Responses to cellular injury

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Chapter 6 Responses to cellular injury

Cellular injury 102
Commonly confused conditions and entities relating to cellular injury 122

CELLULAR INJURY

image Numerous causes: physical and chemical agents including products of micro-organisms
image Various mechanisms: disruption, membrane failure, metabolic interference (respiration, protein synthesis, DNA), free radicals
image May be reversible, or end in cell death

Cell survival depends upon several factors: a constant supply of energy, intact plasma membrane, biologically safe and effective function of generic and specific cellular activities, genomic integrity, controlled cell division, and internal homeostatic mechanisms. Cell death may result from significant disturbance of these factors. However, cell replication proceeds in a human body at a rate of c. 10 000 new cells per second; so, although eventually some will be lost to the environment via the skin or gut surfaces, many will inevitably need to be deleted. Thus, cell death is a normal physiological process as well as a reaction to injury. Similarly, failure or poor regulation of death processes may underlie some diseases.

Causative agents and processes

A wide range of possible agents or circumstances result in cellular injury (Fig. 6.1). These could be categorised according to the nature of the injurious agent, the cellular target, the pattern of cellular reaction or mode of cell death. The sequence of agent, target and mode will be uniform, but some injurious agents have variable effects depending on concentration, duration or other contributory influences such as co-existent disease. Some examples are given in Table 6.1. Major types of cellular injury include:

trauma
thermal injury, hot or cold
poisons
drugs
infectious organisms
ischaemia and reperfusion
plasma membrane failure
DNA damage
loss of growth factors
ionising radiation.
image

Fig. 6.1 Mechanisms of cellular injury. Different agents can injure the various structural and functional components of the cell. Some cells with specific function are selectively prone to certain types of injury.

Table 6.1 Examples of causes of cellular injury and their mode of action

Example agent Mode of action
Trauma (e.g. road traffic accident) Mechanical disruption of tissue
Carbon monoxide inhalation Prevents oxygen transport
Contact with strong acid Coagulates tissue proteins
Paracetamol overdose Metabolites bind to liver cell proteins and lipoproteins
Bacterial infections Toxins and enzymes
Ionising radiation (e.g. X-rays) Damage to DNA

Physical agents

Most physical agents cause passive cell destruction by gross membrane disruption or catastrophic functional impairment. Trauma and thermal injury cause cell death by disrupting cells and denaturing proteins, and also cause local vascular thrombosis with consequent tissue ischaemia or infarction (Ch. 8). Freezing damages cells mechanically because their membranes are perforated by ice crystals. Missile injury combines the effects of trauma and heat; much energy is dissipated into tissues around the track. Blast injuries are the result of shearing forces, where structures of differing density and mobility are moved with respect to one another; traumatic amputation is a gross example. Microwaves (wavelengths in the range from 1 mm to 1 m) cause thermal injury. Laser light falls into two broad categories: relatively low energy produces tissue heating, with coagulation for example; higher energy light breaks intramolecular bonds by a photochemical reaction, and effectively vaporises tissue. Ionising radiation is considered on p. 115.

Chemical and biological agents

Cells may be injured by contact with drugs and other chemicals; the latter may include enzymes and toxins secreted by micro-organisms. This category of agents can give rise to the full range of modes of death.

Drugs and poisons

Many naturally occurring and synthetic chemicals cause cellular injury; the effect is usually dose-related, but in a few instances the effect is exacerbated by constitutional factors.

Some are highly toxic systemic metabolic poisons, while others exert their damage locally; the latter includes caustic liquids applied to skin or mucous membranes, or gases that injure the lung. Furthermore, some substances produce one effect locally and another systemically. For example, some drugs are potentially caustic, and care needs to be taken to avoid extravasation into soft tissues when giving them by intravenous injection.

Caustic agents cause rapid local cell death due to their extreme alkalinity or acidity, in addition to having a corrosive effect on the tissue by digesting proteins.

