Introduction

Apoptosis is an energy-dependent, biochemically-mediated process of programmed cell death. Apoptosis is essential for many processes, including the elimination of infected or transformed cells, a properly functioning immune system, organismal development, and maintaining homeostasis and normal cell turnover in the body. The two main branches of apoptotic pathways are the intrinsic and extrinsic pathways of apoptosis, in which the signals initiating cell death originate from within or outside the cell, respectively.[1] 

The intrinsic pathway initiates from intracellular sensors that detect DNA damage, the presence of viral pathogens, or in response to the lack of survival signals provided externally from other cells. In contrast, the extrinsic pathway of apoptosis initiates with a pro-death signal originating from outside the cell, most often by Natural Killer (NK) lymphocytes or CD8-positive Cytotoxic T lymphocytes (CTLs).

Fundamentals

Apoptosis represents an ATP-requiring pathway inducing programmed-cell death and is a requirement for viability. The Greek term apoptosis was initially attributed to this pathway to describe the “falling off of petals from flowers, or leaves from trees.”[2]

Hallmarks of apoptosis include degradation of DNA, disassembly of the cytoskeleton and nuclear lamina, and cellular blebbing. Importantly, the apoptotic pathway is anti-inflammatory and does not induce cell lysis, in contrast to cell death by necrosis, which lysis cellular contents that subsequently leads to inflammation.[1]

Cellular Level

The primary cells that induce the extrinsic pathway of apoptosis are NK and CTL lymphocytes. NK cells are lymphocytes, yet are part of the innate immune system, whereas CTLs are T lymphocytes of the adaptive immune system, and as such, undergo clonal selection in response to an antigen and can form memory lymphocytes.[3] Both NK cells and CTLs are designed to eradicate either infected or altered cells that are potentially tumorigenic.

NK cells mature in the bone marrow, and in response to viral infections or transformed cells, NK cells provide a first line of defense by inducing apoptosis in human cells that have altered cell surface properties. CTLs mature and emerge from the thymus as naïve CD8-positive T lymphocytes, which subsequently search for antigen presented by dendritic cells in secondary lymphoid tissues such as lymph nodes. Upon specific recognition of an antigen, CTLs undergo clonal expansion, producing thousands of CTL effector lymphocytes that can return to the tissue where the infected cells reside. This process takes approximately a week upon first exposure to the antigen, and the NK cells thus have an essential role in eradicating potentially dangerous cells while the CTL adaptive response is forming.

Molecular Level

NK and CTL lymphocytes both scan somatic cells within the body for altered and potentially dangerous cells, yet use different mechanisms by which they recognize dangerous cells that should be eliminated by the extrinsic pathway of apoptosis. CTLs create their antigen receptors through somatic recombination in the thymus. CTL effector lymphocytes that have been stimulated by antigen presentation and clonally expanded migrate throughout the body in search of the foreign or altered peptide, which it can specifically recognize. CD8-positive CTLs only recognize linear peptides presented by MHC class I molecules, using CD8 as a co-receptor.

Cytosolic antigens of all nucleated cells are presented by the MHC class I pathway. This pathway involves cleaving a portion of all cytosolic proteins into small peptides approximately ten amino acids in length by the proteasome. These peptides are then loaded onto MHC class I molecules in the lumen of the rough endoplasmic reticulum, MHC class I molecules that bind tightly to a peptide are then permitted to translocate to the plasma membrane. A human nucleated cell thus expresses MHC class I molecules presenting peptides representing all of the cytosolic proteins the cell is making. If a cell becomes infected by a virus, or transformed and expressing abnormal human proteins, these altered-self peptides will also be presented on the surface of nucleated cells. A CTL effector lymphocyte that recognizes a peptide presented by MHC class I for which it is specific will initiate the extrinsic pathway of apoptosis. Thus CTLs can recognize cells for the destruction that exhibit altered-self properties.

NK cells recognize altered cells to kill by the extrinsic pathway by a different mechanism, and since NK cells are part of the innate immune system, they use germline-encoded receptors, rather than created by somatic recombination. NK cells express a variety of receptors that are either activating, and induce apoptosis, or are inhibitory, and prevent the induction of the extrinsic pathway on a target cell.[4] Cells that are stressed express stress ligands on the surface of the cell, such as MICA and MICB. NK cells have receptors such as NKG2D that are capable of recognizing these stress ligands. NK cells possess additional receptors capable of recognizing stress ligands on host cells that are referred to as KIRs, as they kill cells, and also are members of the immunoglobulin (Ig) superfamily, as their receptors contain the same Ig motifs found in antibodies and T cell receptors.

In addition to containing many receptors that recognize altered host cells directly, NK cells also express CD16, which is a low-affinity receptor for the Fc portion of the IgG isotypes IgG1 and IgG3. The presence of CD16 (also known as FcγRIII) permits NK cells with an additional mechanism for recognizing and killing altered cells, as IgG antibodies can bind to viral or tumor antigens on the surface of a host cell. Recognition of these bound antigens by CD16 on NK cells triggers NK cells to kill the target cell through the extrinsic pathway, a process known as antibody-dependent cell-mediated cytotoxicity (ADCC).

