the discovery in the 1980s that the death of cells during development of the nematode Caenorhabditis elegans was under strict genetic control. Remarkably, the death of these cells could be perturbed by mutations of a small number of genes called ced (for cell death abnormal) genes.⁴ Horvitz and colleagues determined that two ced genes, ced3 and ced4, encoded death effectors, whereas ced9 was an antiapoptotic gene.⁴ Most of the remaining ced genes were responsible for engulfment and removal of “corpses.” This simple model in which CED-3 is the main death protease that is activated by CED-4 and inhibited by CED-9 has served as a paradigm for defining apoptotic pathways in mammalian cells (Figure 27-2). In 2002, Robert Horvitz was awarded the Nobel Prize for discoveries concerning the genetic regulation of programmed cell death.

Figure 27-2 Caenorhabditis elegans paradigm of apoptosis. Genes involved in the regulation, execution, and clearance of apoptotic cells during development of C. elegans and their mammalian homologues are shown. In this figure, only the most closely related homologues are indicated, but as described in the text and shown in the figures, the complexity in mammalian cells is much greater. Note that at least half of the cell death abnormal (CED) proteins are involved in engulfment and removal of apoptotic corpses. Two distinct but partially overlapping pathways of engulfment are present in C. elegans: CED-2, -12, -5, and -10, which most likely regulate cytoskeletal changes, and CED-1 and -6, which may be involved in recognition (CED-1) and upstream signal transduction (CED-6). CED-7 is homologous to the mammalian adenosine triphosphate (ATP)-binding cassette transporter-1 (ABC-1), which likely affects membrane dynamics. The lower right panel shows a schematic of the two proposed pathways for apoptotic cell ingestion in mammalian cells. In pathway (1), an unknown receptor(s) triggers activation of the GTPase, RhoG, which leads to transport of the scaffolding protein, ELMO, to the cell membrane. There, Dock 180, a guanine exchange factor, promotes Rac activation, leading to activation of the cytoskeleton and phagocytosis. In pathway (2), activation of the CED-1 homologue (most likely CD91) interacts with the adapter protein, GULP, and downstream activation of the cytoskeleton.

Mammalian cells are much more complex and, as will be discussed in detail, have multiple defined pathways that follow the basic C. elegans model. The molecules within these pathways, the downstream effectors of apoptosis, the caspases (cysteine aspartate proteases), and the proteins implicated in the clearance of apoptotic cells are discussed in detail here. Regulation of cell death is of seminal importance in a number of diseases, including cancers, autoimmune diseases, and degenerative disorders.^5,6 The relevance of apoptosis to rheumatic disorders is summarized at the end of the chapter.

Programmed Cell Death

Whereas apoptosis originally referred to the appearance of dying cells in certain contexts, as explained earlier, the concepts of atrophy, cellular or tissue involution or regression, and degeneration had been appreciated for hundreds of years, yet the two phenomena were not associated until relatively recently. Perhaps the most precise descriptions of cells that died in an orderly and apparently programmed fashion were documented in developmental biology. Examples included the involution of cells between digits, the metamorphosis of insect larvae, and the death of specific cells during development of C. elegans.

Autophagy

Autophagy, which means “to eat oneself,” is essentially a protective process that occurs after growth factor withdrawal or nutrient depletion that may or may not result in cell death. The process is highly conserved from yeast through humans. Under circumstances of nutrient depletion, cells switch to a catabolic program in which cellular constituents are degraded for energy production as a survival mechanism. In these cells, a double membrane vesicle (autophagosome) forms around organelles or protein aggregates and encapsulates them (see Figure 27-1D). A characteristic feature of this process is the lipidation of LC3 (microtubule-associated protein 1 light chain 3) to form LC3-II, which can be identified as coarse dots on the autophagosomes seen by microscopy (see Figure 27-6I, later), or by changes in molecular mass on Western blot analysis. The autophagosomes then fuse with the lysosomes, leading to degradation of their contents (Figure 27-3).

Figure 27-3 Pathways of autophagy (ATG). Autophagy is initiated by starvation or by activation of intracellular immune sensors. ATG proteins are activated, leading to the nucleation of an isolation membrane (green rectangle) and conjugation of phosphatidylethanolamine to LC3. Continued concerted activities of ATG proteins lead to formation of the autophagosome from the isolation membrane, which then fuses with lysosomes to form autolysosomes. The contents of the autolysosomes are degraded, and the products recycled for energy utilization.

