Robbins and Cotran Pathologic Basis of Disease PDF

See TARGETED THERAPY available online at www.studentconsult.com C H A P T E R Cell Injury, Cell Death, and Adaptations...

See TARGETED THERAPY available online at www.studentconsult.com C H A P T E R Cell Injury, Cell Death, and Adaptations Scott A. Oakes 2 CHAPTER CONTENTS Introduction to Pathology 33 Mitochondrial Damage 49 Hyperplasia 59 Overview of Cellular Responses to Membrane Damage 51 Mechanisms of Hyperplasia 59 Stress and Noxious Stimuli 34 Damage to DNA 51 Atrophy 59 Causes of Cell Injury 36 Oxidative Stress: Accumulation of Mechanisms of Atrophy 60 Oxygen Deprivation 36 Oxygen-Derived Free Radicals 52 Metaplasia 61 Physical Agents 36 Generation of Free Radicals 52 Mechanisms of Metaplasia 61 Chemical Agents and Drugs 36 Removal of Free Radicals 53 Intracellular Accumulations 62 Infectious Agents 36 Pathologic Effects of Free Radicals 53 Lipids 62 Immunologic Reactions 36 Disturbance in Calcium Homeostasis 54 Steatosis (Fatty Change) 62 Genetic Abnormalities 36 Endoplasmic Reticulum Stress: the Cholesterol and Cholesterol Esters 62 Nutritional Imbalances 36 Unfolded Protein Response 54 Proteins 63 The Progression of Cell Injury and Clinicopathologic Correlations: Hyaline Change 64 Death 36 Selected Examples of Cell Injury Glycogen 64 Reversible Cell Injury 37 and Death 55 Pigments 64 Cell Death 37 Hypoxia and Ischemia 55 Exogenous Pigments 64 Necrosis 39 Mechanisms of Ischemic Cell Injury 55 Endogenous Pigments 64 Patterns of Tissue Necrosis 40 Ischemia-Reperfusion Injury 56 Pathologic Calcification 65 Apoptosis 42 Chemical (Toxic) Injury 56 Dystrophic Calcification 65 Causes of Apoptosis 42 Adaptations of Cellular Growth and Metastatic Calcification 66 Morphologic and Biochemical Changes in Differentiation 57 Cellular Aging 66 Apoptosis 43 Hypertrophy 57 Mechanisms of Apoptosis 44 Mechanisms of Hypertrophy 57 Other Mechanisms of Cell Death 47 Autophagy 48 Mechanisms of Cell Injury 49 General Mechanisms of Cell Injury and Intracellular Targets of Injurious Stimuli 49 the fundamental mechanisms that underlie various forms INTRODUCTION TO PATHOLOGY of cell injury and death. Traditionally, the study of pathology is divided into Pathology is the study of the structural, biochemical, and general pathology and systemic pathology. General functional changes in cells, tissues, and organs that underlie pathology is concerned with the common reactions of cells disease. By the use of morphologic, microbiologic, immu- and tissues to injurious stimuli. Such reactions are often nologic, and molecular techniques, pathology attempts to not tissue specific: thus, acute inflammation in response explain the whys and wherefores of the signs and symptoms to bacterial infections produces a very similar reaction manifested by patients while providing a rational basis for in most tissues. On the other hand, systemic pathology clinical care and therapy. It thus serves as the bridge between examines the alterations and underlying mechanisms the basic sciences and clinical medicine, and is the scientific in diseases of particular organ systems. In this book, foundation for all of medicine. In Chapter 1 we examined we first cover the principles of general pathology and the cellular and molecular properties of healthy cells. In then proceed to specific disease processes as they affect this chapter, we will build on that knowledge to discuss different organs. 33 34 CHAPTER 2 Cell Injury, Cell Death, and Adaptations The four aspects of a disease process that form the on the basis of the presence (or absence) of specific core of pathology are causation (etiology), biochemical molecular alterations. and molecular mechanisms (pathogenesis), the associated Clinical manifestations. The end results of genetic, biochemi- structural (morphologic changes) and functional alterations cal, and structural changes in cells and tissues are in cells and organs, and the resulting clinical consequences functional abnormalities that lead to the clinical manifesta- (clinical manifestations). tions (symptoms and signs) of disease, as well as its Etiology is the initiating cause of a disease. Although there progression (clinical course and outcome). Hence, clini- are myriad factors that cause disease, all can be grouped copathologic correlations are very important in the study into two broad classes: genetic (e.g., inherited or acquired of disease. mutations, and disease-associated gene variants, or polymorphisms) and environmental (e.g., infectious, Virtually all diseases start with molecular or structural nutritional, chemical, physical). The idea that one etiologic alterations in cells. This concept of the cellular basis of disease agent is the cause of one disease arose from the study was first put forth in the nineteenth century by Rudolf of infections and inherited disorders caused by single Virchow, known as the father of modern pathology. Virchow gene anomalies, but the majority of diseases are not this emphasized the idea that individuals are sick because their simple. In fact, most common afflictions, such as athero- cells are sick. We therefore begin our consideration of sclerosis and cancer, arise from the effects of various pathology with the study of the causes, mechanisms, and environmental insults on a genetically susceptible morphologic and biochemical correlates of cell injury. Injury individual and hence are referred to as being multifacto- to cells and to the extracellular matrix ultimately leads to rial. The relative contribution of inherited susceptibility tissue and organ injury, which determines the morphologic and environmental influences varies in different diseases, and clinical patterns of disease. and it is challenging to precisely define their roles in most multifactorial diseases. Pathogenesis refers to the sequence of molecular, bio- OVERVIEW OF CELLULAR chemical, and cellular events that lead to the development of disease. Thus, pathogenesis explains how the underly- RESPONSES TO STRESS AND ing etiologies produce the morphologic and clinical NOXIOUS STIMULI manifestations of the disease. The study of pathogenesis is a central focus of pathology. Even when the initial The normal cell is confined to a fairly narrow range of cause is known (e.g., infection or mutation), it is many function and structure dictated by its state of metabolism, steps removed from the expression of the disease. For differentiation, and specialization; by constraints imposed example, to truly understand the disorder cystic fibrosis by neighboring cells; and by the availability of metabolic it is essential to know not only the defective gene and substrates. It is nevertheless able to handle physiologic gene product, but also the biochemical and morphologic demands, maintaining a healthy steady state called homeo- events that lead to clinically significant disease in the stasis. Adaptations are reversible functional and structural lungs, pancreas, and other organs. New technological responses to changes in physiologic states (e.g., pregnancy) advances, particularly the use of so-called “omics” and some pathologic stimuli, during which new but altered technologies (genomics, proteomics, metabolomics) to interrogate diseases, hold great promise for elucidating pathogenic mechanisms. Hopefully, the application of these methods and the analysis of mounds of “big NORMAL CELL REVERSIBLE data” so generated will lead not only to better under- (homeostasis) INJURY standing of pathogenesis but also to the identification of biomarkers that predict disease progression and Injurious Mild, Stress therapeutic responses. This, of course, is the goal of stimulus transient precision medicine. Morphologic changes refer to the structural alterations in cells or tissues that are characteristic of a disease and hence diagnostic ADAPTATION CELL INJURY of an etiologic process. Traditionally, diagnostic pathology Inability has used morphology to determine the nature of disease to adapt and to follow its progression. Although morphology Severe, progressive remains a cornerstone of diagnosis, it is now routinely supplemented by analysis of protein expression and genetic alterations. Nowhere is this more striking than IRREVERSIBLE in the study of neoplasms; breast cancers that are indis- INJURY tinguishable morphologically may result from different genetic abnormalities that result in widely different courses, therapeutic responses, and prognoses. Molecular analysis by techniques such as next-generation sequencing CELL NECROSIS APOPTOSIS (Chapter 7) has revealed genetic differences that predict DEATH the behavior of tumors as well as their response to therapies, an increasing number of which are now chosen Figure 2.1 Stages of the cellular response to stress and injurious stimuli.  