Tutorial Day 2 Patho Book PDF

1 Cell Injury, Cell Death, and Adaptations OUTLINE Introduction to Pathology, 1 Membrane Damage, 13 Overview of Cellular Responses to Stress and Noxious Stimuli, 1 Disturbance in Calcium H...

1 Cell Injury, Cell Death, and Adaptations OUTLINE Introduction to Pathology, 1 Membrane Damage, 13 Overview of Cellular Responses to Stress and Noxious Stimuli, 1 Disturbance in Calcium Homeostasis, 13 Causes of Cell Injury, 2 Endoplasmic Reticulum Stress, 13 Sequence of Events in Cell Injury and Cell Death, 2 DNA Damage, 14 Reversible Cell Injury, 2 Clinicopathologic Examples of Cell Injury and Necrosis, 14 Cell Death, 3 Hypoxia and Ischemia, 14 Necrosis, 4 Ischemia-Reperfusion Injury, 15 Morphologic Patterns of Tissue Necrosis, 5 Cell Injury Caused by Toxins, 15 Apoptosis, 7 Cellular Adaptations to Stress, 16 Causes of Apoptosis, 7 Hypertrophy, 16 Mechanisms of Apoptosis, 8 Hyperplasia, 16 Autophagy, 10 Atrophy, 17 Mechanisms of Cell Injury and Cell Death, 10 Metaplasia, 18 Mitochondrial Dysfunction and Damage, 10 Intracellular and Extracellular Depositions, 18 Oxidative Stress, 11 Intracellular Accumulations, 19 Generation and Removal of Reactive Oxygen Species, 11 Extracellular Deposits: Pathologic Calcification, 20 Cell Injury Caused by Reactive Oxygen Species, 12 Cellular Aging, 21 INTRODUCTION TO PATHOLOGY epigenetic modifications) from case to case. This realization has The field of pathology is dedicated to understanding the causes of launched the field of precision (or personalized) medicine, in which disease and the changes in cells, tissues, and organs that are associated therapies are designed for each individual’s disease rather than the with development of disease. Thus, pathology provides the scientific diseases as a whole. foundation for the practice of medicine. There are two important To render diagnoses and guide therapy in clinical practice, pa- terms that students will encounter throughout their study of pathology thologists identify changes in the gross or microscopic appearance and medicine: (morphology) of cells and tissues and in their constituents (e.g., genes Etiology is the origin of a disease, including the underlying causes and proteins), as well as biochemical alterations in body fluids (such as and modifying factors. Notably, many common diseases, such as blood and urine). Defining these alterations in diseased tissues aids in hypertension, diabetes, and cancer, are caused by a combination diagnosis as well as in predicting outcomes and optimal therapies. of inherited genetic susceptibility and various environmental trig- gers. Elucidating the genetic and environmental factors underlying OVERVIEW OF CELLULAR RESPONSES TO STRESS diseases is a major goal of modern medicine. AND NOXIOUS STIMULI Pathogenesis refers to the steps in the development of disease, from the initial etiologic trigger to the cellular and molecular changes Cells actively interact with their environment, constantly adjusting that give rise to the specific functional and structural abnormalities their structure and function to accommodate changing demands and which characterize any particular disease. Thus, etiology refers to extracellular stresses in order to maintain a steady state, a process why a disease arises and pathogenesis describes how a disease de- called homeostasis. As cells encounter physiologic stresses or injurious velops (Fig. 1.1). stimuli, they can undergo adaptation, achieving a new steady state and preserving viability and function. If the adaptive capability is exceeded Defining the etiology and pathogenesis of disease is essential not or if the external stress is inherently harmful, cell injury occurs only for understanding disease but is also the basis for developing (Fig. 1.2). Within certain limits, injury is reversible, and homeostasis is rational treatments. It is now appreciated that even diseases that restored; however, if the stress is severe or persistent, it results in present with similar morphologic features (e.g., cancer of a particular irreversible injury and death of the affected cells. Cell death is a crucial organ) show important molecular differences (e.g., mutations, event in the development of many diseases. 1 2 CHAPTER 1 Cell Injury, Cell Death, and Adaptations substances, and excessive or chronic immune responses to mi- Hypoxia and ischemia crobes (Chapter 5). In all these situations, the immune responses Toxins elicit inflammatory reactions, and inflammation is often the cause Infections of damage to cells and tissues. Abnormal ETIOLOGY: Genetic abnormalities. Some chromosomal abnormalities or muta- immune reactions Causes of tions can result in pathologic changes as conspicuous as the disease congenital malformations associated with Down syndrome or as Genetic abnormalities Nutritional subtle as the single amino acid substitution in hemoglobin that imbalances gives rise to sickle cell anemia (Chapter 4). Mutations may cause Physical agents cell injury as a consequence of a decrease (e.g., enzymes in inborn PATHOGENESIS: errors of metabolism) or an increase in the function of a protein, or Biochemical changes Mechanisms of through the accumulation of damaged DNA or misfolded proteins, Structural changes disease which can trigger cell death. Mutations also have a central role in cancer development (Chapter 6). Nutritional imbalances. Protein-calorie insufficiency remains a major ABNORMALITIES IN cause of cell injury, and specific vitamin deficiencies occur frequently CELLS AND TISSUES: even in countries with plentiful resources (Chapter 7). On the other Molecular hand, excessive dietary intake may result in obesity, which is an Functional important underlying factor in many common diseases, such as Morphologic type 2 diabetes and atherosclerosis. Physical agents. Trauma, extremes of temperature, radiation, elec- tric shock, and sudden changes in atmospheric pressure all have CLINICAL MANIFESTATIONS: damaging effects on cells (Chapter 7). Signs and symptoms of disease With this introduction, we proceed to a discussion of the process and FIG. 1.1 Steps in the development of disease. Only some of the major morphologic manifestations of cell injury and then the biochemical etiologies are shown. mechanisms of injury caused by different noxious stimuli. SEQUENCE OF EVENTS IN CELL INJURY AND Cell injury is the basis of all disease, and in this chapter we discuss the causes, mechanisms, and consequences of reversible injury and cell CELL DEATH death. We then consider cellular adaptations to stress and conclude Although injurious stimuli damage cells through diverse biochemical with two other processes that affect cells and tissues, the deposition of mechanisms, all tend to induce a stereotypic sequence of morphologic abnormal substances and cell aging. and structural alterations in most cell types. Reversible Cell Injury CAUSES OF CELL INJURY Reversible cell injury is defined as a derangement of function and The major causes of cell injury can be grouped into the following morphology that cells can recover from if the damaging stimulus categories. is removed (Fig. 1.3). In reversible injury, cells and intracellular Hypoxia and ischemia. Hypoxia refers to oxygen deficiency, and ischemia means reduced blood supply. These are among the most common causes of cell injury. Both deprive tissues of oxygen, HEALTHY CELL the essential molecule for generating energy for cell function and (homeostasis) survival, and ischemia also reduces the supply of nutrients. The most common cause of hypoxia is ischemia resulting from blockage Injurious stimulus of an artery, but it can also result from inadequate oxygenation of the blood, as in diseases of the lung, or from reduction in the oxygen-carrying capacity of the blood, as with anemia of any cause. REVERSIBLE Toxins. Potentially toxic agents are encountered daily in the envi- INJURY ronment; these include air pollutants, insecticides, carbon monox- ide, asbestos, cigarette smoke, ethanol, and drugs. Many therapeutic Severe, progressive drugs can cause cell or tissue injury in a susceptible patient or if used excessively or inappropriately (Chapter 7). IRREVERSIBLE Infectious agents. All types of infectious pathogens, such as viruses, INJURY bacteria, fungi, and parasites, can injure cells by diverse mecha- nisms, including liberation of toxins and eliciting harmful immune responses. Immunologic reactions. Although the immune system defends the CELL body against pathogenic microbes, immune reactions can also NECROSIS DEATH APOPTOSIS result in cell and tissue injury. Examples are autoimmune reactions against one’s own tissues, allergic reactions against environmental FIG. 1.2 Sequence of reversible cell injury and cell death.  