The biochemical pathways by which autophagy is initiated and executed are complex and are schematically outlined in Figure 27-3. In brief, lack of nutrients leads to a reduction in phosphoinositide (PI)-3 kinase activity, resulting in loss of suppression of mTOR (mammalian target of rapamycin) and activation of autophagic (ATG) proteins. The ATG gene family comprises at least 20 members and includes beclin1 (ATG6), a protein that is critically involved in the regulation of autophagic cell death.⁷ Beclin1 is monoallelically deleted in a variety of human cancers, and may function in control of cell growth and in tumor suppression.⁸ Autophagic death shares a number of morphologic features with necrotic cell death⁹ and may be seen in neuronal cell death associated with polyglutamine repeats. Many reports indicate that autophagy is important in immune function. Examples include intracellular host response to pathogens, survival of lymphocytes after growth factor withdrawal, Toll-like receptor (TLR) stimulation in plasmacytoid dendritic cells (pDCs), and major histocompatibility complex (MHC) class II presentation of antigen.^10,11 The strongest link between autophagy and autoimmunity or autoinflammatory disease is the association between a genetic variant of ATG16L1 and another protein that regulates autophagy, the immunity-related guanosine triphosphatase gene (IRGM), and Crohn’s disease. The precise mechanisms that account for this association remain to be determined.¹¹

Necrosis

Necrosis (from the Greek nekros, meaning “corpse”) is distinguished from apoptosis predominantly by morphologic appearance.¹² Necrotic cells are swollen, and electron microscopy reveals disorderly fragmentation of chromatin and severe damage to the mitochondria (see Figure 27-1). The cellular membrane loses integrity and becomes permeable to vital dyes such as trypan blue and propidium iodide (see Figure 27-6, later). The distinction between apoptosis and necrosis remains important from a number of perspectives. In contrast to the genetic and biochemical programs that regulate apoptosis, necrotic cells usually result from death “by accident,” that is, from thermal or drug injury, infection, or infarction of an organ. Because of uncontrolled release of intracellular contents, necrotic cells induce a proinflammatory immune response, whereas apoptotic cells usually elicit an anti-inflammatory response.

The same inducers (e.g., ischemia, hydrogen peroxide) may produce apoptosis or necrosis, depending on the severity of the injury and the rapidity of cell death. The fate of the cell is determined in part by cellular energy reserves, especially adenosine triphosphate (ATP).¹³ ATP is generated by oxidative phosphorylation in mitochondria, as well as by glycolysis in the cytosol. Some inducers initially may cause apoptosis followed by necrosis (postapoptotic necrosis). This is likely to occur when removal of apoptotic cells is delayed and is thought to be important in stimulating an inflammatory response to self-antigens. A unique type of necrosis in neutrophils, called NETosis, is implicated in SLE^13a (see Defective Uptake and Processing of Apoptotic Cells).

Pyroptosis

Pyroptosis (from the Greek pyro, meaning “fire”) is distinguished from other forms of cell death first and foremost by the activation of caspase 1 (interleukin [IL]-1β–converting enzyme) and secretion of the inflammatory cytokine, IL-1β. Pyroptosis is most strongly associated with infection by intracellular bacteria such as Salmonella, Yersinia, and Legionella, although it may also be seen following tissue infarction.¹⁴ Cells undergoing pyroptosis demonstrate nuclear condensation associated with DNA damage, cell swelling, and ultimately cell lysis associated with release of IL-1β.¹⁴ The mechanisms responsible for this process involve intracellular sensors of bacterial products and formation of the inflammasome. These topics are briefly described in the following sections and elsewhere in the text (see Chapter 18).

Death Receptors

The DRs identified, including Fas, TNFR I, DR-3 (TRAMP/wsl/APO-3), DR4 (TRAIL, receptor for the Apo2 ligand), DR5, and DR6, share homology in their intracellular domains over a 70 amino acid region called the death domain (DD).¹⁹ Three decoy receptors have been identified—two (DcR1 and DcR2) that bind and inhibit their ligand, TRAIL, and one (DcR3) that binds Fas ligand. These decoy receptors presumably modulate the cytotoxic function of the ligands, but their biologic contexts remain to be fully defined. Use of alternative splice forms and shedding of the receptors and ligands also downmodulate their function.

