Programmed Cell Death
Classical example of internally programmed cell death is apoptosis discussed in detail below.
However, cell death having combined features of necrosis and apoptosis (necroptosis), and a few other variant forms are also briefly discussed. Besides, an account of autophagy is given below, a process which has come to be regarded as such an important form of programmed cell death in certain diseases that it is called type II programmed cell death, apoptosis being type I.
Read And Learn More: General Pathology Notes
Apoptosis:
Apoptosis is a form of ‘coordinated and genetically programmed cell suicide’ in which unwanted host cells are eliminated by activation of intrinsic enzymes in a variety of physiologic and pathologic conditions (apoptosis = falling off or dropping off, as that of leaves or petals).
The term was first introduced and described in 1972 distinct from necrosis by being controlled and regulated cell death, and by not accompanied by any inflammation and collateral tissue damage.
Apoptosis In Biologic Processes:
Apoptosis plays a role invarious
- Physiologic and
- Pathologic processes.
1. Physiologic Conditions:
- Maintenance of cell population by physiologic elimination of unwanted cells is seen in following
examples: - Organised cell destruction in sculpting of tissues and organs during development of embryo.
- Physiologic involution of cells in hormone-dependent tissues e.g. endometrial shedding, ovarian
- follicles near menopause, regression of lactating breast after withdrawal of breastfeeding.
- Normal cell destruction followed by replacement proliferation such as in intestinal epithelium.
- Loss of immature B and T cells in the bone marrow and thymus by clonal deletion and cytotoxicity due to failure of these lymphocytes to express antigen receptor.
- Elimination of blood cells after they have performed their role during their lifespan for example Neutrophils in acute inflammation, lymphocytes in immune reaction etc.
2. Pathologic Conditions:
Host cells are eliminated by apoptopsis in the following examples since they are damaged beyond repair and have not elicited host reaction such as inflammation or tissue destruction.
- Damage to host cell DNA for example, In therapy for cancer by irradiation or by chemotherapy, mild thermal injury or hypoxia.
- Endoplasmic stress due to accumulation of large excess of misfolded proteins formed by free radical injury or from mutations for example In degenerative diseases of CNS for example, Alzheimer’s disease
- Parkinson’s disease, chronic infective dementias.
- Certain viral infections for example,Councilman bodies in viral hepatitis, progressive depletion of CD4+T cells in HIV infection.
- Cell death by cytotoxic T cells in transplant rejection reaction.
- Pathologic atrophy of organs and tissues on withdrawal of stimuli for example,Prostatic atrophy after orchiectomy, atrophy of kidney or salivary gland on obstruction of ureter or ducts, respectively.
Disorders With Abnormal Apoptosis:
Pathogenesis of a few diseases has been explained on the basis of speed of apoptosis resulting in either increased cell survival or exaggerated cell death:
Conditions with defective apoptosis and increased cell survival:
- These are such disorders in which apoptosis is too little and favours survival of abnormal cells for example,
- While TP53 is called the protector of human genome, mutation of TP53 is most common in various human cancers which lowers rate of apoptosis and thus mutated forms of cells fail to get eliminated due to increased cell survival.
- Mechanism of some autoimmune diseases is explained due to accumulation of defective lymphocytes due to reduced apoptosis of lymphocytes which react against ‘self-antigens’.
Conditions with exaggerated apoptosis and higher cell death:
In these conditions, there is increased speed of cell death due to apoptosis, for example:
- In acute myocardial infarction in which there is 20% necrosis and 80% apoptosis of infarcted myocardium (page 454).
- In various neurodegenerative diseases (for example, Alzheimer’s disease Parkinson’s disease), misfolded proteins and mutations cause increased apoptosis.
Morphologic Features of Abnormal Apoptosis:
The characteristic morphologic changes in apoptosis by light microscopy and electron microscopy are as under:
- There is involvement of single cells or small clusters of cells in the background of viable cells.
- Apoptotic cells are round to oval shrunken masses of intensely eosinophilic cytoplasm (mummified cell) containing shrunken or almost-normal organelles.
- Nuclear chromatin is condensed under the nuclear membrane i.e. pyknosis.
- The cell membrane may show blebs or projections on the surface.
- Apoptotic bodies are cytoplasmic membrane-bound spherical structures containing condensed cell organelles but generally no nucleus.
- Characteristically, unlike necrosis, there is no acute inflammatory reaction around
apoptosis. - Phagocytosis of apoptotic bodies by macrophages takes place at varying speed.
- Accordingly, there may be loosely floating apoptotic single cells after they have lost contact with each other and basement membrane, or there may be major cell loss in the tissue without significant change in the overall tissue structure.
Techniques to identify and count apoptotic cells Apoptotic cells can be identified and counted by following methods:
- Staining of condensed chromatin by haematoxylin, Feulgen stain.
