Apoptosis

The modern understanding of apoptosis began with the 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 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 11-1).¹ 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.”¹

FIGURE 11-1 Electron-microscopic morphology of cell death.

A, A cytotoxic T cell (lower left) conjugated to its target, P815, a murine mast cell before the initiation of cell death. B, Induction of apoptotic changes in P815. Note the reduction in target cell size, nuclear condensation, and vacuoles with the relative preservation of organelles. C, Osmotic lysis and necrosis in P815 induced by antibody and complement. Note the increased size of the nucleus and apparently random fragmentation of the chromatin. Organelles are severely disrupted. D, Autophagic cell death of L929 cells treated for 12 hours with caspase inhibitor (zVAD). Arrows show membrane-bound vacuoles characteristic of autophagosomes. C, dense chromatin; M, mitochondria; P, nuclear pore; V, vacuoles.

(Autophagy image courtesy of Dr. Mike Lenardo. 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.)

Further developments in our understanding of apoptosis paralleled advances in molecular biology, genetics, and biochemistry. The detection of a nucleosomal ladder² 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. Studies in the 1980s demonstrated that the death of cells during nematode development was under strict genetic control. Remarkably, the death of these cells could be perturbed by mutation of a small number of genes called ced (for cell death abnormal) genes.³ Horvitz determined that two ced genes, ced3 and ced4, encoded death effectors, whereas ced9 was an antiapoptotic gene. Most of the remaining ced genes were responsible for engulfment and removal of the “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. Mammalian cells are more complex and, as discussed here in detail, have multiple defined pathways that follow the basic Caenorhabditis 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. The control of cell death is of seminal importance in a number of diseases, including cancers, autoimmune diseases, and degenerative disorders.⁴ Regulation of apoptosis and handling of dying cells are especially relevant to SLE, as also discussed here.

Necrosis

Necrosis has traditionally been viewed as a passive form of cell death resulting from toxic or physical insults leading to adenosine triphosphate (ATP) depletion, although later studies indicate that some changes can be biochemically mediated. Morphologically, necrosis is notable for plasma cell membrane disruption leading to cellular swelling and cytoplasmic vacuolation (see Figure 11-1). In contradistinction to apoptosis, necrotic death induces inflammation around the dying cell as a result of the release of various intracellular components (see Figure 11-1). Although chromatin degradation is evident in necrotic cells, the condensation and organized DNA fragmentation seen in apoptotic cells are usually absent. Notably, 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 cell’s fate is determined in part by cellular energy reserves such as ATP. When removal of the apoptotic cells is delayed, some inducers initially cause apoptosis followed by necrosis (postapoptotic necrosis). This transition from apoptotic to necrotic cells is likely to be important in the context of autoimmunity (see later).

Autophagy

Autophagy, which means to eat oneself, occurs during nutrient stress when cells switch to a catabolic program and degrade cytoplasmic constituents as a survival mechanism.⁵ It does not necessarily lead to cell death. During autophagy, more than 20 members of the ATG family of proteins orchestrate the initiation, elongation, and closure of the characteristic double-membrane vesicle called the autophagosome. The autophagosome then fuses with lysosomes (see Figure 11-1), the contents are degraded, and the products recycled for energy utilization. Autophagy has been implicated in a wide spectrum of diseases ranging from degenerative neurologic diseases associated with polyglutamine repeats to cancer and immunologic diseases such as Crohn disease.⁵

Autophagy is important in immune functions, such as the intracellular host response to pathogens, survival of lymphocytes following growth factor withdrawal, Toll-like receptor (TLR) stimulation in plasmacytoid dendritic cells (pDCs), and major histocompatibility complex (MHC) class II presentation of antigen.^5,6 The association between a genetic variant of ATG16L1 as well as IRGM (immunity-related guanosine triphosphatase gene), another protein that regulates autophagy, and Crohn disease is unclear but could potentially involve thymic selection, macrophage function, or intestinal immunity.⁵ Of relevance to SLE is the identification in patients with the disease of an increased frequency of single-nucleotide polymorphisms) (SNPs) in ATG5 that could potentially predispose to disease through alterations in T-cell selection, clearance of apoptotic cells, or regulation of type I interferon (IFN).⁵

Other Forms of Cell Death

Pyroptosis (from the Greek, “pyro” meaning fire) is distinguished from the other forms of cell death by the activation of caspase 1 (interleukin-1beta [IL-1β]–converting enzyme) and secretion of the inflammatory cytokine IL-1β. Pyroptosis is most strongly associated with infections by intracellular bacteria such as Salmonella, Yersinia, and Legionella, although it may also be seen following tissue infarction.⁷ Cells undergoing pyroptosis demonstrate nuclear condensation associated with DNA damage, cell swelling, and, ultimately, cell lysis associated with release of IL-1β. The mechanisms responsible for this process involve intracellular sensors of bacterial products and formation of the inflammasome.

