Apoptosis: a review of programmed cell death

Abstract

The process of programmed cell death, or apoptosis, is generally characterized by distinct morphological characteristics and energy-dependent biochemical mechanisms. Apoptosis is considered a vital component of various processes including normal cell turnover, proper development and functioning of the immune system, hormone-dependent atrophy, embryonic development and chemical-induced cell death. Inappropriate apoptosis (either too little or too much) is a factor in many human conditions including neurodegenerative diseases, ischemic damage, autoimmune disorders and many types of cancer. The ability to modulate the life or death of a cell is recognized for its immense therapeutic potential. Therefore, research continues to focus on the elucidation and analysis of the cell cycle machinery and signaling pathways that control cell cycle arrest and apoptosis. To that end, the field of apoptosis research has been moving forward at an alarmingly rapid rate. Although many of the key apoptotic proteins have been identified, the molecular mechanisms of action or inaction of these proteins remain to be elucidated. The goal of this review is to provide a general overview of current knowledge on the process of apoptosis including morphology, biochemistry, the role of apoptosis in health and disease, detection methods, as well as a discussion of potential alternative forms of apoptosis.

Figure 1

Figure 1A is a photomicrograph of a section of exocrine pancreas from a B6C3F1 mouse. The arrows indicate apoptotic cells that are shrunken with condensed cytoplasm. The nuclei are pyknotic and fragmented. Note the lack of inflammation. Figure 1B is an image of myocardium from a 14 week-old rat treated with ephedrine (25 mg/kg) and caffeine (30 mg/kg). Within the interstitial space there are apoptotic cells with condensed cytoplasm, condensed and hyperchromatic chromatin and fragmented nuclei (long arrows). Admixed with the apoptotic bodies are macrophages, some with engulfed apoptotic bodies (arrowheads) (Howden et al., 2004, by permission of the American Journal of Physiology). Figure 1C is a photomicrograph of normal thymus tissue from a control Sprague–Dawley rat for comparison. Figure ID illustrates sheets of apoptotic cells in the thymus from a rat that was treated with dexamethasone to induce lymphocyte apoptosis. Under physiological conditions, apoptosis typically affects single cells or small clusters of cells. However, the degree of apoptosis in this treated rat is more severe due to the amount of dexamethasone administered (1 mg/kg bodyweight) and the time posttreatment (12 hours). The majority of lymphocytes are apoptotic although there are a few interspersed cells that are morphologically normal and most likely represent lymphoblasts or macrophages. The apoptotic lymphocytes are small and deeply basophilic with pyknotic and often-fragmented nuclei. Macrophages are present with engulfed cytoplasmic apoptotic bodies (arrows). H&E.

Figure 1

Figure 1A is a photomicrograph of a section of exocrine pancreas from a B6C3F1 mouse. The arrows indicate apoptotic cells that are shrunken with condensed cytoplasm. The nuclei are pyknotic and fragmented. Note the lack of inflammation. Figure 1B is an image of myocardium from a 14 week-old rat treated with ephedrine (25 mg/kg) and caffeine (30 mg/kg). Within the interstitial space there are apoptotic cells with condensed cytoplasm, condensed and hyperchromatic chromatin and fragmented nuclei (long arrows). Admixed with the apoptotic bodies are macrophages, some with engulfed apoptotic bodies (arrowheads) (Howden et al., 2004, by permission of the American Journal of Physiology). Figure 1C is a photomicrograph of normal thymus tissue from a control Sprague–Dawley rat for comparison. Figure ID illustrates sheets of apoptotic cells in the thymus from a rat that was treated with dexamethasone to induce lymphocyte apoptosis. Under physiological conditions, apoptosis typically affects single cells or small clusters of cells. However, the degree of apoptosis in this treated rat is more severe due to the amount of dexamethasone administered (1 mg/kg bodyweight) and the time posttreatment (12 hours). The majority of lymphocytes are apoptotic although there are a few interspersed cells that are morphologically normal and most likely represent lymphoblasts or macrophages. The apoptotic lymphocytes are small and deeply basophilic with pyknotic and often-fragmented nuclei. Macrophages are present with engulfed cytoplasmic apoptotic bodies (arrows). H&E.

Figure 2

Figure 2A is a transmission electron micrograph (TEM) of the normal thymus tissue depicted in Figure 1C. The lymphocytes are closely packed, have large nuclei and scant cytoplasm. Figure 2B is a TEM of apoptotic thymic lymphocytes in an early phase of apoptosis with condensed and peripheralized chromatin. The cytoplasm is beginning to condense and the cell outlines are irregular. The arrow indicates a fragmented section of nucleus and the arrowhead most likely indicates an apoptotic body that seems to contain predominantly cytoplasm without organelles or nuclear material. Figure 2C illustrates an apoptotic lymphocyte in the process of “budding” or extrusion of membrane-bound cytoplasm containing organelles (arrow). Once the budding has occurred, this extruded fragment will be an “apoptotic body.” These apoptotic bodies are membrane-bound and thus do not release cytoplasmic contents into the interstitium. Macrophages or other adjacent healthy cells subsequently engulf the apoptotic bodies. For these reasons, apoptosis does not incite an inflammatory reaction. Figure 2D is a TEM of a section of thymus with lymphocytes in various stages of apoptosis. The large cell in the center of the photomicrograph is a macrophage with engulfed intracytoplasmic apoptotic bodies. This macrophage is also called a “tingible body macrophage.” The arrowhead indicates a lymphocyte in an advanced stage of apoptosis with nuclear fragmentation.