- DNA changes detected by in situ techniques or by gel electrophoresis.
- Immunohistochemical stain with active caspase-3 antibody.
- Immunofluorescence stain with antibody specific for active caspase-3.
- Fluorescent staining with annexin V to locate phosphatidylserine on the outer surface of the cell membrane in apoptosis.
- Fluorochrome-based TUNEL (terminal deoxynuceotidyl transferase dUTP end-labelling) assay by flow cytometry for detecting DNA breakages.
Biochemical Changes:
Sequential biochemical processes underlying the morphologic changes are as under:
- Protein cleavage by proteolytic enzymes Breakage of nuclear proteins and cleavage of cytoskeletal proteins.
- Protein-protein cross-linkages Activated transglutaminases form cross-linkages with cytoplasmic proteins forming shrunken apoptotic bodies.
- DNA condensation and fragmentation Following initial pyknosis of nucleus, activated endonucleases cause fragmentation of chromatin forming oligonucleosomes.
- Recognition and removal of dead cells by phagocytosis Surface receptors on macrophages recognise apoptotic cells due to their expression of phosphatidylserine and thrombospondin on the outer surface of cell membrane from their normal location on the inner layer.
- Features distinguishing apoptosis from necrosis are illustrated and summarised.
Molecualr Mechanisms Of Apoptosis:
Several physiologic and pathologic processes activate apoptosis in a variety of ways. However, in general the following molecular events sum up the sequence involved in apoptosis .
1. Initiation of apoptosis:
All cells have inbuilt effector mechanisms for cell survival and signals of cell death; it is the loss of this balance that determines survival or death of a cell.
Accordingly, a cell may be initiated to programmed cell death by extracellular signals as follows:
- Withdrawal of normal cell survival signals for example, Absence of certain hormones, growth factors, cytokines.
- Agents of cell injury for example, Heat, Radiation, Hypoxia, Toxins, Free radicals.
2. Initiation of death-signalling pathways:
After the cell has been initiated into self-destruct mode, cell death signalling mechanisms are initiated by activation of caspases by one of the two pathways—intrinsic (mitochondrial) and extrinsic (cell death receptor initiated), discussed below.
The term ‘caspase’ is derived from: cb for cystein protease; asp for aspartic acid; and ase is used for naming an enzyme.
Caspases are like other proteases and are normally in inactive form; their activation by either of the following pathways causes a series of proteolytic or protein-splitting actions on nuclear proteins and organelles containing protein components.
Intrinsic (mitochondrial) pathway:
This pathway of cell death signalling is due to increased mitochondrial permeability and is a major mechanism. A mitochondrial protein, cytochrome c, is lifeline of intact mitochondria.
But release of cytochrome c protein from mitochondria into the cytoplasm of the cell initiates the cell into apoptosis. The major mechanism of regulation of this mitochondrial protein is by anti- and pro-apoptotic members of BCL family of oncogenes (the name BCL stands for on B-cell lymphoma; BCL-2 oncogene was first detected this lymphoma).
While anti- and pro-apoptotic BCL members contain four BCL2 homology (BH4) domains; a third group of regulatory proteins of BCL family contain fewer than four BH domains:
Antiapoptotic BCL family (BH4 domains) BCL2, BCL-XL, MCL1 are the main members of this group. These proteins lie on the outer surface of mitochondria, endoplasmic reticulum and cell membrane and do not permit the leakage of mitochondrial protein to the cytosol.
Contrasting features of necrosis and apoptosis:
Proapoptotic BCL family (BH4 domains) BAX and BAK are the principal members of this group. Activated forms of BAX and BAK proteins promote mitochondrial leakage of cytochrome c protein into the cytosol.
BCL family with BH3-only domains This group includes BAD, BIM, BID, Puma, Noxa. These BCL family members are sensor proteins of cellular stress and consequent damage. They maintain a balance for initiation of anti- or proapoptotic mitochondrial pathway of caspase activation in the cell.
Extrinsic (cell death receptor-initiated) pathway:
This signalling pathway of cell death is by activation of death receptors on the cell membrane. An important cell death receptor is type 1 tumour necrosis factor receptor (TNFR1), and a related transmembrane protein called Fas (CD95) and its ligand (FasL).
FasL is expressed on cytotoxic (CD 8+) T cells having role in killing of virus-infected cells and tumour cells. Binding of Fas and FasL activates
Fas-associated death domain (FADD) in the cytoplasm of the cell that activates cascade of caspases.
3. Activation of initiator caspases:
The final culmination of either of the above two pathways is activation of caspases:
- In the mitochondrial pathway,
- Mitochondrial protein cytochrome c liberated into cytosol binds to another protein, apoptosisactivating factor (APAF-1) and forms a complex called apoptosome.