Necroptosis is a programmed form of necrosis that has so far mainly been described in response to certain death ligands such as Fas ligand and tumor necrosis factor alpha (TNF-α; see Biochemistry of Apoptosis). It has long been known that in addition, a stimulation of inflammation through activation of nuclear factor kappa B (NF-κb), TNF-α can induce necrosis (tumor necrosis factor). Only recently has the pathway become clear, and it is described in the section Initiation and Pathways of Apoptosis.

Caspases

The cysteine protease family of caspases plays a central role in apoptosis, and the orthologs in C. elegans and Drosophila demonstrate evolutionary conservation. Caspases are produced as catalytically inactive zymogens and function as homodimers, with each monomer composed of a large subunit and a small subunit. Caspases can be divided into two categories reflecting both structural and functional differences; “initiator” and “effector” caspases (Table 11-1; for a comprehensive review, see reference 11).

TABLE 11-1 Families of Intracellular Proteins Involved in Apoptosis

The “initiator” caspases, which in mammals include caspases 2, 8, 9, and 10, have extended N-terminal prodomains that allow for clustering and autoactivation of the zymogens. The clustering of the initiator caspases depends on adaptor proteins that utilize homophilic interactions between conserved nonenzymatic domains present on both the adaptor and the caspase. These adaptors include Fas-associating protein with death domain (FADD), which recruits caspase 8 via homophilic interactions between DEDs in both proteins (see later and Figures 11-2 and 11-3), and apoptotic protease activating factor 1 (Apaf-1), which recruits caspase 9 through a homophilic interaction in CARD (caspase recruitment domain). The recruitment and activation of various initiator caspases occur in response to unique sets of stimuli and in separate cellular compartments. Autoactivation of initiator caspases likely occurs through oligomerization, ultimately leading to the activation of downstream effector caspases (caspases 3, 6, and 7). As discussed later, caspase 8, considered a pro-apoptotic protease, functions both in Fas-induced apoptosis and in lymphocyte activation and protective immunity.

The “effector” caspases are necessary for the execution of apoptosis. They cleave specific substrates, such as the structural proteins fodrin, gelsolin, and lamins, key intracellular enzymes involved in DNA repair (e.g., poly[ADP]ribose polymerase, DNA-dependent protein kinase [DNA-PK]). These changes facilitate inactivation of synthetic functions of the cell, dissolution of the nuclear membrane, and packaging of cellular proteins into apoptotic blebs on the cell surface. Caspases also cleave regulatory proteins such as Bcl family members and the inhibitor of caspase-activated DNase (ICAD). Cleavage of ICAD leads to the release of active CAD, which enters the nucleus and cleaves nucleosomes at the linker region, yielding the characteristic “DNA ladder.”

Not all caspases are involved in the execution of apoptosis. Human caspases 1, 4, and 5 are most likely involved in inflammation. Caspase 1 was originally defined as the enzyme that cleaves IL-1 (interleukin-1–converting enzyme [ICE]) into its active form. It has been shown that caspases 1 and 5 interact to form a multiprotein complex that has been called the inflammasome (analogous to the apoptosome).¹² Caspases 1 and 5 bind to the adaptor proteins ASC (pyrin CARD [PYCARD]) and NALP1 (DECAP), respectively, by their CARD domains. The protein, pyrin, which is mutated in familial Mediterranean fever (FMF), binds to ASC. Mutation of pyrin therefore most likely leads to inflammation through uncontrolled activation of caspase 1, generating active IL-1 and IL-18.

Inhibition of Caspases—Intracellular Inhibitors of Apoptosis

Intracellular inhibitors of apoptosis (IAPs) are a family of antiapoptotic proteins that are highly conserved through evolution. Members of the family (cellular IAPs c-IAP-1 and c-IAP-2, X-linked IAP [X-IAP], survivin, IAP-like protein 2 [ILP2], ML-IAP (melanoma IAP), Bruce, and neuronal apoptosis inhibitory protein [NAIP]) share a baculovirus IAP repeat (BIR) domain and most contain a RING domain that functions as an E3 ligase. IAPs such as X-IAP directly inhibit effector caspases, especially caspase 9, whereas c-IAPs modulate cell survival by ubiquitylation of substrates such as ribosome-inactivating protein (RIP) and proteins in the NF-κB pathway. IAPs block apoptosis induced by a variety of stimuli, including Fas, TNF-α, ultraviolet (UV) irradiation, and serum withdrawal. IAPs themselves are inhibited by two mitochondrial proteins named Smac/Diablo and HtrA2/Omi, which are released into the cytosol during the intrinsic and some extrinsic apoptotic programs (see Figure 11-3).