Overview of cellular responses to stress and noxious stimuli 35 steady states are achieved, allowing the cell to survive The removal of damaged, unneeded, and aged cells and continue to function (Fig. 2.1). The adaptive response through cell death is a normal and essential process in may consist of an increase in the size (hypertrophy) and embryogenesis, the development of organs, and the functional activity of cells, an increase in cell number maintenance of homeostasis into adulthood. Conversely, (hyperplasia), a decrease in the size and metabolic activity excessive cell death as a result of progressive injury is one of cells (atrophy), or a change in the phenotype of cells of the most crucial events in the evolution of disease in (metaplasia). If the stress is eliminated, the cell can return any tissue or organ. It results from diverse causes, includ- to its original state without having suffered any harmful ing ischemia (reduced blood flow), infection, and toxins. consequences. There are two principal pathways of cell death, necrosis If the limits of adaptive responses are exceeded or if and apoptosis. Nutrient deprivation triggers an adaptive cells are exposed to damaging insults, deprived of critical cellular response called autophagy that may also culminate nutrients, or compromised by mutations that affect essential in cell death. A detailed discussion of these and some cellular functions, a sequence of events follows that is other, less common pathways of cell death follows later in termed cell injury (see Fig. 2.1). Cell injury is reversible up to the chapter. a point, but if the injurious stimulus is persistent or severe, Stresses of different types may induce changes in cells the cell suffers irreversible injury and ultimately undergoes and tissues other than typical adaptations, cell injury, and cell death. Adaptation, reversible injury, and cell death may death. Metabolic derangements and chronic injury may be be stages of progressive impairment following different associated with intracellular accumulations of a number of types of insults. For instance, in response to increased substances, including proteins, lipids, and carbohydrates. hemodynamic loads, the heart muscle becomes enlarged, Calcium may be deposited at sites of cell death, resulting a form of adaptation, which because of increased metabolic in pathologic calcification. Finally, the normal process of aging demands is more susceptible to injury. If the blood supply to is accompanied by characteristic morphologic and functional the myocardium is compromised or inadequate, the muscle changes in cells. first suffers reversible injury, manifested by certain cytoplas- This chapter discusses first the causes, mechanisms, and mic changes (described later). Unless the blood supply is consequences of the various forms of cell damage, including quickly restored, the cells suffer irreversible injury and die reversible cell injury and cell death. We conclude with cellular (Fig. 2.2). adaptations to stress, and three other processes that affect Normal myocyte Cell Adaptation: injury response to increased Reversibly injured load myocyte Adapted myocyte (hypertrophy) Cell death Figure 2.2 The relationship among normal, adapted, reversibly injured, and dead myocardial cells. All three transverse sections of the heart have been stained with triphenyltetrazolium chloride, an enzyme substrate that colors viable myocardium magenta. The cellular adaptation shown here is myocardial hypertrophy (lower left), caused by increased blood pressure requiring greater mechanical effort by myocardial cells. This adaptation leads to thickening of the left ventricular wall (compare with the normal heart). In reversibly injured myocardium (illustrated schematically, right), there are functional alterations, usually without any gross or microscopic changes but sometimes with cytoplasmic changes such as cellular swelling and fat accumulation. In the specimen showing necrosis, a form of cell death (lower right), the light area in the posterolateral left ventricle represents an acute myocardial infarction caused by reduced blood flow (ischemia). 36 CHAPTER 2 Cell Injury, Cell Death, and Adaptations cells and tissues: intracellular accumulations, pathologic important causes of cell and tissue injury (Chapters 3 calcification, and cell aging. and 6). Causes of Cell Injury Genetic Abnormalities As described in Chapter 5, genetic aberrations as extreme The causes of cell injury range from the mechanical trauma as an extra chromosome, as in Down syndrome, or as subtle of an automobile accident to subtle cellular abnormalities, as a single base pair substitution leading to an amino acid such as a mutation causing lack of a vital enzyme that impairs substitution, as in sickle cell anemia, may produce highly normal metabolic function. Most injurious stimuli can be characteristic clinical phenotypes ranging from congenital grouped into the following broad categories. malformations to anemias. Genetic defects may cause cell injury because of deficient protein function, such as enzyme Oxygen Deprivation defects in inborn errors of metabolism, or accumulation of Hypoxia is a deficiency of oxygen, which causes cell injury damaged DNA or misfolded proteins, both of which trigger by reducing aerobic oxidative respiration. Hypoxia is an cell death when they are beyond repair. DNA sequence extremely important and common cause of cell injury and variants that are common in human populations (polymor- cell death. Causes of hypoxia include reduced blood flow phisms) can also influence the susceptibility of cells to injury (ischemia); inadequate oxygenation of the blood due to by chemicals and other environmental insults. cardiorespiratory failure; and decreased oxygen-carrying capacity of the blood, as in anemia or carbon monoxide Nutritional Imbalances poisoning and severe blood loss. Depending on the severity Nutritional imbalances continue to be major causes of of the hypoxic state, cells may adapt, undergo injury, or cell injury. Protein-calorie deficiencies cause an appalling die. For example, if an artery is narrowed, the tissue supplied number of deaths, chiefly among lower-income populations. by that vessel may initially shrink in size (atrophy), whereas Deficiencies of specific vitamins are found throughout more severe or sudden hypoxia induces cell injury and cell the world (Chapter 9). Nutritional shortages can be self- death. imposed, as in anorexia nervosa (a psychological disorder of inadequate food consumption) or stem from food Physical Agents shortages or poor diet. Ironically, nutritional excess also Physical agents capable of causing cell injury include is an important cause of cell injury. Obesity is rampant mechanical trauma, extremes of temperature (burns and in the United States and is associated with an increased deep cold), sudden changes in atmospheric pressure, radia- incidence of several important diseases, such as diabetes and tion, and electric shock (Chapter 9). cancer. In addition to the problems of undernutrition and overnutrition, the composition of the diet makes a significant Chemical Agents and Drugs contribution to a number of diseases. For example, diets The list of chemicals that may produce cell injury defies high in certain lipids lead to elevated serum cholesterol compilation. Simple chemicals such as glucose or salt