CHAPTER 1 Cell Injury, Cell Death, and Adaptations 3 organelles become swollen because they take in water as a result of the failure of energy-dependent ion pumps in the plasma mem- brane. In some forms of injury, degenerated organelles and lipids accumulate inside the injured cells. HEALTHY CELL MORPHOLOGY The two most consistent morphologic correlates of reversible cell injury are cellular swelling and fatty change. Reversible Cellular swelling (Fig. 1.4B) is commonly seen when cells are Recovery Increased cell size injury injured by hypoxia, toxins, and other causes. It may be difficult to Clumping of chromatin appreciate with the light microscope (since fluid from cells is extracted Swelling of ER and mitochondria during tissue processing) but is often apparent grossly when the whole Small amorphous deposits organ is examined. When many cells in an organ are affected, there can in mitochondria be pallor (due to compression of capillaries), increased turgor, and Membrane blebs increased organ weight. Microscopic examination may reveal small, clear vacuoles within the cytoplasm; these represent distended and Myelin figures pinched-off segments of the endoplasmic reticulum (ER). This pattern of Intracytoplasmic vacuoles nonlethal injury is sometimes called hydropic change or vacuolar (pinched-off segments degeneration. of ER) Fatty change is manifested by the appearance of lipid vacuoles in the Detachment of ribosomes cytoplasm. It is principally encountered in organs that are involved in lipid from ER metabolism, such as the liver, and hence it is discussed in Chapter 14. Progressive injury The cytoplasm of injured cells also may become redder (eosinophilic, meaning stained red by the dye eosindthe E in the hematoxylin and eosin Breakdown of plasma membrane, organelles; [H&E] stain)da change that becomes more pronounced with progression to and nucleus necrosis (described later). Other intracellular changes associated with cell injury, which are best seen by electron microscopy (eFig. 1.1), include: (1) plasma membrane alterations such as blebbing, blunting, or distortion of Large amorphous deposits microvilli, and loosening of intercellular attachments; (2) mitochondrial in mitochondria changes such as swelling and the appearance of phospholipid-rich amorphous Leakage of contents densities; (3) dilation of the ER with detachment of ribosomes and dissociation Inflammation of polysomes; and (4) nuclear alterations, with clumping of chromatin. The (host reaction) cytoplasm may contain so-called myelin figures, collections of phos- pholipids resembling myelin sheaths that are derived from damaged cellular NECROSIS membranes. FIG. 1.3 Reversible cell injury and necrosis. The principal cellular alterations that characterize reversible cell injury and necrosis are illus- trated. If an injurious stimulus is not removed, reversible injury culmi- In some situations, potentially injurious insults induce specific nates in necrosis. alterations in cellular organelles, like the endoplasmic reticulum (ER). The smooth ER is involved in the metabolism of various chemicals, including alcohol and drugs such as barbiturates (Chapter 7). Cells exposed to these chemicals show hypertrophy of the smooth ER as an injury to lysosomal membranes results in the enzymatic digestion of adaptive response that may have important functional consequences. the injured cell, which is the culmination of necrosis. Cells adapted to one drug may have increased capacity to metabolize other compounds handled by the same system. Thus, if patients taking Cell Death phenobarbital for epilepsy increase their alcohol intake, they may When cells are injured, they die by different mechanisms, depending experience a drop in blood concentration of the antiseizure medication on the nature and severity of the insult (Table 1.1). to subtherapeutic levels because of increased activity of the smooth ER Necrosis. Severe disturbances, such as loss of oxygen and nutrient in response to the alcohol. supply and the actions of toxins, cause a rapid and uncontrollable Persistent or excessive injury causes injured cells to pass the form of death that has been called “accidental” cell death. The nebulous “point of no return” and undergo cell death, typically by morphologic manifestation of accidental cell death is necrosis the process of necrosis. Although there are no definitive morpho- (Greek, necros ¼ death). Necrosis is the major pathway of cell logic or biochemical correlates of irreversible injury, it is consis- death in many commonly encountered injuries, such as those tently characterized by three phenomena: the inability to restore resulting from ischemia, exposure to toxins, various infections, mitochondrial function (oxidative phosphorylation and adenosine and trauma. Necrosis is considered the inevitable end result of se- triphosphate [ATP] generation) even after resolution of the original vere damage that is beyond salvage and is not thought to be regu- injury; altered structure and loss of function of the plasma mem- lated by specific signals or biochemical mechanisms; necrosis brane and intracellular membranes; and the loss of structural occurs because the injury goes beyond what a cell can repair or integrity of DNA and chromatin. As discussed in more detail later, survive.  CHAPTER 1 Cell Injury, Cell Death, and Adaptations 3.e1 L L mv mv N M M M A B C eFIG. 1.1 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 that contain precipitated calcium and proteins. Higher magnification micrographs of the cell would show disrupted plasma membrane and swelling and fragmentation of organelles. N, Nucleus. (A, Courtesy Dr. Brigitte Kaissling, Institute of Anatomy, University of Zurich, Switzerland. B and C, Courtesy Dr. M.A. Ven- katachalam, University of Texas Health Sciences Center, San Antonio, TX.) 4 CHAPTER 1 Cell Injury, Cell Death, and Adaptations A B C FIG. 1.4 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 cyto- plasm, and swelling of occasional cells. (C) Necrosis (irreversible injury) of epithelial cells, with loss of nuclei and fragmentation of cells and leakage of contents. (Courtesy of Drs. Neal Pinckard and M.A. Venkatachalam, University of Texas Health Sciences Center, San Antonio, TX.) Table 1.1 Features of Necrosis and Apoptosis Feature Necrosis Apoptosis Cell size Enlarged (swelling) Reduced (shrinkage) Nucleus Pyknosis / karyorrhexis / karyolysis Fragmentation into nucleosome-sized fragments Plasma membrane Disrupted Intact; altered structure, especially orientation of lipids Cellular contents Enzymatic digestion; may leak out of cell Intact; may be released in apoptotic bodies Adjacent inflammation Frequent Absent Physiologic or Invariably pathologic (culmination of irreversible Often physiologic; means of eliminating unnecessary cells; pathologic role cell injury) may be pathologic after some forms of cell injury, especially DNA and protein damage DNA, Deoxyribonucleic acid. Apoptosis. By contrast, when cells must be eliminated without elic- It is important to recognize that cellular function may be lost long iting a host reaction, a precise