Infectious organisms

The mechanisms of tissue damage produced by infectious organisms are varied, but with many bacteria it is their metabolic products or secretions that are harmful (Ch. 3). Thus, the host cells receive a chemical insult that may be toxic to their metabolism or membrane integrity. The mode of cell death generally induces an acute inflammatory response, which may be damaging to adjacent cells; organisms that do this are called pyogenic. In contrast, bacterial endotoxin (lipopolysaccharide) induces apoptosis with different pathological consequences. Intracellular agents such as viruses often result in the physical rupture of infected cells, but with some viruses such as hepatitis B (Ch. 16) local tissue damage may result from host immune reactions. Therefore, the cellular response to injury caused by infections will depend on a combination of the damage inflicted directly by the agent and indirectly as a result of the host response to the agent.

Blockage of metabolic pathways

Cell injury may result from specific interference with intracellular metabolism, effected usually by relative or total blockage of one or more pathways.

Cellular respiration

Prevention of oxygen utilisation results in the death of many cells due to loss of their principal energy source. Cyanide ions act in this way by binding to cytochrome oxidase and thus interrupting oxygen utilisation. Cells with higher metabolic requirements for oxygen (e.g. cardiac myocytes) are most vulnerable.

Glucose deprivation

Glucose is another important metabolite and source of energy. Some cells, cerebral neurones for example, are highly dependent. In diabetes mellitus there is inadequate utilisation of glucose due to an absolute or relative lack of insulin.

Protein synthesis

Cell function and viability will also be compromised if protein synthesis is blocked at the translational level because there is a constant requirement to replace enzymes and structural proteins. Ricin, a potent toxin from the castor oil plant, acts in this manner at the ribosomal level. Many antibiotics, such as streptomycin, chloramphenicol and tetracycline, act by interfering with protein synthesis, although toxic effects by this mechanism are fortunately rare.

Loss of growth factor or hormonal influence

Many cells rely on growth factors for their survival. Typically, these bind to growth factor receptors spanning the plasma membrane, triggering an intracellular cascade, often via a tyrosine kinase. This pathway can fail or be blocked at many points including growth factor deficit, receptor loss or blockade, or tyrosine kinase inhibitor (e.g. imatinib) (Ch. 4); affected cells may undergo apoptosis. Similar consequences can follow hormone withdrawal, as either a physiological response or part of a disease process. If widespread in an organ, it will shrink (atrophy).

Ischaemia and reperfusion injury

Impaired blood flow (Ch. 8) causes inadequate oxygen delivery to cells. Mitochondrial production of ATP will cease, and anaerobic glycolysis will result in acidosis due to the accumulation of lactate. The acidosis promotes calcium influx. Cells in different organs vary widely in their vulnerability to oxygen deprivation; those with high metabolic activity such as cortical neurones and cardiac myocytes will be most affected.

When the blood supply is restored, the oxygen results in a burst of mitochondrial activity and excessive release of reactive oxygen species (free radicals).

Free radicals

Free radicals are atoms or groups of atoms with an unpaired electron (symbolised by a superscript dot); they avidly form chemical bonds. They are highly reactive, chemically unstable, generally present only at low concentrations, and tend to participate in or initiate chain reactions.

Free radicals can be generated by two principal mechanisms:

Deposition of energy, e.g. ionisation of water by radiation. An electron is displaced, resulting in free radicals. This is discussed further under the mode of action of ionising radiation (p. 117).
Interaction between oxygen, or other substances, and a free electron in relation to oxidation–reduction reactions. In this instance the superoxide radical (O2•−) could be generated. Mitochondria are the main source, and in pathological circumstances can produce toxic quantities of reactive oxygen species.

The consequences of free radical formation include the following:

A chain reaction may be initiated in which other free radicals are also formed. Common final events are damage to polyunsaturated fatty acids, which are an essential component of cell membranes, or damage to DNA.
The free radical may be scavenged by endogenous or exogenous anti-oxidants, e.g. sulphydryl compounds such as cysteine.
Superoxide radicals may be inactivated by the copper-containing enzyme, superoxide dismutase, which generates hydrogen peroxide; catalase then converts this to water.