- This complex binds to precursor caspases, in particular caspase-9, and cleaves them to form their activated proteins.
- This process is further internally regulated by certain physiologic inhibitors of apoptosis in the cytoplasm which block activation of caspases.
- In the death receptor-initiated pathway:
- FADD formed after receptor-ligand interactions (TNF-TNFR1 and Fas-Fas-L) binds to inactive caspases-10 and cleaves it to form its active form, caspase-8.
- The activation process of caspase-10 to active form caspase 8 in this pathway is inhibited by a protein called FLIP (FLICE-inhibitory proteins) (FLICE is FADD-like interleukin-1β converting enzyme).
4. Activation of apoptosis-executing caspases:
Although different protein molecules are involved in initial steps in mitochondrial (active caspase-9) and death receptor-mediated (caspase-8 and 10) pathways, subsequent events in completion of apoptosis are similar:
- Both mechanisms lead to activation of cascade of caspases, eventually converging to active form of caspase-3 and caspase-6 in both pathways in executing apoptosis
- These executioner caspases (3 and 6) act on various components of the cell such as DNAase and nuclear matrix proteins and lead to proteolytic actions on nucleus, chromatin clumping, cytoskeletal damage, disruption of endoplasmic reticulum, mitochondrial damage, and disturbed cell membrane.
5. Removal of dead cells:
Dead apoptotic cells express certain protein molecules on their surface which facilitate their recognition and engulfment by macrophages:
Phosphatidylserine molecule which is normally present on the inside of the cell membrane, is expressed on the outer surface of the cell membrane in cells undergoing apoptosis, which facilitate identification of apoptotic bodies by the receptors present on macrophages and promotes phagocytosis.
A few apoptotic bodies are coated with thrombospondin, an adhesive glycoprotein molecule, which also helps in recognition of apoptotic cells by macrophage receptors.
Some apoptotic bodies are coated with natural antibodies and components of complement system (for example, C1q) which are recognised by receptors on macrophages.
Following apoptosis, phagocytosis is rather brisk and is unaccompanied by any inflammatory cells.
Variants Of Programmed Cell Death:
Here, a few conditions with combined mechanism of cell death (necrosis plus apoptosis), or nonclassical forms of apoptosis (for example,Pyroptosis, ferroptosis) are briefly discussed.
Necroptosis Of Programmed Cell Death:
As the name indicates, necroptosis is a cross between necrosis and apoptosis; in other words it is a form of programmed necrosis. In usual forms of necrosis, there is leakage of cell’s contents into extracellular compartment after cell death, while in necroptosis the process of leaking of the membrane is internally regulated or programmed.
Necroptosis is biochemically and morphologically similar to necrosis. However, at molecularlevel, necroptosis has some similarity with apoptosis in being triggered by ligation of TNFR1, Fas, and by proteins of viral DNA and RNA but differs from apoptosis in not involving caspase
activation.
The examples of necroptosis are infollowing conditions:
- Physiologic conditions such as in the formation of bony growth plate
- Viral defense mechanism Steatohepatitis
- Inflammatory diseases for example,Crohn’s disease, pancreatitis.
Pyroptosis Of Programmed Cell Death:
Pyroptosis is a form of apoptosis in which there is release of fever-producing cytokine interleukin-1 (pyro = fever). Pyroptosis is triggered by cytoplasmic entry of microbial products which activates inflammasomes.
Inflammasomes are multi-protein cytosolic protein formed in cell death that regulate the activation of caspases and induce inflammation by expression of proinflammatory cytokines when exposed to microbes.
In pyroptosis, cell death is brought about by activation of caspase-1 (also called interleukin-1β converting enzyme or ICE) and caspase-11 which also generates interleukin-1.
Pyroptosis differs from classic apoptosis in having:
- Cellular swelling (instead of cell shrinkage)
- Damage to plasma membrane, and by release of fever-producing cytokine interlukin-1.
Ferroptosis Of Programmed Cell Death:
Ferroptosis is cell death triggered by iron-dependent (i.e. by Fenton reaction) accumulation of reactive oxygen species in the cell. Cell death in these cases can be suppressed by iron-chelating agent, desferrioxamine.
Autophagy:
Autophagy (auto = self, phagy = eating) is a form of catabolic mechanism by which the cell degrades its own dysfunctional and worn out components by way of cannabilism.
These include: outlived proteins, malformed proteins synthesised by lysosomes, and worn out organelles. Autophagic cell death is also called as type II programmed cell death.
Mechanism of Autopagy:
Autophagy involves formation of double-membrane vesicle that encloses cytoplasm and constituents of the cells to be eliminated (long-lived proteins, malformed proteins and damaged organelles), followed by delivery of this vesicle along with its contents to lysosome forming autophagosome.