set of molecular pathways is acti- before cell death occurs and that the morphologic changes of cell vated in the cells that produce a form of cell death called injury (or death) lag behind loss of function and viability (Fig. 1.5). For apoptosis (see Table 1.1). Apoptosis relies on defined genes and example, myocardial cells become noncontractile after 1 to 2 minutes of biochemical pathways and must be tightly controlled because ischemia but may not die until 20 to 30 minutes of ischemia have elapsed. once it starts, it is irreversible; thus, it is referred to as “regulated” Morphologic features indicative of the death of ischemic myocytes appear cell death. The discovery of regulated cell death was a revelation, by electron microscopy within 2 to 3 hours after the death of the cells but since it showed that cell death can be an intentional, highly are not evident by light microscopy until 6 to 12 hours later. controlled process. Apoptosis serves to eliminate cells with a variety of intrinsic abnormalities and promotes clearance of the fragments Necrosis of the dead cells without eliciting an inflammatory reaction. This In necrosis, cellular membranes fall apart, cellular enzymes leak out “clean” form of cell suicide occurs in pathologic situations when and ultimately digest the cell, and there is an accompanying in- a cell’s DNA or proteins are damaged beyond repair or the cell is flammatory reaction (see Fig. 1.3). The local host reaction, called deprived of necessary survival signals. But unlike necrosis, which inflammation, is induced by substances released from dead cells and is always an indication of a pathologic process, apoptosis also oc- serves to eliminate debris and start the subsequent repair process curs in healthy tissues and is not necessarily associated with (Chapter 2). The enzymes responsible for digestion of the dead cells pathologic cell injury. For example, it serves to eliminate un- come from leukocytes that are recruited as part of the inflammatory wanted cells during development and to maintain constant cell reaction and from the disrupted lysosomes of the dying cells themselves. numbers. This type of physiologic cell death is also called pro- The biochemical mechanisms of necrosis vary with different grammed cell death. injurious stimuli and are described later.  CHAPTER 1 Cell Injury, Cell Death, and Adaptations 5 inflammatory reactions. There are several morphologically distinct pat- Reversible Irreversible terns of tissue necrosis, which may provide clues about the underlying cell injury cell injury Ultrastructural Light changes microscopic cause. Although the terms that describe these patterns do not reflect changes underlying mechanisms, such terms are commonly used and their im- Cell Cell death plications are understood by pathologists and clinicians. Most of the types function of necrosis have distinctive gross appearances; fibrinoid necrosis is detected only by microscopic examination. Gross EFFECT morphologic changes MORPHOLOGY In coagulative necrosis, the underlying tissue architecture is pre- served for at least several days after the injury (Fig. 1.6). The affected tissues take on a firm texture. Presumably, the injury denatures not only structural proteins but also enzymes, limiting the proteolysis of the dead cells; as a result, eosinophilic, anucleate cells may persist for days or weeks. Ultimately, the dead cells are digested by the lysosomal enzymes DURATION OF INJURY of recruited leukocytes and the cellular debris is removed by phagocy- FIG. 1.5 The relationship of cellular function, cell death, and the tosis. Coagulative necrosis is characteristic of infarcts (areas of necrosis morphologic changes of cell injury. Note that cells may rapidly become caused by ischemia) in all solid organs except the brain. nonfunctional after the onset of injury yet still be viable, with potentially Liquefactive necrosis is seen at sites of bacterial or, occasionally, reversible damage; with a longer duration of injury, irreversible injury and fungal infections, because microbes stimulate the accumulation of in- cell death may result. Cell death typically precedes ultrastructural, light flammatory cells and the enzymes of leukocytes digest (“liquefy”) the microscopic, and grossly visible morphologic changes. tissue. For obscure reasons, hypoxic death of cells within the central nervous system often causes liquefactive necrosis (Fig. 1.7). In this form MORPHOLOGY of necrosis, the dead cells are completely digested, transforming the Necrosis is characterized by changes in the cytoplasm and nuclei of the tissue into a viscous liquid that is eventually removed by phagocytes. injured cells (see Figs. 1.3 and 1.4C). When the process is initiated by acute inflammation, as in a bacterial Cytoplasmic changes. Necrotic cells show increased eosino- infection, the material is frequently creamy yellow and is called pus. A philia, attributable in part to increased binding of eosin to denatured localized collection of pus is called an abscess (Chapter 2). cytoplasmic proteins and in part to loss of basophilic ribonucleic acid Although gangrenous necrosis is not a distinctive pattern of cell death, (RNA) in the cytoplasm (basophilia stems from binding of the blue dye the term is still commonly used in clinical practice. It usually refers to the hematoxylindthe H in “H&E”). Compared with viable cells, necrotic condition of a limb (generally the lower leg) that has lost its blood supply and cells may have a glassy, homogeneous appearance, mostly due to the has undergone coagulative necrosis involving multiple tissue layers. When loss of glycogen particles. When enzymes have digested cytoplasmic bacterial infection is superimposed, the morphologic appearance is often liq- organelles, the cytoplasm becomes vacuolated and appears “moth- uefactive because of destruction mediated by the contents of the bacteria and eaten.” By electron microscopy, necrotic cells are characterized by the attracted leukocytes (resulting in so-called wet gangrene). discontinuities in plasma and organelle membranes, marked dilation of Caseous necrosis is encountered most often in foci of tuberculous mitochondria associated with large amorphous intramitrochondrial infection. Caseous means “cheese-like,” referring to the friable yellow-white densities, disruption of lysosomes, and intracytoplasmic myelin figures, appearance of the area of necrosis (Fig. 1.8). On microscopic examination, the which are more prominent in necrotic cells than in cells with reversible necrotic focus appears as a collection of cellular debris with an amorphous injury (eFig. 1.1). granular pink appearance in H&E-stained tissue sections. Unlike coagulative Nuclear changes. Nuclear changes assume one of three patterns, all necrosis, the tissue architecture is obliterated and cellular outlines cannot be caused by breakdown of DNA and chromatin. Pyknosis is characterized discerned. Caseous necrosis is often surrounded by a collection of macro- by nuclear shrinkage and increased basophilia; the DNA condenses into a phages and other inflammatory cells; this appearance is characteristic of a dark, shrunken mass. The pyknotic nucleus can subsequently undergo nodular inflammatory lesion called a granuloma (Chapter 2). fragmentation; this change is called karyorrhexis. At the same time, Fat necrosis refers to focal areas of fat destruction, which can be due the nucleus may undergo karyolysis, in which the basophilia fades due to abdominal trauma or acute pancreatitis (Chapter 15), in which enzymes to digestion of deoxyribonucleic acid (DNA) by DNase. In 1 to 2 days, the leak out of damaged pancreatic acinar cells and ducts and digest perito- nucleus in a dead cell may undergo complete dissolution. neal fat cells and their contents, including stored triglycerides. The Fates of necrotic cells. Necrotic cells may persist for some time or released fatty acids combine with calcium