The clinicopathological events involving free radicals include:

toxicity of some poisons (e.g. carbon tetrachloride)
oxygen toxicity
tissue damage in inflammation
intracellular killing of bacteria.

Cells irreversibly damaged by free radicals are deleted, generally by apoptosis.

Failure of membrane integrity

Cell membrane damage is an important mode of cellular injury for which there are several possible mechanisms:

complement-mediated cytolysis
perforin-mediated cytolysis
specific blockage of ion channels
failure of membrane ion pumps
free radical attack.

Cell membrane damage is one of the consequences of complement activation (Ch. 9); some of the end products of the complement cascade have cytolytic activity. Another effector of cytolysis is perforin, a mediator of lymphocyte cytotoxicity that causes damage to the cell membrane of the target cells such as those infected by viruses. Incidental membrane tears or perforations can be repaired very quickly, so do not necessarily result in cell death.

Intramembrane channels permit the controlled entry and exit of specific ions. Blockage of these channels is sometimes used therapeutically. For example, verapamil is a calcium channel blocker used in the treatment of hypertension and ischaemic heart disease. Used in inappropriate circumstances or at high dosage, however, the calcium channel blockage may have toxic effects.

Membrane ion pumps that are responsible for maintaining intracellular homeostasis, for example calcium, potassium and sodium concentrations within cells, are dependent on an adequate supply of ATP. Any chemical agents that deplete ATP, either by interfering with mitochondrial oxidative phosphorylation or by consuming ATP in their metabolism, will compromise the integrity of the membrane pumps and expose the cell to the risk of lysis. The Na/K ATPase in cell membranes can be directly inhibited by ouabain. Failure of membrane ion pumps frequently results in cell swelling, also called oncosis or hydropic change (see below), which may progress to cell death.

Just as disastrous for the cell is biochemical alteration of the lipoprotein bilayer forming the cell membrane. This can result from reactions with either the phospholipid or protein moieties. Membrane phospholipids may be altered through peroxidation by reactive oxygen species and by phospholipases. If the membrane damage results in lysosome permeability, release of its contents precipitates further cell damage or death. Membrane proteins may be altered by cross-linking induced by free radicals.

DNA damage or loss

Damage to DNA results primarily from reactive oxygen species attack, for example following ionising radiation (p. 117). Damage may not be evident immediately; dividing cells are more susceptible. Cell populations that are constantly dividing (i.e. labile cells such as intestinal epithelium and haemopoietic cells) are soon affected by a dose of radiation sufficient to alter their DNA. Other cell populations may require a growth or metabolic stimulus before the DNA damage is revealed. Since non-lethal DNA damage may be inherited by daughter cells, a clone of transformed cells with abnormal growth characteristics may be formed; this is the process of neoplastic transformation that results in tumours (Ch. 11).

Normal erythrocytes are particularly sensitive to injury because they lack a nucleus and, therefore, the means for inducing repair mechanisms. This will also be the fate of any cell in which the nucleus is severely damaged, or when mitosis is attempted but its completion is blocked. The latter is the result of DNA strand breaks or cross-linkages; ionising radiation and some cytotoxic drugs used in cancer therapy have this effect. Damaged cells are deleted, usually by apoptosis.

Cellular appearances following injury

The agents and mechanisms mentioned above cause a variety of histological abnormalities, although very few are specific for each agent. Two patterns of sublethal cellular alteration seen fairly commonly are hydropic change and fatty change.

Hydropic change (oncosis)

In hydropic change the cytoplasm becomes pale and swollen due to accumulation of fluid. Hydropic change generally results from disturbances of metabolism such as hypoxia or chemical poisoning. These changes are reversible, although they may herald irreversible damage if the causal injury is persistent.

Fatty change (steatosis)

Vacuolation of cells is due often to the accumulation of lipid droplets as a result of a disturbance to ribosomal function and uncoupling of lipid from protein metabolism. The liver is commonly affected in this way by several causes, such as hypoxia, alcohol or diabetes. Moderate degrees of fatty change are reversible, but severe fatty change may not be.