in and predispose to atherosclerosis, a leading risk factor for hypertonic concentrations may cause cell injury directly or cardiovascular disease, the number one killer of adults in the by deranging electrolyte and fluid balance in cells. Even United States. oxygen at high concentrations is toxic. Trace amounts of poisons, such as arsenic, cyanide, or mercury, may damage The Progression of Cell Injury and Death sufficient numbers of cells within minutes or hours to cause death. Other potentially injurious substances are our daily It is useful to describe the basic alterations that occur in companions: environmental pollutants, insecticides, and damaged cells before discussing the mechanisms that bring herbicides; industrial and occupational hazards, such as about these changes. All stresses and noxious influences carbon monoxide and asbestos; recreational drugs such as exert their effects first at the molecular or biochemical level. alcohol; and the ever increasing variety of therapeutic drugs, There is a time lag between the stress and the morphologic many of which have toxic side effects. These are discussed changes of cell injury or death; understandably, the early further in Chapter 9. changes are subtle and are only detected with highly sensitive methods of examination (Fig. 2.3). With histochemical, Infectious Agents ultrastructural, or biochemical techniques, changes may be These agents range from submicroscopic viruses to tape- seen in minutes to hours after injury, whereas changes visible worms several feet in length. In between are rickettsiae, by light microscopy or the naked eye may take considerably bacteria, fungi, and higher forms of parasites. The ways longer (hours to days) to appear. As would be expected, by which these biologic agents cause injury are diverse the morphologic manifestations of necrosis take more time (Chapter 8). to develop than those of reversible damage. For example, in ischemia of the myocardium, cell swelling is a reversible Immunologic Reactions morphologic change that may occur in a matter of minutes, The immune system serves an essential function in defense and is an indicator of ongoing cellular damage that may against infectious pathogens, but immune reactions may progress to irreversibility within 1 or 2 hours. Unmistakable also cause cell injury. Injurious reactions to endogenous light microscopic evidence of cell death, however, may not self antigens are responsible for autoimmune diseases be seen until 4 to 12 hours after onset of ischemia. (Chapter 6). Immune reactions to many external agents, The sequential structural changes in cell injury progressing such as viruses and environmental substances, are also to cell death are illustrated in Fig. 2.4 and described later.  Reversible cell injury 37 Table 2.1 Features of Necrosis and Apoptosis Reversible Irreversible Feature Necrosis Apoptosis cell injury cell injury Cell size Enlarged (swelling) Reduced (shrinkage) Ultrastructural Light changes microscopic Nucleus Pyknosis, karyorrhexis, Fragmentation into Biochemical changes karyolysis nucleosome-size alterations Cell cell death fragments function Plasma Disrupted Intact; altered membrane structure, especially EFFECT orientation of lipids Gross morphologic Cellular Enzymatic digestion; Intact; may be released changes contents may leak out of cell in apoptotic bodies Adjacent Frequent No inflammation Physiologic or Usually pathologic Often physiologic, pathologic (culmination of means of eliminating role irreversible cell unwanted cells; may injury) be pathologic after some forms of cell DURATION OF INJURY injury, especially DNA damage Figure 2.3 Sequential development of biochemical and morphologic changes in cell injury. Cells may become rapidly nonfunctional after the onset of injury, although they may still be viable, with potentially reversible damage; a longer duration of injury may lead to irreversible damage and cell death. Note that irreversible biochemical alterations may cause cell MORPHOLOGY death, and typically this precedes ultrastructural, light microscopic, and grossly visible morphologic changes. Cellular swelling is the earliest manifestation of almost all forms of injury to cells (Fig. 2.5B). When it affects many cells, it causes pallor, increased turgor, and increased weight of the affected organ. On microscopic examination, small clear vacuoles may be seen Within limits, the cell can repair the alterations seen in within the cytoplasm; these represent distended and pinched-off reversible injury and if the injurious stimulus abates, may segments of the ER. This pattern of nonlethal injury is sometimes return to normalcy. Persistent or excessive injury, however, called hydropic change or vacuolar degeneration. The causes cells to pass the rather nebulous “point of no return” cytoplasm of injured cells appears red (eosinophilc) when stained into irreversible injury and cell death. Different injurious with hematoxylin and eosin (H&E) due to loss of RNA, which stimuli induce death mainly by necrosis and/or apoptosis binds the blue hematoxylin dye. The eosinophilia becomes more (see Fig. 2.4 and Table 2.1). pronounced with progression toward necrosis. The ultrastructural changes of reversible cell injury, visible by electron microscopy (Fig. 2.6B), include the following: REVERSIBLE CELL INJURY 1. Plasma membrane alterations, such as blebbing, blunting, and loss of microvilli Reversible cell injury is characterized by functional and 2. Mitochondrial changes, including swelling and the appearance structural alterations in early stages or mild forms of injury, of small amorphous densities which are correctable if the damaging stimulus is removed. 3. Accumulation of “myelin figures” in the cytosol composed of Two features are consistently seen in reversibly injured cells. phospholipids derived from damaged cellular membranes Early alterations in reversible injury include generalized 4. Dilation of the ER, with detachment of polysomes swelling of the cell and its organelles, blebbing of the plasma 5. Nuclear alterations, with disaggregation of granular and fibrillar membrane, detachment of ribosomes from the endoplas- elements mic reticulum (ER), and clumping of nuclear chromatin. Swelling of cells results from influx of water. This is usually caused by failure of the adenosine triphosphate (ATP)-dependent Na+-K+ plasma membrane pump due to depletion of ATP resulting from oxygen deficiency, CELL DEATH which interferes with mitochondrial oxidative phosphory- lation, or mitochondrial damage by radiation or toxins There are two principal types of cell death, necrosis and (discussed later). apoptosis, which differ in their mechanisms, morphology, Fatty change occurs in organs that are actively involved and roles in physiology and disease (see Table 2.1). Severe in lipid metabolism (e.g., liver). It results when toxic mitochondrial damage with depletion of ATP and rupture injury disrupts metabolic pathways and leads to rapid of lysosomal and plasma membranes are typically associated accumulation of triglyceride-filled lipid vacuoles. This with necrosis. Necrosis occurs in many commonly encoun- is discussed in Chapter 18. tered injuries, such as those following ischemia, exposure Other alterations are described in the following to toxins, various infections, and trauma. Apoptosis has sections. many unique features (see later). 38 CHAPTER 2 Cell Injury, Cell Death, and Adaptations NORMAL CELL Reversible injury Recovery Swelling of Condensation endoplasmic of chromatin reticulum and mitochondria Membrane blebs Myelin figure Membrane blebs Breakdown of plasma membrane, Progressive injury organelles, and nucleus; leakage of contents Cellular fragmentation Myelin figure Apoptotic APOPTOSIS body Phagocytosis Phagocyte of apoptotic cells and fragments NECROSIS Inflammation Amorpous densities in mitochondria Figure 2.4 Schematic illustration of the morphologic changes in cell injury culminating in necrosis or apoptosis. A B C Figure 2.5 Morphologic changes in reversible cell injury and necrosis. (A) Normal kidney tubules with viable epithelial cells. (B) Early (reversible) ischemic injury showing surface blebs, increased eosinophilia of cytoplasm, and swelling of occasional cells. (C) Necrosis (irreversible injury) of epithelial cells, with loss of nuclei, fragmentation of cells, and leakage of contents. The ultrastructural features of these stages of cell injury are shown in Fig. 2.6. (Courtesy Drs. Neal Pinckard and M.A. Venkatachalam, University of Texas Health Sciences Center, San Antonio, Tex.)  