The inhibitory receptors predominantly recognize MHC class I molecules, HLA-A, HLA-B, and HLA-C, which are expressed by nucleated host cells. Absence of MHC class I expression will tip the balance in favor of activating receptors if they have recognized a stress ligand, and therefore NK cells will be more likely to kill a cell that exhibits an absence of self with regards to MHC class I expression. Many virally infected or cancerous cells downregulate MHC class I as a strategy to inhibit the presentation of non-self antigens at the cell surface and therefore avoid being killed by CTLs. Therefore, NK cells complement CTLs very well, as CTLs are designed to kill altered-self cells as recognized by MHC class I. If a virally infected or transformed cell attempts to avoid killing by a CTL by downregulating MHC class I expression, it will be more susceptible to being killed by an NK cell.

Unfortunately, this complementary relationship is not always perfect as viruses and cancer cells have evolved additional mechanisms to avoid being killed by the extrinsic pathway of apoptosis. For example, cytomegalovirus both blocks expression of MHC class I, yet also expresses viral proteins designed to mimic the presence of MHC class I. Several viruses have adapted to reduce expression of the stress ligand NKG2D. Intracellular viruses can also avoid detection from CTLs by establishing a latent phase, in which few viral proteins are produced. Therefore less viral antigens will be presented on MHC class I. Cancer cells or persistent viral infections can also induce T cell exhaustion, in which CTLs lose the ability to kill altered self-cells in response to continual exposure to a foreign antigen.[5] Finally, antigenic variation is a mechanism that can help rapidly mutating cancer cells, and viruses evade detection by antigen-specific CTLs.

Function

As discussed in the previous section, the extrinsic pathway of apoptosis is critical for the eradication of infected or potentially cancerous cells. Concerning virally infected cells, the extrinsic apoptotic pathway is essential for eradicating the reservoir of viral infections. Although antibodies are effective at neutralizing extracellular viruses and preventing an inhaled virus from initiating an infection, they are not as effective at eliminating intracellular viruses that have already established infection. The extrinsic apoptotic pathway is thus crucial for eradicating cells that are harboring an intracellular infection or are transformed and potentially malignant. Furthermore, by inducing apoptosis in virally infected cells, the dying cell does not lyse and release its contents into surrounding tissues, including intact infective virions that would be capable of infecting neighboring cells.

Both the extrinsic and intrinsic pathways of apoptosis cooperate to maintain T cell homeostasis. Antigen-specific lymphocytes are rapidly clonally expanded in response to the presence of a foreign antigen. Yet, after clearing an infection, the majority of clonally expanded lymphocytes die by apoptosis, leaving a small percentage of cells that function as memory or effector lymphocytes. Both death receptors of the extrinsic pathway and mitochondrial proteins of the intrinsic pathway contribute to the contraction phase of lymphocytes after successful clearance of a pathogen, thus preventing additional host damage induced by cytokines, and also permitting more energy expenditure if a subsequent pathogen is encountered.[6] Similarly, damaging inflammation in response to infection must subside after clearance of a pathogen to achieve homeostasis, and both the extrinsic and intrinsic pathways induce apoptosis in neutrophils after clearance of a pathogen.[7]

The elimination of self-reactive thymocytes in the thymus is essential to achieve central tolerance and prevent autoimmune disease. Developing thymocytes that bind too strongly to MHC molecules presenting self-antigens in the thymus are eliminated in a process called negative selection. Negative selection requires the actions of caspases to initiate apoptosis in self-reactive thymocytes.[8] 

Negative selection is an imperfect process, and some self-reactive T lymphocytes will escape to the periphery. Peripheral tolerance occurs via multiple mechanisms, including the induction of apoptosis in self-reactive T cells in a process termed activation-induced cell death (AICD).[9] Repeated stimulation of self-reactive T cells in the periphery results in the upregulation of the death receptor Fas, therefore making the auto-reactive T cell more susceptible to apoptosis by cells expressing FasL. Although the discussion has focused on apoptosis of T cells, similar processes occur to eliminate developing self-reactive B cells in the bone marrow as well as mature B cells in the periphery.

Immunological privilege is a physiological mechanism of self-tolerance functioning to exclude certain peripheral tissues such as the eye, brain, and testes from the damaging effects of the immune system. However, physical barriers protecting these sites can be compromised, leading to exposure of the sites to lymphocytes. Yet the constitutive expression of FasL on privileged tissues represents a backup mechanism to protect privileged tissues from lymphocyte destruction, as the FasL initiates the extrinsic pathway of apoptosis in infiltrating lymphocytes expressing the Fas receptor.[10]