Mechanisms of cell death

There are two distinct mechanisms by which cells die: necrosis and apoptosis. However, there are also other cellular deaths combining features of both these processes. Discussion of cell death is further complicated by a lack of uniform nomenclature; some authors use the term necrosis to denote cell death by any cellular mechanism, but more often it is used to describe a specific mechanism. Although there are usually particular triggers for one process or another, there are some situations where apoptosis follows a lower dose or shorter duration of insult while necrosis occurs above that threshold. Mechanisms of cell death involve defined metabolic pathways. Consequently, cell death processes may be amenable to therapeutic interventions.

Necrosis

image Necrosis is death of tissues following bioenergetic failure and loss of plasma membrane integrity
image Induces inflammation and repair
image Causes include ischaemia, metabolic, trauma
image Coagulative necrosis in most tissues; firm pale area, with ghost outlines on microscopy
image Colliquative necrosis is seen in the brain; the dead area is liquefied
image Caseous necrosis is seen in tuberculosis; there is pale yellow semi-solid material
image Gangrene is necrosis with putrefaction: it follows vascular occlusion or certain infections and is black
image Fibrinoid necrosis is a microscopic feature in arterioles in malignant hypertension
image Fat necrosis may follow trauma and cause a mass, or may follow pancreatitis visible as multiple white spots

Necrosis is characterised by bioenergetic failure and loss of plasma membrane integrity. The ischaemia–reperfusion model has been the focus of much resear Ch. Failure of ATP production renders plasma membrane ion pumps ineffective with resulting loss of homeostasis, influx of water, oncosis, lysis and cell death, but in many circumstances this sequence may be an oversimplification.

Anaerobic conditions result in acidosis, thus promoting calcium inflow. Calcium uptake by mitochondria eventually exceeds their storage capacity, and contributes to disruption of the inner membrane (mitochondrial permeability transition); ATP production ceases and contents leak into the cytosol. This mitochondrial sequence is particularly exacerbated, if not initiated, by reperfusion causing a burst of reactive oxygen species production.

DNA damage, for example by free radicals or alkylating agents, initiates repair sequences including activation of the nuclear enzyme poly (ADP-ribose) polymerase (PARP). In proliferating cells, as they are dependent on glycolysis, this leads to NAD depletion and thus ATP depletion and consequently necrosis.

Falling ATP levels can open plasma membrane channel-mediated calcium uptake (death channels); large rises in cytosol calcium activate calcium-dependent proteases or lead on to mitochondrial permeability transition. In contrast, free radical damage to endoplasmic reticulum allows calcium stores to leak into the cytosol; smaller rises in calcium tend to cause apoptosis rather than necrosis.

Free radical damage to lysosomal membranes releases proteases, such as cathepsins which damage other membranes and can cause cell death. By a similar mechanism, binding of tumour necrosis factor to its cell surface receptor stimulates excessive mitochondrial reactive oxygen species with the results noted above and hence necrosis.

All these pathways eventually lead to rupture of the plasma membrane and spillage of cell contents, but this is not the end of the sequence. Some of the contents released are immunostimulatory, for example heat shock proteins and purine metabolites. These provoke the inflammatory response (Ch. 10), which paves the way for repair.

Several distinct morphological types of necrosis are recognised:

coagulative
colliquative
caseous
gangrene
fibrinoid
fat necrosis.

The type of tissue and nature of the causative agent determine the type of necrosis.

Necrosis must be distinguished from apoptosis, in which cell death results from a different mechanism. The cell membrane remains intact and there is no inflammatory reaction.

Coagulative necrosis

Coagulative necrosis is the commonest form of necrosis and can occur in most organs. Following devitalisation, the cells retain their outline as their proteins coagulate and metabolic activity ceases. The gross appearance will depend partly on the cause of cell death, and in particular on any vascular alteration such as dilatation or cessation of flow. Initially, the tissue texture will be normal or firm, but later it may become soft as a result of digestion by macrophages. This can have disastrous consequences in necrosis of the myocardium following infarction, as there is a risk of ventricular rupture (Ch. 13).