Nucleases and the Degradation of Cellular DNA and RNA

Nucleic acids are of high relevance to SLE as they are prominent targets of autoantibodies in this disease and can potently stimulate the production of type I IFN. RNase 1 and DNase I are the predominant serum nucleases thought to be crucial for the degradation of nucleic acids released from dead and dying cells into blood or tissue. Mice deficient in DNase I, at least on some strain backgrounds, demonstrate a lupus-like phenotype,¹³ but whether altered DNase activity represents a genetic susceptibility factor in human lupus remains controversial.

A hallmark of programmed cell death is internucleosomal cleavage of DNA, which results in nuclear condensation and a nucleosomal “ladder.” Numerous DNases have been implicated in this process, including caspase-activated DNase (CAD/DFF40) and apoptosis-inducing factor (AIF)/endonuclease G (Endo G) (located in mitochondria) (see Table 11-1). Initially, degradation of chromatin into large DNA fragments to produce large (50-200 kb) fragments occurs through cooperative interaction between endo G and AIF. Subsequently, while the cell membrane remains intact, CAD/DFF40 cleaves DNA into internucleosomal units, producing a characteristic 180-bp ladder. Following phagocytic ingestion of apoptotic cells with partially processed DNA, DNase II, another DNase located in lysosomes, completes DNA digestion. Mice deficient in both CAD/DFF40 and DNase II have defects in thymic development associated with production of inflammatory cytokines, supporting a model in which DNA degradation during apoptosis is a sequential process that requires initiation in a cell-autonomous way and is then fully executed by the phagocyte. Of interest, mice doubly deficient in DNAseII and IFN receptors demonstrate a disease similar to rheumatoid arthritis.¹⁴

A third category of nucleases are those involved in processing of normal or abnormal (e.g., virus) nucleic acids. TREX1 (three prime repair enxonuclease 1) is a 3-5′ exonuclease that appears to be necessary for degradation of cytoplasmic single-stranded DNA.¹⁵ Loss of TREX1 function stimulates the production of type I IFN through an unknown DNA sensor.

The Bcl-2 Family: Central Regulators of Apoptosis

There are at least 20 known Bcl-2 family members in mammals that have diverse cellular localization and function and are broadly divided into three interacting groups (see Table 11-1; and reviewed in reference 16). All Bcl-2 family members possess one or more BH (Bcl-2 homology) domains, which allow for interaction with a variety of proapoptotic or antiapoptotic proteins. Multidomain prosurvival members Bcl-2, and closely related Bcl-xL, Mcl-1, and A1, can protect cells from a wide range of potentially death-inducing stimuli, including irradiation (UV and gamma), growth factor withdrawal, and chemotherapy. The proapoptotic multidomain group that includes Bax, Bak, and Bok likely functions by altering the permeability or conductance of mitochondrial and other membranes, resulting in the release of additional proapoptotic mediators (see later). Finally, the “BH3-only” group of proapoptotic proteins contains at least eight members and functions primarily by antagonizing the protective effects of the Bcl-2 family. Some BH3-only proteins may sensitize cells for apoptosis, whereas others likely function more directly in activating apoptosis.⁸ The BH3-only members act as sentinels in various organelles, integrating proximal death or survival signals and ultimately facilitating Bax/Bak-induced apoptosis.

The cellular localization of the Bcl-2 family members is diverse and likely reflects the complexity of networks that sense cellular damage and govern life or death. The antiapoptotic Bcl-2 subfamily is associated with membranes, including the cytoplasmic face of the mitochondrial membrane. Although Bcl-2 itself inserts into membranes in healthy cells, other related proteins must undergo allosteric changes prior to membrane association, allowing for the unique response to cytotoxic stressors. Similarly, the proapoptotic Bax family has both cytosolic (e.g., Bax) and membrane-associated (e.g., Bak) members in healthy cells that translocate to the outer mitochondrial membrane after appropriate stimuli. The BH3-only family adds to the complexity of apoptosis regulation because individual members are expressed in a cell-type specific manner and may allow for the response to organelle-specific signals. Constitutively expressed BH3-only members remain latent until released by a variety of stimuli. Examples are Bad (sequestered to 14-3-3 scaffold proteins) and Bid (undergoes cleavage). Finally, some BH3-only members are transcriptionally regulated so that certain forms of apoptosis resulting from cellular stress increase the expression of proapoptotic proteins.

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