to produce grossly identifiable may be digested by enzymes and disappear. Dead cells may be replaced by chalky white lesions (Fig. 1.9). On histologic examination, the foci of ne- myelin figures, which are either phagocytosed by other cells or further crosis contain shadowy outlines of necrotic fat cells surrounded by granular degraded into fatty acids. These fatty acids bind calcium salts, which may basophilic calcium deposits and an inflammatory reaction. result in the dead cells ultimately becoming calcified (dystrophic Fibrinoid necrosis is a special form of necrosis, visible by light micro- calcification, see later). scopy. It may be seen in immune reactions in which complexes of antigens and antibodies are deposited in the walls of blood vessels, and in severe hyper- tension. Deposited immune complexes and plasma proteins that have leaked into the walls of injured vessels produce a bright pink, amorphous appearance on H&E preparations called fibrinoid (fibrin-like) by pathologists (Fig. 1.10). Morphologic Patterns of Tissue Necrosis Fibrinoid necrosis is seen most often in certain forms of vasculitis (Chapter 3) Some severe injuries result in the death of many or all cells in a tissue or and in transplanted organs undergoing rejection (Chapter 5). even an entire organ. This may happen in severe ischemia, infections, and 6 CHAPTER 1 Cell Injury, Cell Death, and Adaptations I N A B FIG. 1.6 Coagulative necrosis. (A) A wedge-shaped kidney infarct (yellow) with distinct margins. (B) Micro- scopic view of the edge of the infarct, with normal kidney (N) and necrotic cells in the infarct (I). The necrotic cells show preserved outlines with loss of nuclei, and an inflammatory infiltrate is present (difficult to discern at this magnification). FIG. 1.7 Liquefactive necrosis. An infarct in the brain showing disso- FIG. 1.8 Caseous necrosis. Tuberculosis of the lung, with a large area lution of the tissue. of caseous necrosis containing yellow-white (cheesy) debris. FIG. 1.9 Fat necrosis in acute pancreatitis. The areas of white chalky FIG. 1.10 Fibrinoid necrosis in an artery in a patient with polyarteritis deposits represent foci of fat necrosis with calcium soap formation nodosa, a form of vasculitis (Chapter 3). The wall of the artery shows a (saponification) at sites of lipid breakdown in the mesentery. circumferential bright pink area of necrosis with protein deposition and inflammation.  CHAPTER 1 Cell Injury, Cell Death, and Adaptations 7 Leakage of intracellular proteins through the damaged cell membrane provides a means of detecting tissue-specific necrosis using blood or serum samples. Cardiac muscle, for example, contains a unique isoform of the contractile protein troponin, HEALTHY CELL whereas hepatic bile duct epithelium contains a temperature- resistant isoform of the enzyme alkaline phosphatase, and hepato- cytes contain transaminases. These proteins leak out of necrotic cells into the blood, where they serve as clinically useful markers of damage in the corresponding tissues. Reduced cell size Apoptosis Peripheral condensation Apoptosis is a pathway of cell death in which cells activate enzymes of chromatin that degrade the cells’ own nuclear DNA and nuclear and cyto- Tightly packed organelles plasmic proteins (Fig. 1.11). Fragments of the apoptotic cells then break off, giving the appearance that is responsible for the name Membrane blebs (apoptosis, “falling off”). The plasma membrane of the apoptotic cell remains intact, but the membrane is altered in such a way that the fragments, called apoptotic bodies, are recognized and rapidly phagocytosed by macrophages. In contrast to necrosis (see Table 1.1), the apoptotic cell and its fragments are cleared before cellular contents have leaked out, so apoptotic cell death does not elicit an inflammatory Cellular fragmentation reaction in the host. Nuclear fragmentation Causes of Apoptosis Apoptotic body Apoptosis occurs in many physiologic conditions and serves to eliminate potentially harmful cells and cells that have outlived their APOPTOSIS usefulness (Table 1.2). It also occurs as a pathologic event when cells are damaged beyond repair, especially when the damage affects the cells’ DNA or proteins. Physiologic apoptosis. During the normal development of an or- Phagocytosis of apoptotic ganism, some cells die and are replaced by new ones. In mature cells and fragments organisms, highly proliferative and hormone-responsive tissues undergo cycles of proliferation and cell loss that are often determined by the levels of growth factors or survival sig- nals. In these situations, the cell death is always by apoptosis, ensuring that unwanted cells are eliminated without eliciting Phagocyte potentially harmful inflammation. In the immune system, FIG. 1.11 Apoptosis. The cellular alterations in apoptosis are illustrated. apoptosis removes excess leukocytes following immune Contrast these with the changes that characterize necrotic cell death, shown in Fig. 1.3. Table 1.2 Physiologic and Pathologic Conditions Associated With Apoptosis Condition Mechanism of Apoptosis Physiologic During embryogenesis Loss of growth factor signaling (presumed mechanism) Turnover of proliferative tissues (e.g., intestinal Loss of growth factor signaling or survival signals (presumed mechanism) epithelium, lymphocytes in lymph nodes and thymus) Involution of hormone-dependent tissues Decreased hormone levels lead to reduced survival signals (e.g., endometrium) Decline of leukocyte numbers at the end of immune and Loss of survival signals as stimulus for leukocyte activation is eliminated inflammatory responses Elimination of potentially harmful self-reactive Strong recognition of self antigens induces apoptosis by both the mitochondrial lymphocytes and death receptor pathways Pathologic DNA damage Activation of proapoptotic BH3-only proteins Accumulation of misfolded proteins Activation of proapoptotic BH3-only proteins, possibly direct activation of caspases Infections, especially certain viral infections Activation of proapoptotic proteins or caspases by viral proteins; killing of infected cells by cytotoxic T lymphocytes (CTLs), which activate caspases 8 CHAPTER 1 Cell Injury, Cell Death, and Adaptations responses, B lymphocytes in germinal centers that fail to produce Mechanisms of Apoptosis high-affinity antibodies, and lymphocytes that recognize self Apoptosis is regulated by biochemical pathways that control the antigens that could cause autoimmune diseases if they were to balance of death- and survival-inducing signals and ultimately the survive (Chapter 5). activation of enzymes called caspases, so named because they are Apoptosis in pathologic conditions. Apoptosis eliminates cells with cysteine proteases that cleave proteins after aspartic acid residues. Two certain types of irreparable damage, such as severe DNA damage, distinct pathways converge on caspase activation: the mitochondrial e.g., after exposure to radiation and cytotoxic drugs. The accumu- pathway and the death receptor pathway (Fig. 1.12). Although these lation of misfolded proteins also triggers apoptotic death; the un- pathways can intersect, they are generally induced under different derlying mechanisms of this cause of cell death and its conditions, involve different molecules, and serve distinct roles in significance in disease are discussed later, in the context of ER physiology and disease. stress. Certain infectious agents, particularly some viruses, also The mitochondrial (intrinsic) pathway is responsible for induce apoptotic death of infected cells. apoptosis in most physiologic and pathologic situations. MITOCHONDRIAL (INTRINSIC) DEATH RECEPTOR (EXTRINSIC) PATHWAY PATHWAY FasL Cross-linking between Fas and FasL Fas (CD95) or type 1 TNF receptor Cytochrome c inside intermembranous space of mitochondria Death domain BAX (or BAK) Fas binding dimerize to form to adaptor channel protein BCL-2 family Cytochrome c effectors and other (BAX, BAK) Regulators proapoptotic Cofactors (BCL-2, BCL-XL) proteins Caspase-9 Caspase-8 BH3-only proteins Downstream caspases Growth factor withdrawal Activation of enzymes Absent survival including endonuclease signal Protein misfolding Nuclear DNA damage by fragmentation Breakdown of proteins radiation, toxins and cytoskeleton Secretion of and free radicals soluble factors by apoptotic cell APOPTOTIC CELL Membrane alteration and formation of ‘eat me’ signals for phagocytes Apoptotic body Phagocyte FIG. 1.12 Mechanisms of apoptosis. The two pathways of apoptosis differ in their induction and regulation, but both culminate in the activation of caspases. In the mitochondrial pathway, BH3-only proteins sense a lack of survival signals or DNA or protein damage and activate effector molecules that increase mitochondrial permeability. In concert with a deficiency of BCL-2 and other proteins that oppose mitochondrial permeability, the mitochondria become leaky and various substances, such as cytochrome c, enter the cytosol and activate caspases. Activated caspases induce the changes that culminate in cell death and fragmentation. 