TNFR family members are characterized by two to six cysteine-rich domains (CRDs) in their extracellular regions.¹⁷ The co-crystal structure of TNFR I and lymphotoxin-α indicates that CRDs project from the cell surface in a linear array, making distinct contact with ligands at subunit interfaces. The first CRD may also be responsible for preassembly of the receptor as trimers that undergo further conformational alteration upon ligand engagement.

The three-dimensional structure of the DD has been solved by nuclear magnetic resonance spectroscopy and has been shown to consist of six amphipathic α-helices that create a unique fold.²⁰ Functionally, the DD appears to be a novel protein-protein association motif that facilitates homotypic interactions. For example, the DD of Fas and TNFR I self-associate, thereby recruiting adapter proteins that also contain DD and that directly or indirectly mediate receptor signal transduction (see Figure 27-4).

Death Receptor Signal Transduction

This section will focus predominantly on signaling from Fas and TNFR, because these are the best-characterized members of the death receptor subfamily, and it is likely that other death receptors signal through similar pathways. As illustrated in Figure 27-4, Fas and TNFR I share a common death pathway. Binding of Fas ligand to Fas causes conformational changes in the receptor cluster, leading to recruitment of intracellular adapter molecules. Initially, aggregation of Fas induces uptake of the adapter protein, FADD, to the DD of Fas. FADD has two structural domains: a C-terminal DD, which mediates Fas binding, and an N-terminal death effector domain (DED). The FADD DED allows recruitment of procaspase 8 and procaspase 10 through DED-DED interactions. Procaspases 8 and 10 have a bipartite structure comprising a DED and an enzymatic caspase domain, the latter linking Fas aggregation with the execution phase of apoptosis. Apposition of procaspases 8 and 10 to the activated Fas complex leads to autocatalytic cleavage and conversion of the proenzymes to activated proteases, which are released and are able to initiate a proteolytic cascade, leading to programmed cell death. In some cell lines, caspase 8 cleavage also results in cleavage of the proapoptotic molecule Bid, which activates the mitochondrial amplification cascade (type II pathway²¹; Supplemental Figure 27-2 on www.expertconsult.com; see also Figure 27-4).

Supplemental Figure 27-2 Inhibitors of apoptosis. Inhibitors of death pathways are shown in brown. Note that a number of tumor necrosis factor (TNF) family members (e.g., CD40L, BAFF/ BLyS/TALL, receptor activator of NFκB [RANK] ligand) activate the nuclear factor κB (NFκB) pathway, resulting in increased transcription of antiapoptotic proteins, inhibitors of apoptosis (IAPs), Bcl-2 family members, and A1.¹⁸¹ In some cases, inhibitors of apoptosis are blocked or destroyed during apoptosis. For example, the protein Smac/Diablo is released from mitochondria and eliminates the antiapoptotic effect of the IAP family. Growth factors impart survival signals, in part through the phosphoinositide (PI)-3 kinase → Akt pathway (right side of diagram). Activation of the kinase, Akt, directly inactivates certain proapoptotic proteins such as Bad and caspase 9, and promotes survival by inhibiting the forkhead family (FKRL) of transcription factors and enhancing the expression of the antiapoptotic protein, NFκB.¹⁸² Inhibitor of caspase-activated DNase (ICAD) is a specific inhibitor of the DNAse, CAD, which is degraded by caspases. Tetrapeptide inhibitors of specific caspases are shown in blue.

Although the six DD-containing receptors initiate cell death in certain contexts, all may signal cell survival/proliferation in different cell types and/or in different contexts. The ability to signal an opposite cell fate depends on the recruitment of proteins such as tumor necrosis factor receptor–associated factors (TRAFs), which activate NFκB, thereby promoting cell survival (see later).