Cell death 39 L L mv mv M M M A B C Figure 2.6 Ultrastructural features of reversible and irreversible cell injury (necrosis) in a rabbit kidney. (A) Electron micrograph of a normal epithelial cell of the proximal kidney tubule. Note abundant microvilli (mv) lining the luminal surface (L). (B) Epithelial cell of the proximal tubule showing early cell injury resulting from reperfusion following ischemia. The microvilli are lost and have been incorporated in apical cytoplasm; blebs have formed and are extruded in the lumen. Mitochondria (M) would have been swollen during ischemia; with reperfusion, they rapidly undergo condensation and become electron-dense. (C) Proximal tubular cell showing late injury, expected to be irreversible. Note the markedly swollen mitochondria containing electron- dense deposits, expected to contain precipitated calcium and proteins. Higher magnification micrographs of the cell would show disrupted plasma membrane and swelling and fragmentation of organelles. (A, Courtesy Dr. Brigitte Kaisslin, Institute of Anatomy, University of Zurich, Switzerland. B, C, Courtesy Dr. M.A. Venkatachalam, University of Texas Health Sciences Center, San Antonio, Tex.) Necrosis has historically been thought of as “accidental” of the lethally injured cell. When damage to membranes is cell death, reflecting severe injury that irreparably damages severe, lysosomal enzymes enter the cytoplasm and digest so many cellular components that the cell simply “falls the cell. Cellular contents also leak through the damaged apart.” If early (reversible) injury progresses because the plasma membrane into the extracellular space, where they injurious stimulus persists, the end result is death by elicit a host reaction (inflammation). Some specific substances necrosis. When cells die by necrosis, there is a local inflam- released from injured cells have been called damage-associated matory response that clears the “scene of the accident.” molecular patterns (DAMPs). These include ATP (released In contrast, apoptosis is “regulated” cell death, mediated from damaged mitochondria), uric acid (a breakdown by defined molecular pathways that are activated under product of DNA), and numerous other molecules that are specific circumstances and kill cells with surgical precision, normally confined within healthy cells and whose release without inflammation or the associated collateral damage. is an indicator of severe cell injury. These molecules are The separation of necrosis and apoptosis is not always so recognized by receptors present in macrophages and most clear, however, and some forms of necrosis are genetically other cell types, and trigger phagocytosis of the debris as controlled through a defined molecular pathway, called well as the production of cytokines that induce inflam- “necroptosis” (discussed later). Moreover, in some situa- mation (Chapter 3). Inflammatory cells produce more tions, cell death may show morphologic features of both proteolytic enzymes, and the combination of phagocytosis apoptosis and necrosis, or progress from one to the other, and enzymatic digestion usually leads to clearance of the so the distinctions may not be absolute. Nevertheless, it is necrotic cells. useful to consider these as largely non-overlapping pathways Necrosis-associated leakage of intracellular proteins of cell death because their principal mechanisms, morpho- through damaged plasma membranes and ultimately logical features, and functional consequences are usually into the circulation is the basis for blood tests that detect different. tissue-specific cellular injury. Cardiac muscle cells, for example, express cardiac-specific variants of the contractile Necrosis protein troponin, while bile duct epithelium expresses a specific isoform of the enzyme alkaline phosphatase and Necrosis is a pathologic process that is the consequence hepatocytes express transaminases. Necrosis of these cell of severe injury. The main causes of necrosis include loss types and associated loss of membrane integrity is reflected of oxygen supply (ischemia), exposure to microbial toxins, in increased serum levels of these proteins, which serve as burns and other forms of chemical and physical injury, and biomarkers that are used clinically to assess and quantify unusual situations in which active proteases leak out of tissue damage. Cardiac-specific troponins can be detected in cells and damage surrounding tissues (as in pancreatitis). the blood as early as 2 hours after myocardial cell necrosis, All of these initiating triggers lead to irreparable damage well before histologic evidence of myocardial infarction of numerous cellular components. becomes apparent. Because of their sensitivity and specific- Necrosis is characterized by denaturation of cellular ity, serial measurement of serum cardiac troponins has a proteins, leakage of cellular contents through damaged central role in the diagnosis and management of patients membranes, local inflammation, and enzymatic digestion with myocardial infarction (Chapter 12). 40 CHAPTER 2 Cell Injury, Cell Death, and Adaptations phosphorylation and ATP generation) even after resolution MORPHOLOGY of the original injury, and profound disturbances in membrane Necrotic cells show increased eosinophilia in H&E stains, function. As mentioned earlier, injury to lysosomal mem- attributable in part to the loss of cytoplasmic RNA and in part branes results in the enzymatic dissolution of the injured to accumulation of denatured cytoplasmic proteins (which bind cell that is characteristic of necrosis. the red dye eosin). The necrotic cell may have a glassy homoge- neous appearance relative to normal cells, mainly as a result of Patterns of Tissue Necrosis the loss of glycogen particles (see Fig. 2.5C). When enzymes have The discussion of necrosis has focused so far on changes digested the cell’s organelles, the cytoplasm becomes vacuolated in individual cells. When large numbers of cells die, the and appears moth-eaten. Dead cells may be replaced by large tissue or organ is said to be necrotic; thus, a myocardial whorled phospholipid precipitates called myelin figures, which infarct is necrosis of a portion of the heart caused by death are either phagocytosed by other cells or further degraded into of many myocardial cells. Necrosis of tissues has several fatty acids; calcification of such fatty acid residues results in morphologically distinct patterns, which are important to deposition of calcium-rich precipitates. By electron microscopy, recognize because they provide clues about the underlying necrotic cells are characterized by discontinuities in plasma and cause. Although the terms that describe these patterns are organelle membranes, marked dilation of mitochondria with the somewhat dated, they are often used and their implications appearance of large amorphous densities, intracytoplasmic myelin are understood by pathologists and clinicians. figures, amorphous debris, and aggregates of fluffy material rep- resenting denatured protein (see Fig. 2.6C). Nuclear changes appear in one of three patterns, all due MORPHOLOGY to breakdown of DNA. The basophilia of the chromatin may fade Coagulative necrosis is a form of necrosis in which the (karyolysis), a change that presumably reflects loss of DNA architecture of dead tissue is preserved for a span of at least because of enzymatic degradation by endonucleases. A second some days (Fig. 2.7).The affected tissue has a firm texture. Presum- pattern (also seen in apoptotic cell death) is pyknosis, character- ably, the injury denatures not only structural proteins