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.  CHAPTER 1 Cell Injury, Cell Death, and Adaptations 9 Mitochondria contain several proteins that are capable of MORPHOLOGY inducing apoptosis, including cytochrome c. When mitochon- drial membranes become permeable, cytochrome c leaks into In H&E-stained tissue sections, the nuclei of apoptotic cells show various the cytoplasm, triggering caspase activation and apoptotic death. stages of chromatin condensation, aggregation, and, ultimately, karyorrhexis The permeability of mitochondria is controlled by a family of (Fig. 1.13). At the molecular level this is reflected in fragmentation of DNA into more than 20 proteins, the prototype of which is BCL-2. In nucleosome-sized pieces. The cells rapidly shrink, form cytoplasmic buds, and healthy cells, BCL-2 and the related protein BCL-XL are produced fragment into apoptotic bodies that are composed of membrane-bound pieces in response to growth factors and other stimuli that keep cells of cytosol and organelles (eFig. 1.2; also see Fig. 1.11). Because these frag- viable. These antiapoptotic proteins maintain the integrity of ments are quickly shed and phagocytosed without eliciting an inflammatory mitochondrial membranes, in large part by holding two proapo- response, even substantial apoptosis may be histologically undetectable. ptotic members of the family, BAX and BAK, in check. When cells are deprived of growth factors and survival signals, are exposed to agents that damage DNA, or accumulate unacceptable amounts of misfolded proteins, a number of sensors are acti- Other pathways of cell death, in addition to necrosis and vated. The most important of these sensors are called BH3-only apoptosis, have been described. Necroptosis is a form of cell death proteins because they contain the third homology domain of caused by the cytokine tumor necrosis factor (TNF) that shows the BCL-2 family. These sensors shift the balance in favor of features of both necrosis and apoptosis, hence its name. Pyroptosis BAK and BAX, which dimerize, insert into the mitochondrial (pyro, fever) is induced by activation of inflammasomes (Chapter membrane, and form channels through which cytochrome c 5), which releases the cytokine interleukin-1 (IL-1), which cause and other mitochondrial proteins escape into the cytosol. At inflammation and fever. Ferroptosis depends on levels of cellular the same time, the deficiency of survival signals leads to iron. The roles of these mechanisms of cell death in normal phys- decreased levels of BCL-2 and BCL-XL, further compromising iology and pathologic states are not clearly established and remain mitochondrial permeability. Once in the cytosol, cytochrome c topics of investigation. interacts with certain cofactors and activates caspase-9, leading to the activation of a caspase cascade. The death receptor (extrinsic) pathway of apoptosis. Many cells express surface molecules, called death receptors, which trigger apoptosis. Most of these are members of the tumor necro- sis factor (TNF) receptor family, which contain in their cyto- plasmic regions a conserved “death domain,” so named because it mediates interaction with other proteins involved in cell death. The prototypic death receptors are the type I TNF re- ceptor and Fas (CD95). Fas ligand (FasL) is a membrane protein expressed mainly on activated T lymphocytes. When these T cells recognize Fas-expressing targets, Fas molecules are cross- linked by FasL and bind adaptor proteins via the death domain (see Fig. 1.12). These recruit and activate caspase-8, which in turn activates downstream caspases. The death receptor pathway is involved in elimination of self-reactive lymphocytes and in killing of target cells by some cytotoxic T lymphocytes that ex- press FasL. Terminal phase of apoptosis. Activated caspase-8 and caspase-9 act through a final common series of reactions that first involve the activation of additional caspases, which through numerous substrates ultimately activate enzymes that degrade the cell’s proteins and nucleus. The end result is the characteristic cellular fragmentation of apoptosis. Clearance of apoptotic cells. Apoptotic cells and their fragments entice phagocytes by producing a number of “eat-me” signals. For instance, in normal cells phosphatidylserine is present on the inner leaflet of the plasma membrane, but in apoptotic cells this phospholipid “flips” to the outer leaflet, where it is recognized FIG. 1.13 Morphologic appearance of apoptotic cells. Apoptotic cells (some indicated by arrows) in a normal crypt in the colonic epithelium are by tissue macrophages. Cells that are dying by apoptosis also shown. (The preparative regimen for colonoscopy frequently induces secrete soluble factors that recruit phagocytes. Numerous macro- apoptosis in epithelial cells, which explains the abundance of dead cells phage receptors are involved in the binding and engulfment of in this normal tissue.) Note the fragmented nuclei with condensed apoptotic cells. This process is so efficient that the dead cells chromatin and the shrunken cell bodies, some with pieces falling off. disappear without leaving a trace, and there is no accompanying (Courtesy of Dr. Sanjay Kakar, Department of Pathology, University of inflammation. California San Francisco, San Francisco, CA.)  CHAPTER 1 Cell Injury, Cell Death, and Adaptations 9.e1 A B eFIG. 1.2 Morphologic features of apoptosis. (A) 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. (B) 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). (A, 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 5e29; B, Courtesy Dr. Zheng Dong, Medical College of Georgia, Augusta, GA.) 10 CHAPTER 1 Cell Injury, Cell Death, and Adaptations Autophagy pathways that can initiate the sequence of events that lead to cell injury Autophagy (“self-eating”) refers to lysosomal digestion of the cell’s and culminate in cell death. Before discussing individual pathways of own components. It is a survival mechanism in times of nutrient cell injury and their mechanisms, some general principles should be deprivation that enables the starved cell to live by eating its own contents emphasized. and recycling these contents to provide nutrients and energy. In this The cellular response to injurious stimuli depends on the type of process, intracellular organelles and portions of cytosol are first injury and its duration and severity. Thus, low doses of toxins or a sequestered within an ER-derived double membrane (phagophore), brief duration of ischemia may lead to reversible cell injury, which matures into an autophagic vacuole. The formation of this auto- whereas larger toxin doses or longer ischemia times may result in phagosome is initiated by cytosolic proteins that sense