Defective function of caspase 8 leads to a form of cell death called NECROPTOSIS that combines features of apoptosis and necrosis resulting in activation of NFκB and production of inflammatory cytokines such as TNF.^21a

Death Ligands

FasL (CD178) is a 40-kD type II transmembrane protein that shares 15% to 35% amino acid identity with the TNF superfamily of ligands. FasL is expressed constitutively in the anterior chamber of the eye and in the testis but is induced when CD8, T helper-1 (Th1) CD4⁺ T cell, and some natural killer (NK) cell populations become activated.²² In lymphocytes, expression of ligand is tightly regulated and activity on the cell surface short-lived, because metalloproteases cleave the extracellular portion of the ligand into soluble, functional molecules. The zinc metalloprotease, TNF-converting enzyme (TACE), is a membrane-anchored member of the disintegrin family of proteases that cleave active TNF from the cell surface.^23,24

Function in Immune Regulation

Although Fas and FasL interactions in the thymus are not thought to play a major role in negative selection, this pathway is involved in the maintenance of immune privilege in the eye and the testis, in the pathogenesis of graft-versus-host disease, and in immune evasion by tumor.²² The major physiologic function of Fas and FasL in the immune system is the preservation of peripheral tolerance. This is achieved by the phenomenon of activation-induced cell death (AICD), whereby CD8 T cells, Th1 CD4⁺ T cells, and possibly NK cells induce apoptosis of activated T cells, B cells, and macrophages. Deletion of activated immune cells removes the source of proinflammatory molecules, prevents the continued presentation of self-peptides by primed (high levels of co-stimulatory molecules) antigen-presenting cells, and eliminates B cells that have mutated to self-specificity within the germinal centers.²⁵ More recently, it has been shown that Fas-FasL interactions promote a variety of additional functions, including early T cell proliferation, tumor survival, and cell migration. These topics are discussed in greater detail elsewhere,^26,27 and the consequences of Fas deficiency are described later in this chapter.

It should be noted that, whereas TRAIL signals apoptosis through DR4 and DR5 predominantly in tumor cells, evidence suggests that TRAIL also plays a role in negative selection of thymocytes.²⁸ Similarly, DR3 (TWEAK, receptor for the Apo3 ligand) has been implicated in negative selection.²⁹ Finally, DR6 plays a role in immunologic homeostasis, as evidenced by enhanced T and B cell proliferation in DR6-deficient mice.³⁰

Genotoxic Injury

Mutations occur frequently in mammalian DNA and usually are promptly repaired. However, if repair fails or DNA is severely damaged by radiation or drugs, the transcription factor, p53 (“guardian of the genome”), is upregulated and phosphorylated by DNA damage sensors such as ATR and ATM. Activated p53 induces cell cycle arrest through induction of the cyclin-dependent kinase inhibitor, p21. If DNA damage is repaired, cell cycle arrest is abrogated, whereas if the injury cannot be repaired, the cell undergoes apoptosis. The critical importance of p53 as a tumor suppressor is illustrated by the high frequency of p53 mutations in cancers.³² p53 induces apoptosis, in part through transcription of death effectors such as Bax that cause mitochondrial stress. In addition, activation of the transcription factor Foxo-1 upregulates the expression of Bim, FasL, and TRAIL.³³

Mitochondrial Stress

Mitochondria are cytoplasmic organelles that contain their own 16-kb genome encased by inner and outer membranes, with a number of proteins, including cytochrome-c, situated between these membranes (see Figure 27-4). Mitochondria help to maintain redox potential and serve as the energy powerhouse of the cell through the generation of ATP by oxidative phosphorylation. These biochemical pathways create an electrochemical gradient (Δψ) that is positive and acidic on the outside and alkaline on the inner side of the mitochondrial membrane. Spanning the inner membrane is the adenine nuclear translocator (ANT), which mediates ATP transport (with VDAC, see later) to the cytosol. On the outer mitochondrial membrane (OMM) is situated the voltage-dependent anion channel (VDAC), which is permeable to solutes of approximately 5000 kD.³⁴

Genotoxic injury, reduced supply of nutritional or growth factors, raised intracellular calcium, reactive oxygen intermediates (ROIs), and exposure to certain chemicals such as staurosporine cause mitochondrial stress. These initiating factors lead to selective mitochondrial membrane permeabilization (MMP) with resulting dissipation of the proton gradient responsible for the Δψ permeability transition, permeabilization of the outer membrane, and loss of ATP production. Mitochondria themselves are the major producers of ROIs, which, in excess, damage nucleic acids, proteins, and membrane lipids.