but also ized by nuclear shrinkage and increased basophilia. Here the enzymes and so blocks the proteolysis of the dead cells; as a chromatin condenses into a dense, shrunken basophilic mass. In result, intensely eosinophilic cells with indistinct or reddish nuclei the third pattern, known as karyorrhexis, the pyknotic nucleus may persist for days or weeks. Ultimately, the necrotic cells are undergoes fragmentation. With the passage of time (1 or 2 days), broken down by the action of lysosomal enzymes derived from the nucleus in the necrotic cell totally disappears. infiltrating leukocytes, which also remove the debris of the dead cells by phagocytosis. Ischemia caused by obstruction in a vessel may lead to coagulative necrosis of the supplied tissue in all organs except the brain (see next paragraph for explanation). A It is useful to consider the possible events that determine localized area of coagulative necrosis is called an infarct. when reversible injury becomes irreversible and progresses Liquefactive necrosis, in contrast to coagulative necrosis, is to necrosis. The clinical relevance of this question is obvious— characterized by digestion of the dead cells, resulting in transforma- if we can answer it, we may be able to devise strategies for tion of the tissue into a viscous liquid. It is seen in focal bacterial preventing cell injury from having permanent deleterious or, occasionally, fungal infections, because microbes stimulate the consequences. Although the “point of no return,” at which accumulation of leukocytes and the liberation of enzymes from the damage becomes irreversible, is still largely undefined, these cells. The necrotic material is frequently creamy yellow two phenomena consistently characterize irreversibility—the because of the presence of leukocytes and is called pus. For inability to reverse mitochondrial dysfunction (lack of oxidative I N A B Figure 2.7 Coagulative necrosis. (A) A wedge-shaped kidney infarct (yellow). (B) Microscopic view of the edge of the infarct, with normal kidney (N) and necrotic cells in the infarct (I) showing preserved cellular outlines with loss of nuclei and an inflammatory infiltrate (seen as nuclei of inflammatory cells in between necrotic tubules).  Cell death 41 Figure 2.10 Fat necrosis. The areas of white chalky deposits represent foci of fat necrosis with calcium soap formation (saponification) at sites of lipid breakdown in the mesentery. Figure 2.8 Liquefactive necrosis. An infarct in the brain, showing dissolution of the tissue. Fat necrosis refers to focal areas of fat destruction, typically unknown reasons, hypoxic death of cells within the central nervous resulting from release of activated pancreatic lipases into the system often manifests as liquefactive necrosis (Fig. 2.8). substance of the pancreas and the peritoneal cavity. This occurs Gangrenous necrosis is not a specific pattern of cell death, in the calamitous abdominal emergency known as acute pancreatitis but the term is commonly used in clinical practice. It is usually (Chapter 19). In this disorder, pancreatic enzymes leak out of applied to a limb, generally the lower leg, that has lost its blood damaged acinar cells and liquefy the membranes of fat cells in supply and has undergone necrosis (typically coagulative necrosis) the peritoneum, releasing triglyceride esters that are split by involving multiple tissue planes. When bacterial infection is pancreatic lipases. Fatty acids are generated that combine with superimposed, there is more liquefactive necrosis because of the calcium to produce grossly visible chalky-white areas (fat saponifica- actions of degradative enzymes in the bacteria and the attracted tion), which enable the surgeon and the pathologist to identify leukocytes (giving rise to so-called wet gangrene). the underlying disorder (Fig. 2.10). On histologic examination, Caseous necrosis is encountered most often in foci of the necrotic areas contain the shadowy outlines of necrotic fat tuberculous infection (Chapter 8). The term caseous (cheeselike) cells, basophilic calcium deposits, and an inflammatory reaction. is derived from the friable white appearance of the area of necrosis Fibrinoid necrosis is a special form of vascular damage (Fig. 2.9). On microscopic examination, the necrotic area appears usually seen in immune reactions involving blood vessels. It typically as a structureless collection of fragmented or lysed cells and occurs when complexes of antigens and antibodies are deposited amorphous granular debris enclosed within a distinctive inflam- in the walls of arteries. Deposits of these immune complexes, matory border; this appearance is characteristic of a focus of together with plasma proteins that has leaked out of vessels, inflammation known as a granuloma (Chapter 3). result in a bright pink and amorphous appearance in H&E stains called “fibrinoid” (fibrin-like) by pathologists (Fig. 2.11). The immunologically mediated vasculitis syndromes in which this type of vascular injury is seen are described in Chapter 11. Figure 2.11 Fibrinoid necrosis in an artery. The wall of the artery shows Figure 2.9 Caseous necrosis. Tuberculosis of the lung, with a large area of a circumferential bright pink area of necrosis with inflammation caseous necrosis containing yellow-white and “cheesy” appearing debris. (neutrophils with dark nuclei). 42 CHAPTER 2 Cell Injury, Cell Death, and Adaptations Ultimately, in the living patient most necrotic cells and their contents disappear due to enzymatic digestion and Apoptosis in Physiologic Situations phagocytosis of the debris by leukocytes. If necrotic cells Death by apoptosis is a normal phenomenon that serves and cellular debris are not promptly destroyed and reab- to eliminate cells that are no longer needed, or as a sorbed, they provide a nidus for the deposition of calcium mechanism to maintain a constant number of various cell salts and other minerals and thus tend to become calcified. populations in tissues. It is estimated that humans turn This phenomenon, called dystrophic calcification, is considered over almost 1 million cells per second! Central to this process later in the chapter. is death of cells by apoptosis and their removal by phago- cytes. Apoptosis is important in the following physiologic situations: KEY CONCEPTS The removal of supernumerary cells (in excess of the required number) during development. Cell death is critical for CELL INJURY AND NECROSIS involution of primordial structures and remodeling of Exposure of cells to stress or noxious agents causes cell injury maturing tissues. Apoptosis is a generic term for this that is reversible to a point but may progress to death of the pattern of cell death, regardless of the context, while cells, principally by necrosis. programmed cell death refers only to apoptosis during Reversible cell injury: Characterized by cellular swelling, fatty development. change, plasma membrane blebbing and loss of microvilli, Involution of hormone-dependent tissues on hormone with- mitochondrial swelling, dilation of the ER, and eosinophilia (due drawal, such as endometrial cell breakdown during the to decreased cytoplasmic RNA) menstrual cycle, ovarian follicular atresia in menopause, Necrosis: A pathologic process in which cellular membranes and regression of the lactating breast after weaning. are destroyed, enzymes and other constituents leak out, and Cell turnover in proliferating cell populations, such as local inflammation is induced to clear the damaged cells. immature lymphocytes in the bone marrow and thymus, Morphologic features are: eosinophilia; nuclear shrinkage, B lymphocytes in germinal centers that fail to express fragmentation, and dissolution; breakdown of plasma membrane useful antigen receptors (Chapter 6), and epithelial cells and organellar membranes; abundant myelin figures; and leakage in intestinal crypts, to maintain a constant cell number and enzymatic digestion of cellular contents (homeostasis). Patterns of tissue necrosis: Under different conditions, necrosis Elimination of potentially harmful