Figure 2

Figure 2A is a transmission electron micrograph (TEM) of the normal thymus tissue depicted in Figure 1C. The lymphocytes are closely packed, have large nuclei and scant cytoplasm. Figure 2B is a TEM of apoptotic thymic lymphocytes in an early phase of apoptosis with condensed and peripheralized chromatin. The cytoplasm is beginning to condense and the cell outlines are irregular. The arrow indicates a fragmented section of nucleus and the arrowhead most likely indicates an apoptotic body that seems to contain predominantly cytoplasm without organelles or nuclear material. Figure 2C illustrates an apoptotic lymphocyte in the process of “budding” or extrusion of membrane-bound cytoplasm containing organelles (arrow). Once the budding has occurred, this extruded fragment will be an “apoptotic body.” These apoptotic bodies are membrane-bound and thus do not release cytoplasmic contents into the interstitium. Macrophages or other adjacent healthy cells subsequently engulf the apoptotic bodies. For these reasons, apoptosis does not incite an inflammatory reaction. Figure 2D is a TEM of a section of thymus with lymphocytes in various stages of apoptosis. The large cell in the center of the photomicrograph is a macrophage with engulfed intracytoplasmic apoptotic bodies. This macrophage is also called a “tingible body macrophage.” The arrowhead indicates a lymphocyte in an advanced stage of apoptosis with nuclear fragmentation.

Figure 3

Schematic representation of apoptotic events. The two main pathways of apoptosis are extrinsic and intrinsic as well as a perforin/granzyme pathway. Each requires specific triggering signals to begin an energy-dependent cascade of molecular events. Each pathway activates its own initiator caspase (8, 9, 10) which in turn will activate the executioner caspase-3. However, granzyme A works in a caspase-independent fashion. The execution pathway results in characteristic cytomorphological features including cell shrinkage, chromatin condensation, formation of cytoplasmic blebs and apoptotic bodies and finally phagocytosis of the apoptotic bodies by adjacent parenchymal cells, neoplastic cells or macrophages.

Figure 4

There are many ways of detecting apoptosis at different stages on histological sections. One commonly used method is called TUNEL (Terminal deoxynucleotidyl Transferase Biotin-dUTP Nick End Labeling). One of the major characteristics of apoptosis is the degradation of DNA after the activation of Ca/Mg dependent endonucleases. This DNA cleavage leads to strand breaks within the DNA. However, necrosis can also result in similar DNA cleavage. Therefore an additional method should be used to confirm apoptosis. The TUNEL method identifies cells with DNA strand breaks in situ by using terminal deoxynucleotidyl transferase (TdT) to transfer biotin-dUTP to the cleaved ends of the DNA. The biotin-labeled cleavage sites are then detected by reaction with HRP (horse radish peroxidase) conjugated streptavidin and visualized by DAB (diaminobenzidine), which gives a brown color. The tissue is typically counterstained with Toluidine blue to allow evaluation of tissue architecture. Figure 4A a photomicrograph of a section of testes from a B6C3F1 mouse that was in a National Toxicology Program bioassay. The TUNEL assay was used on this section of tissue to detect apoptotic cells. The seminiferous tubule is cut in cross section and has sperm, spermatogonia and spermatocytes in various stages of development. The arrows point to labeled (dark brown) apoptotic spermatogonia in the basal layer of the seminiferous epithelium. Figure 4B is an image of myocardium from 14 week-old rat treated with ephedrine (25 mg/kg) and caffeine (30 mg/kg). There are multiple apoptotic cells that have stained brown with the TUNEL method (arrows).

Figure 5

When histological changes suggestive of apoptosis are detected in H&E stained tissues (i.e., Figure 1B), confirmation of apoptosis can be obtained with a variety of additional stains. The images in Figure 5 are of myocardium from 14-week-old rats treated with ephedrine (25 mg/kg) and caffeine (30 mg/kg). Figure 5A is the interventricular septum stained by anti-phospho-H2A.X, a histone variant that becomes phosphorylated during apoptosis; note the strongly stained apoptotic bodies (arrows). Figure 5B demonstrates the negative control of the same section shown in (A); nonimmune rabbit IgG was used at equivalent conditions in place of the primary antibody. Note the unstained apoptotic bodies (arrows). Figure 5C illustrates cleaved caspase-3 staining for apoptosis revealing the presence of intracytoplasmic positive myofibers (small arrows) located in the interventricular septum; large arrow indicates an apoptotic body. Figure 5D is a negative control of the same heart demonstrated in 5C. The small arrows demonstrate degenerating myofibers; large arrows indicate apoptotic bodies. Nyska et al., 2005, by permission ² of Oxford University Press.