Molecular mechanism for formation of autophagosome in response to stress is complex and briefly involves following steps:
- Initiation: It begins with nucleation complex of proteins that starts to form membrane of the vesicle. This requires several autophagy-related genes at least 16 Atgs are involved in its formation and regulation.
- Elongation: The vesicle membrane is elongated and closed to enclose the contents. This step involves certain ubiquitin-like conjugation pathways that includes microtubule-associated protein light chain 3 (LC3) and Golgi-associated ATPase-enhancer (GATE-16).
- Maturation: Newly formed vesicle fuses with endosome and then finally with lysosome to form autophagolysosome.
- Degradation: The material enclosed in the autophagosome is degraded by the lysosome, recycled to generate energy and used for building blocks.
Types Of Autophagy:
Depending upon the mechanism by which the intracellular material is delivered to the lysosome for degradation, autophagy is of three types:
- Microautophagy In this, the cytoplasm along with degradable material is sequestered through direct invagination to the lysosomal membrane.
- Chaperone – mediated autophagy Here, the cellular proteins attached with a chaperone pentapeptide protein, KFERQ, are degraded selectively through direct translocation to lysosomal membrane.
- Macroautophagy This is the form which is generally referred to as autophagy. It involves formation of subcellular double-membrane vesicle (autophagosome) that sequesters a portion of cytosol that transports degradable content of the cytoplasm and delivers them to the lysosome for breakdown by lysosomal enzymes.
Significance:
Autophagy is a physiological process but dysregulated autophagy has come to occupy an important role in many diseases. Currently, autophagy is considered important in following conditions:
Physiological role It performs following roles:
- Stress It is an adaptive response and is exaggerated during stress (for example, Oxidative stress, nutrient deficiency, ageing) by allowing the cell to cannibalise its own constituents for prolonging cell survival.
- Ageing There is interrelationship of autophagy pathways with factors affecting ageing.
Role in pathological conditions :
Autophagy has been found involved in mechanisms of following diseases:
- Cancer Autophagy has a role in limiting cancer cell growth, reducing mutagenesis by eliminating damaged mitochondria and by killing developing tumour cells.
- Myopathies Autophagy is physiologically increased in skeletal muscle and neurons.
- A few examples of myopathies are called autophagic vacuolar myopathies (AVMs) due to presence of autophagic vacuoles in the cytoplasm and they have primary defect in lysosomal protein.
- These include Danon disease (from deficiency of lysosome-associated membrane protein-2 or LAMP-2
- And Pompe disease (glycogen storage disease type II from acid maltase deficiency).
- Neuromuscular disorders Some of these conditions have accumulation of inclusion bodies and vacuoles in the cell for example,Inclusion body myositis.
- Neurodegenerative disorders A few examples of this group are due to dysregulated autophagy for example,Accelerated autophagy in Alzheimer’s disease.
- Infectious diseases Many microbial agents are degraded by autophagy for example,Mycobacteria, HSV-1, Shigella.
- Inflammatory bowel disease Studies have shown autophagy-related genes (Atgs) are linked to pathogenesis of ulcerative colitis and Crohn’s disease.
Programmed Cell Death:
- Apoptosis is a classical example of coordinated and genetically programmed cell suicide.
- Apoptosis has significance in a variety of physiologic (for example,Endometrial shedding) and
pathologic conditions (for example,for example,iral infections). - Abnormality in apoptosis may cause too little apoptosis (favouring cell survival) (for example,In TP53 mutation in cancers) or too much apoptosis (exaggerated cell death) (for example,In neurodegenerative diseases).
- Morphologically, apoptotic cells appear as round to oval shrunken masses of intensely eosinophilic cytoplasm (mummified cell) containing pyknotic nucleus; it is not accompanied by any inflammation.
- Special techniques can be employed to identify apoptotic cells by antibody specific for caspase-3.
- Biochemical processes underlying apoptosis are protein cleavage, protein cross-linkages and DNA damage.
- Pathogenetically, apoptosis is initiated by loss of signals of normal cell survival and by action of agents injurious to the cell.
- Molecular mechanism of apoptosis is under genetic control which may be by intrinsic (mitochondrial) pathway (pro- and anti-apoptotic members of BCL-2 family), extrinsic (cell death receptor initiated) pathway (TNFR1, Fas, Fas-L) and finally by activated caspases.
- This is followed by phagocytosis of apoptotic bodies. Necroptosis is a form of programmed necrosis having biochemical and morphologic similarity with necrosis but like apoptosis it is triggered by ligand proteins.
- Other atypical forms of apoptosis are pyroptosis (associated with fever-producing IL1) and ferroptosis (Fenton reaction-dependent).
- Autophagy is a lysosome-mediated mechanism of self-eating of worn out organelles and malformed proteins in the cell.
- Autophagy is involved in response to stress, ageing and in many pathologic conditions for example ,Cancer, neurodegenerative diseases etc.
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