self-reactive lymphocytes in tissues may assume specific patterns: coagulative, liquefactive, to prevent immune reactions against one’s own tissues gangrenous, caseous, fat, and fibrinoid (Chapter 6). Death of host cells that have served their useful purpose, such as neutrophils in an acute inflammatory response, and Apoptosis lymphocytes at the end of an immune response. Apoptosis is a type of cell death that is induced by a In all of these situations, cells undergo apoptosis because tightly regulated suicide program in which cells destined they are deprived of necessary survival signals, such as to die activate intrinsic enzymes that degrade the cells’ growth factors and interactions with the extracellular matrix, genomic DNA and nuclear and cytoplasmic proteins. or they receive pro-apoptotic signals from other cells or the Apoptotic cells break up into plasma membrane–bound surrounding environment. fragments, called apoptotic bodies, which contain portions of the cytoplasm and nucleus. While the plasma membrane Apoptosis in Pathologic Conditions remains intact, its surface components are altered so as to Apoptosis eliminates cells that are injured beyond repair produce “find me” and “eat me” signals for phagocytes, without eliciting a host reaction, thus limiting collateral discussed later. As a result, the dead cell and its fragments tissue damage. Death by apoptosis is responsible for loss are rapidly devoured, before the contents leak out, and of cells in a variety of pathologic states: therefore apoptosis does not elicit an inflammatory reaction. DNA damage. Radiation and cytotoxic anticancer drugs Apoptosis was first recognized in 1972 by the distinctive can damage DNA, either directly or via production morphologic appearance of membrane-bound fragments of free radicals. If repair mechanisms cannot correct derived from cells, and named after the Greek designation the damage, the cell triggers intrinsic mechanisms for “falling off.” It was subsequently discovered in model that induce apoptosis. In these situations, apoptosis organisms such as worms that certain cells undergo apoptosis has a protective effect by preventing the survival of at precise times during development. This phenomenon, cells with DNA mutations that can lead to malignant termed programmed cell death, is controlled by the action of transformation. a small number of genes and is required for normal emby- Accumulation of misfolded proteins. Cell death triggered rogenesis. Thus, apoptosis is a unique mechanism of cell by improperly folded intracellular proteins and the death, distinct from necrosis in many respects (see Fig. 2.4 subsequent endoplasmic reticulum (ER) stress response and Table 2.1). is discussed later. Apoptosis can be induced during certain infections, Causes of Apoptosis particularly viral infections, as a result of the virus Apoptosis occurs in two broad contexts: as part of normal itself (as in adenovirus and HIV infections) or the host physiologic processes, and as a pathophysiologic mechanism immune response (as in viral hepatitis). An important host of cell loss in many different diseases. response to viruses consists of cytotoxic T lymphocytes  Cell death 43 (CTLs) specific for viral proteins, which induce apoptosis relatively normal, are more tightly packed. This contrasts with of infected cells in an attempt to eliminate reservoirs of necrosis, in which an early feature is cell swelling, not shrinkage. infection. During this process, there can be significant Chromatin condensation. This is the most characteristic tissue damage. The same CTL-mediated mechanism is feature of apoptosis. The chromatin aggregates peripherally, under responsible for killing of tumor cells, cellular rejection the nuclear membrane, into dense masses of various shapes and of transplants, and tissue damage in graft-versus-host sizes (see Fig. 2.12B). The nucleus itself may break up into two disease. or more fragments. Apoptosis may also contribute to pathologic atrophy in Formation of cytoplasmic blebs and apoptotic bodies. parenchymal organs after duct obstruction, such as occurs The apoptotic cell first shows extensive surface membrane blebbing, in the pancreas, parotid gland, and kidney. which is followed by fragmentation of the dead cells into membrane- Morphologic and Biochemical Changes in Apoptosis bound apoptotic bodies composed of cytoplasm and tightly packed organelles, with or without nuclear fragments (see Fig. 2.12C). Before discussing underlying mechanisms, the morphologic Phagocytosis of apoptotic cells or cell bodies, usually and biochemical characteristics of apoptosis are described. by macrophages. The apoptotic bodies are rapidly ingested by phagocytes and degraded by the phagocyte’s lysosomal enzymes. In H&E-stained tissue, the apoptotic cell appears as a round MORPHOLOGY or oval mass of intensely eosinophilic cytoplasm with fragments of dense nuclear chromatin (see Fig. 2.12A). Because the cell The following morphologic features, some best seen with the shrinkage and formation of apoptotic bodies are rapid and the electron microscope, characterize cells undergoing apoptosis pieces are quickly cleared by phagocytes, considerable apoptosis (Fig. 2.12; see Fig. 2.4). may occur in tissues before it is apparent in histologic sections. Cell shrinkage. Cell size is reduced, the cytoplasm is dense The absence of an inflammatory response can also make it difficult and eosinophilic (see Fig. 2.12A), and the organelles, although to detect apoptosis by light microscopy. A B C Figure 2.12 Morphologic features of apoptosis. (A) Apoptosis of an epidermal cell in an immune reaction. The cell is reduced in size and contains brightly eosinophilic cytoplasm and a condensed nucleus. (B) This electron micrograph of cultured cells undergoing apoptosis shows some nuclei with peripheral crescents of compacted chromatin, and others that are uniformly dense or fragmented. (C) These images of cultured cells undergoing apoptosis show blebbing and formation of apoptotic bodies (left panel, phase contrast micrograph), a stain for DNA showing nuclear fragmentation (middle panel), and activation of caspase-3 (right panel, immunofluorescence stain with an antibody specific for the active form of caspase-3, revealed as red color). (B, From Kerr JFR, Harmon BV: Definition and incidence of apoptosis: a historical perspective. In Tomei LD, Cope FO, editors: Apoptosis: The Molecular Basis of Cell Death. Cold Spring Harbor, NY, 1991, Cold Spring Harbor Laboratory Press, pp 5–29; C, Courtesy Dr. Zheng Dong, Medical College of Georgia, Augusta, Ga.) 44 CHAPTER 2 Cell Injury, Cell Death, and Adaptations Mechanisms of Apoptosis with consequent release of death-inducing (pro-apoptotic) Apoptosis results from the activation of enzymes called molecules from the mitochondrial intermembrane space into caspases (so named because they are proteases containing a the cytoplasm (Fig. 2.14). Mitochondria are organelles that cysteine in their active site and cleave proteins after aspartic contain remarkable proteins such as cytochrome c, a double- residues). Like many proteases, caspases exist as inactive edged sword that is essential for producing the energy (e.g., proenzymes and must undergo enzymatic cleavage to become ATP) that sustains cell viability, but that when released into active. The presence of active caspases is therefore a marker the cytoplasm (an indication that the cell is not healthy) for cells undergoing apoptosis (see Fig. 2.12C). The process initiates the suicide program of apoptosis. The release of of apoptosis may be divided into an initiation phase, during pro-apoptotic proteins such as cytochrome c is determined which some caspases become catalytically active and unleash by the integrity of the outer mitochondrial membrane, a cascade of other caspases, and an execution phase, during which is tightly controlled by the BCL2 family of proteins. which the terminal caspases trigger cellular fragmentation. This family is named after BCL2, a gene that is frequently Regulation of these enzymes depends on a finely tuned