Microscopic examination of an area of necrosis shows a variable appearance depending on the duration. In the first few hours, there will be no discernable abnormality. Subsequently, there will be progressive loss of nuclear staining until it ceases to be haematoxyphilic; this is accompanied by loss of cytoplasmic detail (Fig. 6.2). The collagenous stroma is more resistant to dissolution. The result is that, histologically, the tissue retains a faint outline of its structure until such time as the damaged area is removed by phagocytosis (or sloughed off a surface), and is then repaired or regenerated. The presence of necrotic tissue usually evokes an inflammatory response; this is independent of the initiating cause of the necrosis.

image

Fig. 6.2 Necrosis. Histology of part of a kidney deprived of its blood supply by an arterial embolus (Ch. 8). This is an example of coagulative necrosis. Cellular and nuclear detail has been lost. The ghost outline of a glomerulus can be seen in the centre, with remnants of tubules elsewhere.

Colliquative necrosis

Colliquative necrosis occurs in the brain because of its lack of any substantial supporting stroma; thus, necrotic neural tissue may totally liquefy. There will be a glial reaction around the periphery, and the site of necrosis will be marked eventually by a cyst.

Caseous necrosis

Tuberculosis is characterised by caseous necrosis, a pattern of necrosis in which the dead tissue is structureless. Histological examination shows an amorphous eosinophilic area stippled by haematoxyphilic nuclear debris. Although not confined to tuberculosis, nor invariably present, caseation in a biopsy should always raise the possibility of tuberculosis.

Gangrene

Gangrene is necrosis with putrefaction of the tissues, sometimes as a result of the action of certain bacteria, notably clostridia. The affected tissues appear black because of the deposition of iron sulphide from degraded haemoglobin. Thus, ischaemic necrosis of the distal part of a limb may proceed to gangrene if complicated by an appropriate infection. As clostridia are very common in the bowel, intestinal necrosis is particularly liable to proceed to gangrene; it can occur as a complication of appendicitis, or incarceration of a hernia if the blood supply is impeded. These are examples of ‘wet’ gangrene. In contrast, ‘dry’ gangrene is usually seen in the toes, as a result of gradual arterial or small vessel obstruction in atherosclerosis or diabetes mellitus, respectively. In time, a line of demarcation develops between the gangrenous and adjacent viable tissues.

In contrast to the above, primary infection with certain bacteria or combinations of bacteria may result in similar putrefactive necrosis. Gas gangrene is the result of infection by Clostridium perfringens, while synergistic gangrene follows infection by combinations of organisms, such as Bacteroides and Borrelia vincenti.

Fibrinoid necrosis

In the context of malignant hypertension (Ch. 13), arterioles are under such pressure that there is necrosis of the smooth muscle wall. This allows seepage of plasma into the media with consequent deposition of fibrin. The appearance is termed fibrinoid necrosis. With haematoxylin and eosin staining, the vessel wall is a homogeneous bright red. Fibrinoid necrosis is sometimes a misnomer because the element of necrosis is inconspicuous or absent. Nevertheless, the histological appearance is distinctive and its close resemblance to necrotic tissue perpetuates the name of this lesion.

Fat necrosis

Fat necrosis may be due to:

direct trauma to adipose tissue and extracellular liberation of fat
enzymatic lysis of fat due to release of lipases.

Following trauma to adipose tissue, the release of intracellular fat elicits a brisk inflammatory response, with polymorphs and macrophages phagocytosing the fat, proceeding eventually to fibrosis. The result may be a palpable mass, particularly at a superficial site such as the breast.

In acute pancreatitis, there is release of pancreatic lipase (Ch. 16). As a result, fat cells have their stored fat split into fatty acids, which then combine with calcium to precipitate out as white soaps. In severe cases, hypocalcaemia can ensue.