Mechanism

The canonical pathway of the extrinsic pathway of apoptosis begins with the binding of members of the tumor necrosis factor receptor superfamily (TNFRSF) to cognate trimeric ligands of the tumor necrosis factor superfamily (TNFSF), which can be either soluble or expressed on the surface of another cell such as a CTL. TNFRSF members oligomerize in response to trimeric ligand binding, although some TNFRSF members exist as oligomers in the absence of ligand. TNFRSF members that induce apoptosis in response to ligand binding contain a “death domain” that is approximately 80 amino acids long. The death domain serves as a docking site for other pro-apoptotic proteins such as Fadd, forming a membrane-bound death-inducing signaling complex (DISC), which ultimately leads to the activation of caspases. Caspases are cysteine-rich proteases that naturally exist as zymogens, and when activated, possess proteolytic activity that functions to cleave other intracellular proteins, ultimately achieving the orderly disassembly of the cell.[11]

Caspase-8, an initiator caspase, is recruited to the DISC complex and is subsequently activated upon binding to the DISC. Activated caspase-8 cleaves several substrates, including caspase-3, resulting in activation of the executioner caspase. Activated caspase-3 cleaves actin, nuclear laminins, and the inhibitor of a DNase, thus promoting DNA degradation. The cleavage of actin disrupts cell division and cell migration, while cleavage of DNA and nuclear laminins prevents nuclear function and expression of survival genes. There are many variations of the canonical pathway; for example, in response to binding by TNF-a, the receptor TNFR1 can activate either apoptosis via the canonical pathway described, or can activate the pro-survival transcription factor NF-kB, which acts as a master regulator of inflammatory cytokine production.[12]

The most relevant TNFSF members expressed by NK cells and CTLs express are Fas ligand and TRAIL. Fas ligand expression is fairly exclusive to NK cells and CTLs, and it initiates apoptosis by binding the TNFRSF member Fas (CD95), which can express in a variety of tissues. In addition to inducing apoptosis through TNFSF ligands, NK cells and CTLs can also induce apoptosis in altered host cells by releasing granzymes and perforin onto the target cell. These cytotoxic substances are synthesized and stored in granules in activated CTLs and NK cells, and recognition of altered cells promotes exocytosis of the granules, releasing the contents onto the target cell.

Perforin functions to promote uptake of the cytotoxic substances into the target cell. Granzyme B is a protease; once released inside the target cell, it promotes apoptosis of the target cell by activating caspases and other proteins. It is important to note that the extrinsic and intrinsic pathways are not distinct, and both granzymes and caspases activated by TNFRSF members can cleave the protein Bid, which subsequently initiates apoptosis by the intrinsic, mitochondrial pathway.

Regardless of how an apoptotic pathway becomes initiated, there are also profound changes induced in the plasma membrane. Phosphatidylserine is normally restricted to the inner leaflet of the plasma membrane. Upon initiation of apoptosis, phospholipid scramblase activity results in phosphatidylserine exposure on the outer leaflet of the cell, serving as a marker for engulfment by phagocytes. Mutations that interfere with the engulfment of apoptotic cells have been implicated in a variety of autoimmune diseases such as lupus (SLE).[13]

Testing

Defective Fas-mediated apoptosis in an in vitro assay and an increased number of double-negative T cells in the peripheral blood are indicative of autoimmune lymphoproliferative syndrome (ALPS), a disease described below.

Pathophysiology

A heterozygous mutation in the gene for Fas, also known as CD95, is the most prominent cause of the condition known as an autoimmune lymphoproliferative syndrome (ALPS). Since the Fas receptor functions as a trimer, heterozygotes suffer from the disease, as one defective copy of the protein will destroy the ability of the Fas receptor to function. Additional mutations that cause ALPS have been identified in FasL and caspase-8, among other proteins. The inability of ALPS patients to clear a large number of clonally expanded lymphocytes after clearance of an infection results in lymphadenopathy and splenomegaly that appear early in childhood.[14]

ALPS patients most often also develop autoimmune disorders and are at increased risk for developing lymphomas, since the extrinsic pathway of apoptosis contributes to the elimination of self-reactive lymphocytes, as well as malignant lymphocytes. Patients may also present with cytopenias, hypergammaglobulinemia, and frequent infections. Management includes corticosteroids and IVIG for first-line therapy with rituximab also used for managing autoimmune symptoms.

Clinical Significance

Increasing the killing capabilities of the extrinsic pathway of apoptosis is desired in treating cancer, and a variety of anticancer drugs increase Fas receptor expression in some tumor cell lines.[15] The TNFRSF member TRAIL has shown some promise in being able to selectively kill cancer cells through binding to multiple receptors (DR4 and DR5). Yet, unfortunately, cancer cells have demonstrated the ability to obtain resistance to death by TRAIL.[16] Conversely, the extrinsic pathway of apoptosis is detrimental in other situations and can be the cause of disease. Extrinsic apoptosis mediated by CTLs has been postulated to cause the damaging effects of liver destruction in chronic viral hepatitis, and CTLs appear to be the cause of Type I diabetes, as the belief is the extrinsic apoptotic pathway initiated by CTLs eliminates the pancreatic beta cells in type I diabetes.[17] Finally, both NK cells and CTLs both contribute to graft-versus-host disease, a significant barrier to the success of bone marrow transplantation.