overexpressed due to chromosomal translocations and other balance between the abundance and activity of pro-apoptotic aberrations in certain B cell lymphomas (Chapter 13). There and anti-apoptotic proteins. are more than 20 members of the BCL family, which can Two distinct pathways converge on caspase activation: be divided into three groups based on their pro-apoptotic the mitochondrial pathway and the death receptor pathway or anti-apoptotic function and the BCL2 homology (BH) (Fig. 2.13). Although these pathways intersect, they are domains they possess. generally induced under different conditions, involve dif- Anti-apoptotic. BCL2, BCL-XL, and MCL1 are the principal ferent initiating molecules, and serve distinct roles in members of this group; they possess four BH domains physiology and disease. (called BH1-4). These proteins reside in the outer mito- chondrial membrane as well as in the cytosol and ER The Mitochondrial (Intrinsic) Pathway of Apoptosis membranes. By keeping the mitochondrial outer mem- The mitochondrial pathway is responsible for apoptosis in brane impermeable, they prevent leakage of cytochrome most physiologic and pathologic situations. It results from c and other death-inducing proteins into the cytosol (see increased permeability of the mitochondrial outer membrane Fig. 2.14A). MITOCHONDRIAL (INTRINSIC) DEATH RECEPTOR (EXTRINSIC) PATHWAY PATHWAY Receptor-ligand interactions Fas TNF receptor Cell injury Mitochondria Growth factor Adaptor proteins withdrawal DNA damage (by radiation, Initiator Cytochrome c Phagocyte toxins, free BCL2 family caspases and other radicals) effectors (BAX, BAK) pro-apoptotic Protein proteins Executioner misfolding Regulators caspases (ER stress) BCL2 (BCL2, BCL-XL) family sensors Endonuclease Breakdown of activation cytoskeleton Nuclear fragmentation Ligands for phagocytic cell receptors Apoptotic body Cytoplasmic bleb Figure 2.13 Mechanisms of apoptosis. Although the two pathways of apoptosis differ in their induction and regulation, they both culminate in the activation of caspases. In the mitochondrial pathway, proteins of the BCL2 family, which regulate mitochondrial permeability, become imbalanced such that the ratio of pro-apoptotic versus anti-apoptotic proteins results in the leakage of various substances from mitochondria that lead to caspase activation. In the death receptor pathway, signals from plasma membrane receptors lead to the assembly of adaptor proteins into a “death-inducing signaling complex,” which activates caspases, and the end result is the same.  Cell death 45 A. VIABLE CELL B. APOPTOSIS thus protecting cells from apoptosis. When cells are deprived of survival signals, suffer DNA damage, or develop ER Survival signal (e.g., growth factor) Lack of Irradiation stress due to the accumulation of misfolded proteins, survival signals BH3-only proteins are upregulated through increased transcription and/or post-translational modifications (e.g., phosphorylation). These BH3-only proteins in turn directly activate the two critical pro-apoptotic family members, BAX and BAK, which form oligomers that DNA damage insert into the mitochondrial membrane and allow proteins Production of from the inner mitochondrial membrane to leak out into anti-apoptotic the cytoplasm. BH3-only proteins may also bind to and proteins Activation of sensors (BH3-only proteins) block the function of BCL2 and BCL-XL. At the same time, (e.g., BCL2) synthesis of BCL2 and BCL-XL may decline because their BCL2 transcription relies on survival signals. The net result of (or BCL-XL) Cytochrome c Antagonism of BCL2 BAX-BAK activation coupled with loss of the protective functions of the anti-apoptotic BCL2 family members is the release into the cytoplasm of several mitochondrial proteins such as cytochrome c that can activate the caspase cascade (see Fig. 2.14). Once released into the cytosol, cytochrome c binds to a protein called APAF-1 (apoptosis-activating factor-1), forming a multimeric structure called the apoptosome. This complex No leakage of Activation of cytochrome c BAX/BAK channel binds to caspase-9, the critical initiator caspase of the mitochondrial pathway, and promotes its autocatalytic Leakage of cytochrome c, cleavage, generating catalytically active forms of the enzyme. other proteins Active caspase-9 then triggers a cascade of caspase activation by cleaving and thereby activating other pro-caspases (such Activation of caspases as caspase-3), which mediate the execution phase of apoptosis (discussed later). Other mitochondrial proteins with arcane names like Smac/DIABLO enter the cytoplasm, where they APOPTOSIS bind to and neutralize cytoplasmic proteins that function as physiologic inhibitors of apoptosis (IAPs). The normal Figure 2.14 The intrinsic (mitochondrial) pathway of apoptosis. (A) Cell function of the IAPs is to block the inappropriate activation viability is maintained by the induction of anti-apoptotic proteins such as BCL2 by survival signals. These proteins maintain the integrity of of caspases, including executioners like caspase-3, and keep mitochondrial membranes and prevent leakage of mitochondrial proteins. cells alive. Thus, IAP inhibition permits initiation of the (B) Loss of survival signals, DNA damage, and other insults activate caspase cascade. sensors that antagonize the anti-apoptotic proteins and activate the pro-apoptotic proteins BAX and BAK, which form channels in the The Extrinsic (Death Receptor–Initiated) Pathway mitochondrial membrane. The subsequent leakage of cytochrome c (and of Apoptosis other proteins, not shown) leads to caspase activation and apoptosis. This pathway is initiated by engagement of plasma membrane death receptors. Death receptors are members of the tumor necrosis factor (TNF) receptor family that Pro-apoptotic. BAX and BAK are the two prototypic contain a cytoplasmic domain involved in protein-protein members of this group; they contain the first three BH interactions. This death domain is essential for delivering domains (BH1-3). On activation, BAX and/or BAK apoptotic signals. (Some TNF receptor family members do oligomerize within the outer mitochondrial membrane not contain cytoplasmic death domains; their function is to and enhance its permeability. The precise mechanism activate inflammatory cascades [Chapter 3], and their role by which BAX-BAK oligomers permeabilize membranes in triggering apoptosis is much less established.) The best- is not settled. According to one model illustrated in Fig. known death receptors are the type 1 TNF receptor (TNFR1) 2.14B, they form a channel in the outer mitochondrial and a related protein called Fas (CD95), but several others membrane that allows cytochrome c leakage from the have been described. The mechanism of apoptosis induced intermembranous space. by these death receptors is well illustrated by Fas, a death Regulated apoptosis initiators. Members of this group, receptor expressed on many cell types (Fig. 2.15). The ligand including BAD, BIM, BID, Puma, and Noxa, contain only for Fas is called Fas ligand (FasL). FasL is expressed on T one BH domain, the third of the four BH domains, and cells that recognize self antigens (and functions to eliminate hence are sometimes called BH3-only proteins. The activity self-reactive lymphocytes that also express the receptor Fas of BH3-only proteins is modulated by sensors of cellular upon recognition of self antigens) and on some CTLs that stress and damage; when upregulated and activated, they kill virus-infected and tumor cells. When FasL binds to Fas, can initiate apoptosis. three or more molecules of Fas are brought together, and their cytoplasmic death domains form a binding site for an Growth factors and other survival signals stimulate adaptor protein called FADD (Fas-associated death domain). the production of anti-apoptotic proteins such as BCL2, Once attached to this complex, FADD binds inactive 46 CHAPTER 2 Cell Injury, Cell Death, and Adaptations an inhibitor of a DNase, making the DNase enzymatically active and allowing DNA degradation to commence. Cas- pases also