Apoptosis

image Individual cell deletion in physiological growth control and in disease
image Activated or prevented by a variety of stimuli
image Reduced apoptosis contributes to cell accumulation, e.g. neoplasia
image Increased apoptosis results in excessive cell loss, e.g. atrophy

Apoptosis is quite different from necrosis (Table 6.2); indeed, it includes suppression of necrosis. It is an energy-dependent process for deletion of unwanted individual cells. Apoptosis is involved in morphogenesis (Ch. 5), and is the mechanism for controlling organ size. Unwanted or defective cells also undergo apoptosis; thus, lymphocyte proliferation in germinal centres and the thymus is followed by apoptosis of unwanted cells. Factors controlling apoptosis include substances outside the cell and internal metabolic pathways:

Inhibitors include growth factors, cell matrix, sex steroids, some viral proteins.
Inducers include growth factor withdrawal, loss of matrix attachment, glucocorticoids, some viruses, free radicals, ionising radiation, DNA damage, Fas ligand.

Table 6.2 Comparison of cell death by apoptosis and necrosis

Feature Apoptosis Necrosis
Induction May be induced by physiological or pathological stimuli Invariably due to pathological injury
Extent Single cells Cell groups
Biochemical events Energy-dependent fragmentation of DNA by endogenous endonucleases Energy failure
Impairment or cessation of ion homeostasis
Lysosomes intact Lysosomes leak lytic enzymes
Cell membrane integrity Maintained Lost
Morphology Cell shrinkage and fragmentation to form apoptotic bodies with dense chromatin Cell swelling and lysis
Inflammatory response None Usual
Fate of dead cells Ingested (phagocytosed) by neighbouring cells Ingested (phagocytosed) by neutrophil polymorphs and macrophages
Outcome Cell elimination Defence, and preparation for repair

Exposure to inducers or withdrawal of inhibitors acts via the bcl-2 protein family, which then inhibit or activate the death pathway, resulting in activation of initiator and executioner caspases. Alternatively, activation of the plasma membrane receptor Fas (CD95) by its ligand bypasses bcl-2 to activate other caspases (Fig. 6.3). These result in degradation of the cytoskeletal framework, fragmentation of DNA and loss of mitochondrial function. The nucleus shrinks (pyknosis) and fragments (karyorrhexis). The cell shrinks, retaining an intact plasma membrane (Fig. 6.4), but alteration of this membrane rapidly induces phagocytosis. Dead cells not phagocytosed fragment into smaller membrane-bound apoptotic bodies. There is no inflammatory reaction to apoptotic cells, probably because the cell membrane is intact. Various diseases are associated with reduced or increased apoptosis.

image

Fig. 6.3 Mechanisms of apoptosis. Apoptosis results from activation of caspases triggered either by the bcl-2 family or by the binding of Fas ligand to its receptor.

image

Fig. 6.4 Apoptosis. Histology of skin from a case of graft-versus-host disease (Ch. 9) in which there is individual cell death (arrowed) in the epidermis as a result of immune injury.

Reduced apoptosis

The product of the p53 gene checks the integrity of the genome before mitosis; defective cells are switched to apoptosis instead. In contrast, bcl-2 protein inhibits apoptosis. Therefore, loss of p53 function or excess bcl-2 expression may result in failure of initiation of apoptosis with resulting cell accumulation; both these defects are important in neoplasia (Ch. 11). Autoimmune disease (Ch. 9) might reflect failure of induction of apoptosis in lymphoid cells directed against host antigens; in systemic lupus erythematosus, alterations are reported in the Fas lymphocyte receptor. Some viruses enhance their survival by inhibiting apoptosis of cells they infect, and latent infection by Epstein–Barr virus upregulates bcl-2.

Increased apoptosis

Diseases in which increased apoptosis is probably important include acquired immune deficiency syndrome (AIDS), neurodegenerative disorders and anaemia of chronic disorders (Ch. 23). In AIDS, human immunodeficiency virus proteins may activate CD4 on uninfected T-helper lymphocytes, inducing apoptosis with resulting immunodepletion. Apoptosis is the usual mode of cell death in exposure to ionising radiation (p. 117).

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