nutrient depri- irreversible injury and necrosis. vation (Fig. 1.14). The vacuole fuses with lysosomes to form an The consequences of an injurious stimulus also depend on the autophagolysosome, and lysosomal enzymes digest the cellular compo- type of cell and its metabolic state, adaptability, and genetic nents. In some circumstances, autophagy may be associated with atrophy makeup. For instance, skeletal muscle in the leg can survive com- of tissues (discussed later) and represent an adaptation that helps cells plete ischemia for 2 to 3 hours, whereas more metabolically active survive lean times. If, however, the starved cell can no longer cope by cardiac muscle dies after only 20 to 30 minutes. Genetically deter- devouring its contents, autophagy may also signal cell death by apoptosis. mined diversity in metabolic pathways can contribute to differences Extensive autophagy is seen in ischemic injury and some types of in responses to injurious stimuli. For instance, when exposed to the myopathies. Autophagic vacuoles may also form around microbes in same dose of a toxin, individuals who inherit variants in genes infected cells, leading to destruction of these infectious pathogens. encoding cytochrome P-450 may catabolize the toxin at different Cancer cells acquire the ability to survive even in times of stress without rates, leading to different outcomes. autophagy (Chapter 6). Thus, a once little-appreciated survival pathway Cell injury usually results from functional and biochemical ab- in cells may prove to have wide-ranging roles in human disease. normalities in one or more essential cellular components (Fig. 1.15). Deprivation of oxygen and nutrients (as in hypoxia MECHANISMS OF CELL INJURY AND CELL DEATH and ischemia) primarily impairs energy-dependent cellular func- tions, while damage to proteins and DNA triggers apoptosis. There are numerous and diverse extrinsic causes of cell injury and cell Because any one injurious insult may trigger multiple, overlapping death, so it is not surprising that there are many intrinsic biochemical biochemical pathways, it has proved difficult to prevent cell injury from any cause by targeting an individual pathway. NUTRIENT DEPLETION In the following sections, we discuss the mechanisms that lead to cell injury and death. While each of the mechanisms tends to cause cell death predominantly by necrosis or apoptosis, the two pathways may Cytoplasmic intersect. For instance, ischemia and the production of free radicals are organelles typically associated with necrotic cell death, but they can also trigger Cytoplasmic INITIATION sensors apoptosis. Phagophore Mitochondrial Dysfunction and Damage Atgs Atg proteins ELONGATION Mitochondria produce life-sustaining energy in the form of ATP. They may be damaged functionally or structurally by many types of inju- NUCLEUS rious stimuli, including hypoxia, chemical toxins, and radiation. There are two major consequences of mitochondrial dysfunction. Failure of oxidative phosphorylation, leading to decreased ATP generation and depletion of ATP in cells. Since ATP is Lysosome the energy source required for virtually all enzymatic and biosyn- Recycling of thetic activities in cells, loss of ATP, which is often a consequence Enzymes metabolites of ischemia (discussed later), has effects on many cellular systems. Reduced activity of plasma membrane ATP-dependent sodium pumps results in intracellular accumulation of sodium and MATURATION OF efflux of potassium. The net gain of solute is accompanied by AUTOPHAGOSOME osmotic gain of water, causing cell swelling and dilation of the ER. The compensatory increase in anaerobic glycolysis leads to lactic DEGRADATION AUTOPHAGOLYSOSOME acid accumulation, decreased intracellular pH, and decreased activity of many cellular enzymes. Prolonged or worsening depletion of ATP causes structural FIG. 1.14 Autophagy. Cellular stresses, such as nutrient deprivation, activate autophagy genes (Atgs), whose products initiate the formation of disruption of the protein synthetic apparatus, manifested as membrane-bound vesicles in which cellular organelles are sequestered. detachment of ribosomes from the rough ER and dissociation These vesicles fuse with lysosomes, in which the organelles are digested, of polysomes, with a consequent reduction in protein synthesis. and the products are used to provide nutrients for the cell. The same Ultimately, there is irreversible damage to mitochondrial and process can trigger apoptosis by mechanisms that are not well defined. lysosomal membranes, and the cell undergoes necrosis.  CHAPTER 1 Cell Injury, Cell Death, and Adaptations 11 Hypoxia/ischemia Radiation Mutations Radiation ROS Mutations Cell stress Other injurious agents Other injurious agents Infections MITOCHONDRIA CELLULAR MEMBRANES NUCLEUS ENDOPLASMIC RETICULUM ATP ROS Damage to lysosomal Damage to plasma membranes membrane Energy- Damage to DNA damage dependent lipids, proteins, Leakage of Impaired transport Accumulation of functions nucleic acids enzymes functions, leakage of misfolded proteins cellular contents Cell cycle Activation of arrest BH3-only sensors Cell injury Unfolded protein response NECROSIS NECROSIS APOPTOSIS APOPTOSIS FIG. 1.15 The principal biochemical mechanisms and sites of damage in cell injury. Note that causes and mechanisms of cell death by necrosis and apoptosis are shown as being independent but there may be overlap; for instance, both may occur as a result of ischemia, oxidative stress, and radiation-induced cell death. ATP, Adenosine triphosphate; ROS, reactive oxygen species. Although necrosis is the principal form of cell death caused by Generation and Removal of Reactive Oxygen Species hypoxia, apoptosis by the mitochondrial pathway is also The accumulation of ROS is determined by their rates of produc- thought to contribute. tion and removal (Fig. 1.16). The properties and pathologic effects of Abnormal oxidative phosphorylation also leads to the formation of the major ROS are summarized in Table 1.3. reactive oxygen species, described later. ROS are normally produced by two major pathways. Damage to mitochondria is often associated with the formation of a ROS are produced in small amounts in all cells during the high-conductance channel in the mitochondrial membrane, called reduction-oxidation (redox) reactions that occur during energy the mitochondrial permeability transition pore. The opening of generation. In this process, molecular oxygen is reduced in mito- this channel leads to the loss of mitochondrial membrane potential chondria by the sequential addition of four electrons to produce and pH changes, further compromising oxidative phosphorylation. water. This reaction is imperfect, however, and when oxygen is only partially reduced, small amounts of highly reactive, short- As discussed earlier, mitochondria contain proteins such as cyto- lived toxic intermediates  are generated. These intermediates include chrome c that, when released into the cytoplasm, alert the cell to superoxide (O2, ), which is converted to hydrogen peroxide (H2O2) internal injury and activate apoptosis. The leakage of these proteins is spontaneously and by the action of the enzyme  superoxide dismut- regulated by other proteins and is a response to loss of survival signals ase (SOD). H2O2 is more stable than O2, and can cross biologic and other proapoptotic triggers. Thus, mitochondria are life sustaining membranes. In the presence of metals, such as Fe2þ, H2O2 is con- when healthy yet capable of activating numerous protective and verted to the highly reactive hydroxyl radical OH. Ionizing radia- pathologic reactions when damaged. tion and high doses of ultraviolet light can increase the production of ROS by hydrolyzing water into hydroxyl ( OH) and hydrogen Oxidative Stress (H ) free radicals. Oxidative stress refers to cellular damage induced by the accumu- ROS are produced in phagocytic leukocytes, mainly