proteolyze structural components of the nuclear matrix and thus promote fragmentation of nuclei. Other FasL steps in apoptosis are less well-defined. For instance, we do not know how membrane blebs and apoptotic bodies Fas are formed. Removal of Dead Cells The formation of apoptotic bodies breaks cells up into “bite-sized” fragments that are edible for phagocytes. Death FADD Apoptotic cells and their fragments also undergo several domain changes in their membranes that actively promote their phagocytosis so they are most often cleared before they lose membrane integrity and release their cellular contents. In Pro caspase-8 healthy cells, phosphatidylserine is present on the inner leaflet of the plasma membrane, but in apoptotic cells this Autocatalytic caspase phospholipid “flips” out and is expressed on the outer layer activation of the membrane, where it is recognized by several macro- Active caspase-8 phage receptors. Cells that are dying by apoptosis also secrete soluble factors that recruit phagocytes, and macrophages themselves may produce proteins that bind to apoptotic cells (but not live cells), leading to their engulfment. Apop- Executioner caspases totic bodies may also become coated with natural antibodies and proteins of the complement system, notably C1q, which are recognized by phagocytes. Thus, numerous ligands APOPTOSIS induced on apoptotic cells serve as “eat me” signals and are recognized by receptors on phagocytes that bind and Figure 2.15 The extrinsic (death receptor–initiated) pathway of engulf these cells. This process of apoptotic cell phagocytosis apoptosis, illustrated by the events following Fas engagement. FADD, is called efferocytosis; it is so efficient that dead cells disappear, Fas-associated death domain; FasL, Fas ligand. often within minutes, without leaving a trace. In addition, production of pro-inflammatory cytokines is reduced in macrophages that have ingested apoptotic cells. Together caspase-8 (or caspase-10), bringing together multiple caspase with rapid clearance, this limits inflammatory reactions, molecules and leading to autocatalytic cleavage and genera- even in the face of extensive apoptosis. tion of active caspase-8. In turn, active caspase-8 initiates the same executioner caspase sequence as in the mitochon- drial pathway. This extrinsic apoptosis pathway can be inhibited by a protein called FLIP, which binds to pro- KEY CONCEPTS caspase-8, thereby blocking FADD binding, but cannot APOPTOSIS activate the caspase. Some viruses and normal cells produce Regulated mechanism of cell death that serves to eliminate FLIP as a mechanism to protect themselves from Fas- unwanted and irreparably damaged cells, with the least possible mediated apoptosis. host reaction The extrinsic and intrinsic pathways of apoptosis are Characterized by enzymatic degradation of proteins and DNA, initiated in fundamentally different ways by distinct mol- initiated by caspases, and by recognition and removal of dead ecules, but there may be interconnections between them. cells by phagocytes For instance, in hepatocytes and pancreatic β cells, caspase-8 Initiated by two major pathways: produced by Fas signaling cleaves and activates the BH3-only Mitochondrial (intrinsic) pathway is triggered by loss protein BID, which then feeds into the mitochondrial of survival signals, DNA damage, and accumulation of pathway. The combined activation of both pathways delivers misfolded proteins (ER stress), which leads to leakage of a fatal blow to the cells. pro-apoptotic proteins from mitochondrial membrane into The Execution Phase of Apoptosis the cytoplasm and subsequent caspase activation; can be inhibited by anti-apoptotic members of the BCL2 family, The intrinsic and extrinsic pathways converge to acti- which are induced by survival signals including growth vate a caspase cascade that mediates the final phase of factors apoptosis. The intrinsic mitochondrial pathway activates Death receptor (extrinsic) pathway eliminates self-reactive the initiator caspase-9, whereas the extrinsic death recep- lymphocytes and is a mechanism of cell killing by cytotoxic tor pathway activates caspase-8 and caspase-10. The active T lymphocytes; is initiated by engagement of death recep- forms of these caspases trigger the rapid and sequential tors (members of the TNF receptor family). The responsible activation of the executioner caspases, such as caspase-3 ligands can be soluble or expressed on the surface of and caspase-6, which then act on many cellular compo- adjacent cells nents. For instance, once activated these caspases cleave  Cell death 47 Other Mechanisms of Cell Death TNF TNFR1 Although necrosis and apoptosis are the best-defined mechanisms of cell death, several other ways by which cells die have been described. Their importance in human diseases remains a topic of investigation, but students should be RIPK1 complex RIPK1 aware of their names and unique features. Necroptosis. As the name indicates, this form of cell death is a hybrid that shares aspects of both necrosis and apoptosis. Morphologically, and to some extent biochemi- P FADD RIPK1 cally, it resembles necrosis, as both are characterized by P loss of ATP, swelling of the cell and organelles, generation RIPK3 Caspase 8 of reactive oxygen species (ROS), release of lysosomal inactive enzymes, and ultimately rupture of the plasma membrane. MLKL Mechanistically, it is triggered by signal transduction P pathways that culminate in cell death, a feature similar MLKL P to apoptosis. Because of these overlapping features, P MLKL necroptosis is sometimes called programmed necrosis to MLKL Plasma membrane distinguish it from forms of necrosis driven passively associated by toxic or ischemic injury to the cell. In sharp contrast to apoptosis, the signals leading to necroptosis do not result in caspase activation, and hence it is also sometimes referred to as “caspase-independent” programmed cell Plasma death. The process of necroptosis starts in a manner similar membrane to that of the extrinsic form of apoptosis, that is, by ligation disruption of a receptor by its ligand. Ligation of TNFR1 is the most widely studied model of necroptosis, but many other signals, including ligation of Fas and yet to be identified TISSUE CELL DEATH BY INFLAMMATION sensors of viral DNA and RNA, can also trigger necrop- DAMAGE NECROPTOSIS tosis. Since TNF can cause both apoptosis and necroptosis, the mechanisms underlying these effects of TNF are Figure 2.16 Molecular mechanism of TNF-mediated necroptosis. especially illustrative (Fig. 2.16). Cross-linking of TNFR1 by TNF initiates the illustrated series of Although the entire set of signaling molecules and downstream events, which ultimately lead to plasma membrane disruption, their interactions are not known, necroptosis involves cell death, and inflammation. See text for details. (Modified from Galluzi L, et al: Programmed necrosis from molecules to health and disease, Int Rev two kinases called receptor-interacting protein kinase 1 and Cell Molec Biol 289:1, 2011.) 3 (RIPK1 and RIPK3). As indicated in Fig. 2.16, ligation of TNFR1 recruits these kinases into a multiprotein complex, and RIPK3 phosphorylates a cytoplasmic protein called MLKL. In response to its phosphorylation, MLKL interleukin-1 (IL-1) and releases its biologically active monomers assemble into oligomers, translocate from the form. IL-1 is a mediator of many aspects of inflamma- cytosol to the plasma membrane, and cause the plasma tion, including leukocyte recruitment and fever (Chapter membrane disruption that is characteristic of necrosis. 3). Caspase-1 and the closely related caspases-4 and -5 This explains the morphologic similarity of necroptosis also induce death of the cells. Unlike classical apoptosis, with necrosis initiated by other injuries. this pathway of cell death is characterized by release Necroptosis is postulated to be an important death of inflammatory mediators. Pyroptosis is thought to pathway both in physiologic and pathologic conditions. be the mechanism by which some microbes cause the For example, p