Once MMP is initiated, cytochrome-c is released from the intermitochondrial space into the cytosol (see Figure 27-4). In the cytosol, cytochrome-c and the cofactors Apaf-1 and ATP or dATP assemble with caspase 9 to form a molecular aggregate called the apoptosome, which promotes the cleavage of procaspase 9 into its active form.³⁵ Caspase 9 acts on effector caspases such as caspase 3, resulting in the caspase cascade that leads to cleavage and inactivation of a wide variety of substrates within the cell (see Figure 27-4). A caspase-independent apoptosis-inducing factor (AIF) is also released from the mitochondria and induces nuclear changes and cell death by less well defined pathways.³⁶

Endoplasmic Reticulum (ER) Stress

The main functions of the ER are to regulate intracellular calcium flux and to promote proper folding of nascent proteins. In the contiguous conduit, the Golgi apparatus, post-translation modifications such as glycosylation and isoprenylation are executed. Elaborate mechanisms are in place to ensure that errors in protein folding do not occur, but if they do, an unfolded protein response (UPR) is initiated. The ER/Golgi initiates apoptosis if calcium flux is excessive, if unfolded proteins persist, or if post-translational protein modification is abnormal.³⁷ Apoptosis is the result of three central players: inositol-requiring protein-1 (IRE1), activating transcription factor-6 (ATF6), and protein kinase RNA-like ER kinase (PERK) (see Figure 27-4). Unfolded proteins may activate these proteins directly, or by binding to the ER chaperone immunoglobulin-binding protein, Bip. Once activated, the three proteins cause selective RNA cleavage, termination of protein synthesis, and induction of proapoptotic responses (see Figure 27-4). In contrast to the death receptor or mitochondrial pathways, apoptosis is executed through caspase 12 in mice and possibly caspase 4 in mammals.³⁸ However, many of the same molecules that regulate apoptosis in the mitochondria, the Bcl-2 family in particular, also influence ER-mediated apoptosis, possibly through control of intraluminal calcium. It is important to point out that induction of the UPR, in certain circumstances, leads to NFκB activation and inflammation, rather than cell death.³⁹

Antiapoptotic Proteins: FLIP, Bcl-2, IAPs, and Akt

(See Supplemental Figure 27-2 on www.expertconsult.com.)

Cellular homeostasis within each tissue is carefully regulated. Excessive cell growth or premature cell death translates into disease, as will be discussed in the last part of this chapter. The death and survival pathways are finely balanced, and each cell death program is regulated, often at multiple levels. Before we discuss how the death program is executed, we will describe the way in which cell death is modulated by inhibitors of apoptosis.

Finding the Dying Cell

During apoptosis, the enzyme, calcium-independent phospholipase A2, is activated by caspase cleavage, leading to the generation of lysophosphatidylcholine (LPC) (see Figure 27-5 for complete apoptosis schema). LPC acts both as a soluble chemoattractant for macrophages and as an epitope on the cell membrane for natural immunoglobulin (Ig)M antibodies.⁵³ Additional “find-me” signals include sphingosine-1-phosphate (S1P) and the nucleotides ATP and UTP, which are released by dying cells, especially as the plasma membrane becomes damaged (necrosis).^54,55 Intravital microscopy coupled with biochemical studies revealed that cell necrosis led to release of ATP, activation of Nrlp3 inflammasomes, and release of IL-1β.⁵⁶ Subsequently, a chemokine gradient attracted neutrophils to the site of cell damage and formylated peptides to the necrotic cell.

Figure 27-5 A, Attraction and keep-out signals released by dying cells. Lactoferrin is a keep-out signal for neutrophils. LPC, lysophosphatidylcholine; S1P, sphingosine-1-phosphate. B, Receptors, ligands, and opsonins (bridging proteins) implicated in recognition or phagocytosis of apoptotic cells (see text for details). CRP, C-reactive protein; CRT, calreticulin; LyPtC, lysophosphatidylcholine; Ox, oxidized form; PS, phosphatidylserine; TSP, thrombospondin. Yellow circles are lipids or oxidized lipids.