neutrophils, lation of reactive oxygen species (ROS), a form of free radical. Cell as a weapon for destroying ingested microbes and other substances injury in many circumstances involves damage by free radicals; these during inflammation (Chapter 2). ROS are generated in the phago- situations include chemical and radiation injury, hypoxia, cellular lysosomes of leukocytes by a process that is similar to mitochon- aging, tissue injury caused by inflammatory cells, and ischemia- drial respiration and is called the respiratory burst (or oxidative reperfusion injury (discussed later). Free radicals are chemical spe- burst). In this process, the enzyme phagocyte oxidase, located in cies with a single unpaired electron in an outer orbital. Such chemical the membranes of phagolysosomes, catalyzes the generation of su- species are extremely unstable and readily react with inorganic and peroxide, which is converted to H2O2. H2O2 is in turn converted to organic compounds, such as nucleic acids, proteins, and lipids. During the highly reactive compound hypochlorite (the major component this reaction, the molecules that are “attacked” by free radicals are of household bleach) by the enzyme myeloperoxidase, which is often themselves converted into other types of free radicals, thereby abundant in leukocytes, especially neutrophils. ROS released from propagating the chain of damage. neutrophils may injure tissues. 12 CHAPTER 1 Cell Injury, Cell Death, and Adaptations Radiation Toxins Pathologic effects Reperfusion Production of ROS Membrane damage Lipid peroxidation SOD O2 H2O2 HO Superoxide Hydrogen Hydroxyl peroxide radical Protein modification Breakdown Misfolding Glutathione DNA damage peroxidase Catalase Mutations, CYTOPLASM H2O Strand breaks Removal of free radicals NUCLEUS FIG. 1.16 The generation, removal, and role of reactive oxygen species (ROS) in cell injury. The production of ROS is increased by many injurious stimuli. These free radicals are removed by spontaneous decay and by specialized enzymatic systems. Excessive production or inadequate removal leads to accumulation of free radicals in cells, which may damage lipids (by peroxidation), proteins, and DNA, resulting in cell injury. SOD, Superoxide dismutase. Table 1.3 Principal Free Radicals Involved in Cell Injury Free Radical Mechanisms of Production Mechanisms of Removal Pathologic Effects  Superoxide (O2, ) Incomplete reduction of O2 during Conversion to H2O2 and O2 by Direct damaging effects on mitochondrial oxidative superoxide dismutase lipids (peroxidation), phosphorylation; by phagocyte proteins, and DNA oxidase in leukocytes Hydrogen peroxide (H2O2) Mostly from superoxide by action of Conversion to H2O and O2 by Can be converted to OH SOD catalase, glutathione peroxidase and ClO, which destroy microbes and cells  Hydroxyl radical ( OH) Produced from H2O, H2O2, and O2, Conversion to H2O by glutathione Direct damaging effects on by various chemical reactions peroxidase lipids, proteins, and DNA  Peroxynitrite (ONOO) Interaction of O2, and NO mediated Conversion to nitrite by enzymes in Direct damaging effects on by NO synthase mitochondria and cytosol lipids, proteins, and DNA ClO, Hypochlorite; NO, nitric oxide; SOD, superoxide dismutase. Cells have evolved mechanisms that remove free radicals and Endogenous or exogenous antioxidants (e.g., vitamins E, A, and C thereby minimize their injurious effects. Free radicals are inherently and b-carotene) may either block the formation of free radicals unstable and decay spontaneously. There are also nonenzymatic and or scavenge them once they have formed. enzymatic systems, sometimes called free radical scavengers, that serve to inactivate free radicals (see Fig. 1.16): Cell Injury Caused by Reactive Oxygen Species Superoxide dismutases (SODs), found in many cell types, Reactive oxygen species cause cell injury by damaging multiple convert superoxide into H2O2, which is degraded by catalase components of cells (see Fig. 1.16): (see below). Peroxidation of membrane lipids. ROS damage plasma membranes Glutathione peroxidases are a family of enzymes whose major as well as mitochondrial and lysosomal membranes because the function is to protect cells from oxidative damage. The most double bonds in membrane lipids are vulnerable to attack by free abundant member of this family, glutathione peroxidase 1, is radicals. The lipideradical interactions yield peroxides, which are found in the cytoplasm of all cells. It catalyzes the breakdown themselves unstable and reactive, and an autocatalytic chain reac- of H2O2 to H2O. tion ensues. Catalase, present in peroxisomes, catalyzes the decomposition of Crosslinking and other changes in proteins. Free radicals promote hydrogen peroxide into O2 and H2O. It is highly efficient, being sulfhydryl-mediated protein crosslinking, resulting in enhanced capable of degrading millions of molecules of H2O2 per second. degradation or loss of functional activity. Free radical reactions  CHAPTER 1 Cell Injury, Cell Death, and Adaptations 13 initially due to release from intracellular stores and later from increased influx across the dysfunctional plasma membrane. Excessive intracellular Injury O2 Cytosolic Ca2+ Ca2þ may cause cell injury by activating various enzymes, e.g., proteases and phospholipases, that damage cellular components. Phospholipase Protease ROS ATP activation activation Endoplasmic Reticulum Stress Lysosome The accumulation of misfolded proteins in a cell can stress compensatory pathways in the ER and lead to cell death by apoptosis. During protein synthesis, chaperones in the ER enhance Lipid Phospholipid Phospholipid Cytoskeletal the proper folding of newly synthesized proteins, but this process is peroxidation synthesis degradation damage imperfect, and some misfolded polypeptides are generated that are Lipid targeted for proteolysis by ubiquitination. When misfolded proteins breakdown accumulate in the ER, they induce a protective cellular response that is products called the unfolded protein response (Fig. 1.18). This adaptive response activates signaling pathways that increase the production of chaperones and retard protein translation, thus reducing the levels of MEMBRANE DAMAGE misfolded proteins in the cell. However, if the quantity of misfolded protein exceeds what can be handled by the adaptive response, addi- FIG. 1.17 Mechanisms of membrane damage. Decreased O2 and increased cytosolic Ca2þ are typically seen in ischemia but may tional signals are generated that activate proapoptotic sensors, leading accompany other forms of cell injury. Reactive oxygen species, which to apoptosis mainly by the mitochondrial (intrinsic) pathway. are often produced on reperfusion of ischemic tissues, also cause membrane damage (not shown). MILD SEVERE may also directly cause fragmentation of polypeptides. Damaged ER STRESS ER STRESS proteins may fail to fold properly, triggering the unfolded protein response, described later. Misfolded proteins Misfolded proteins DNA damage. Free radical reactions produce DNA damage of several (low amount) (large amount) types, notably mutations and DNA breaks. Such DNA damage has ER been implicated in apoptotic cell death, aging, and malignant transfor- lumen mation of cells. In addition to their role in cell injury and killing of microbes, ROS P P P P at low concentrations may be involved in numerous signaling path- P P Sensor of ways in cells and thus in physiologic reactions. misfolded Signaling Signaling proteins Membrane Damage (e.g., IRE1) CYTOSOL Most forms of cell injury that culminate in necrosis are characterized by increased membrane permeability that ultimately leads to overt synthesis of Activation of BH3 membrane damage. Cellular membranes may be damaged by ROS, chaperones proteins decreased phospholipid biosynthesis (due to hypoxia and nutrient protein synthesis Activation of deprivation), increased degradation (e.g., following phosphatase acti- protein degradation caspases vation due to increased intracellular calcium), and cytoskeletal ab- normalities that disrupt the anchors for plasma membranes (Fig. 1.17). The most important sites of membrane damage are the following: Mitochondrial membrane damage, discussed earlier. Plasma membrane damage, which leads to loss of osmotic balance Reduced load of and influx of fluids and ions, as well as loss of cellular contents. misfolded proteins Injury to lysosomal membranes, leading to leakage into the cytoplasm of lysosomal enzymes such as acid hydrolases, which are activated in ADAPTIVE UNFOLDED the acidic intracellular pH of the injured (e.g., ischemic) cell. These en- APOPTOSIS PROTEIN RESPONSE zymes digest numerous cellular components, producing irreversible damage and necrosis. FIG. 1.18 The unfolded protein response and ER stress. The presence of misfolded proteins in the ER is detected by sensors in the ER Disturbance in Calcium Homeostasis membrane, such as the kinase IRE1, which form oligomers and are activated by phosphorylation (the extent of both being proportional to the Calcium ions normally serve as second messengers in several signaling amount of misfolded proteins). This triggers an adaptive unfolded protein pathways but if released into the cytoplasm of cells in excessive amounts response, which can protect the cell from the harmful consequences of are also an important source of cell injury. Cytosolic free Ca2þ is normally the misfolded proteins. When the level of misfolded proteins is too great maintained at much lower concentrations (w0.1 mmol) than extracellular to be corrected, the mitochondrial pathway of apoptosis is induced and Ca2þ (1.3 mmol), and most intracellular Ca2þ is sequestered in mito- the irreparably damaged cell dies; this is also called the terminal unfolded chondria and the ER. Ischemia and certain toxins increase cytosolic Ca2þ, protein response. IRE1, Inositol requiring enzyme-1. 14 CHAPTER 1 Cell Injury, Cell Death, and Adaptations Table 1.4 Diseases Caused by Misfolded Proteins Disease Affected Protein Pathogenesis Diseases Caused by Mutant Proteins That Are Degraded, Leading to Their Deficiency Cystic fibrosisa Cystic fibrosis transmembrane Loss of CFTR leads to defects in ion transport conductance regulator (CFTR) Familial LDL receptor Loss of LDL receptor leading to hypercholesterolemia hypercholesterolemiaa Tay-Sachs diseasea Hexosaminidase a-subunit Lack of the lysosomal enzyme leads to storage of GM2 gangliosides in neurons Diseases Caused by Misfolded Proteins That Result in ER Stress-Induced Cell Loss Retinitis pigmentosaa Rhodopsin Abnormal folding of rhodopsin causes photoreceptor loss and blindness Creutzfeldt-Jakob Prions Abnormal folding of PrPsc causes neuronal cell death disease Diseases Caused by Misfolded Proteins That Result From Both ER Stress-Induced Cell Loss and Functional Deficiency of the Protein a-1-antitrypsin a-1 antitrypsin Storage of nonfunctional protein in hepatocytes causes apoptosis; absence of deficiency enzymatic activity in lungs causes destruction of elastic tissue, giving rise to emphysema a Misfolding is responsible for protein dysfunction and cellular injury in a subset of molecular subtypes. Shown are selected illustrative examples of diseases in which protein misfolding is a mechanism of functional derangement or cell or tissue injury. CFTR, Cystic fibrosis transporter; LDL, low density lipoprotein; PrP, prion protein. Intracellular accumulation of misfolded proteins may be caused Clinicopathologic Examples of Cell Injury and Necrosis by abnormalities that increase the production of misfolded proteins The mechanisms involved in some common causes of cell injury or reduce the ability to eliminate them. This may result from muta- culminating in necrosis are summarized next. tions that lead to the production of abnormal proteins; aging, which is associated with decreased capacity to correct misfolding; infections, Hypoxia and Ischemia especially viral infections, in which microbial proteins are synthesized in Oxygen deprivation is one of the most frequent causes of cell injury such large amounts that they overwhelm the quality control system that and necrotic cell death in clinical medicine. Oxygen is required for normally ensures proper protein folding; and changes in intracellular oxidative phosphorylation and the generation of ATP, the energy pH and redox state. Deprivation of glucose and oxygen, as in ischemia store of cells. Therefore, cells deprived of oxygen are at risk of and hypoxia, may also increase the burden of misfolded proteins. suffering catastrophic failure of many essential functions. In contrast Protein misfolding within cells may cause diseases by creating a to hypoxia, in which blood flow is maintained and during which deficiency of an essential protein or by inducing apoptosis energy production by anaerobic glycolysis can continue, ischemia (Table 1.4). Misfolded proteins often lose their activity and are rapidly compromises the delivery of substrates for glycolysis. Thus, in degraded, both of which can contribute to a loss of function. If this ischemic tissues, not only does aerobic metabolism cease but anaer- function is essential, cellular injury ensues. One example is cystic obic energy generation also fails after glycolytic substrates are fibrosis, which is caused by inherited mutations in a membrane exhausted or glycolysis is inhibited by the accumulation of metabo- transport protein, some of which prevent its normal folding. Cell lites, which otherwise would be washed out by flowing blood. For this injury as a result of protein misfolding is recognized as a feature of a reason, ischemia causes more rapid and severe cell and tissue injury number of diseases (see Table 1.4). than hypoxia. Cells subjected to the stress of hypoxia that do not immediately die DNA Damage activate compensatory mechanisms that are induced by transcription Exposure of cells to radiation or chemotherapeutic agents, intra- factors of the hypoxia-inducible factor (HIF) family. HIF simulates the cellular generation of ROS, and acquisition of mutations may all synthesis of several proteins that help the cell to survive in the face of induce DNA damage, which, if severe, may trigger apoptotic death. low oxygen. Some of these proteins, such as vascular endothelial Damage to DNA is sensed by intracellular sentinel proteins, which growth factor (VEGF), stimulate the growth of new vessels and thus transmit signals that lead to the accumulation of p53 protein. p53 first increase blood flow and the supply of oxygen. Other proteins induced arrests the cell cycle (at the G1 phase) to allow the DNA to be repaired by HIF cause adaptive changes in cellular metabolism by stimulating before it is replicated (Chapter 6). However, if the damage is too great the uptake of glucose and glycolysis. Anaerobic glycolysis can generate to be repaired successfully, p53 triggers apoptosis, mainly by the ATP in the absence of oxygen using glucose derived either from the mitochondrial pathway. When p53 is mutated or absent (as it is in circulation or from the hydrolysis of intracellular glycogen. Tissues certain cancers), cells with damaged DNA that would otherwise un- with a greater glycolytic capacity due to the presence of glycogen dergo apoptosis survive. In such cells, the DNA damage may result in (e.g., the liver and striated muscle) can survive loss of oxygen and various types of genomic alterations (e.g., chromosomal deletions) that decreased oxidative phosphorylation better than tissues with limited lead to neoplastic transformation (Chapter 6). glycogen stores (e.g., the brain).  CHAPTER 1 Cell Injury, Cell Death, and Adaptations 15 Arterial occlusion the situation. ROS generated by infiltrating leukocytes may also contribute to the damage of vulnerable injured cells. Influx of calcium may cause injury by mechanisms described Ischemia earlier.