Cell death is an important feature of development in all multicellular organisms. During fetal life it is used to mold and sculpt, removing unnecessary cells to provide shape and form. It also is an important feature of lymphocyte homeostasis, returning T- and B-cell populations to their appropriate levels after bursts of antigen-induced proliferation. Apoptosis also plays a crucial role in the deletion of potentially autoreactive thymocytes during negative selection and in the removal of developing T cells unable to recognize self (failure to undergo positive selection).
Although the induction of apoptosis involves different signals depending on the cell types involved, the actual death of the cell is a highly conserved process amongst vertebrates and invertebrates. For example, T cells may be induced to die by many different signals, including the withdrawal of growth factor, treatment with glucocorticoids, or TCR signaling. Each of these signals engages unique signaling pathways, but in all cases, the actual execution of the cell involves the activation of a specialized set of proteases known as cuspases. The role of these proteases was first revealed by studies of developmentally programmed cell deaths in the nematode
Active apoptotic effectors
Active apoptotic effectors
Two pathways to apoptosis in T cells. (a) Activated peripheral T cells are induced to express high levels of Fas and FasL. FasL induces the trimerization of Fas on a neighboring cell. FasL can also engage Fas on the same cell, resulting in a self-induced death signal. Trimerization of Fas leads to the recruitment of FADD, which leads in turn to the cleavage of associated molecules of procaspase 8 to form active caspase 8. Caspase 8 cleaves procaspase 3, producing active caspase 3, which results in the death of the cell. Caspase 8 can also cleave Bid to a truncated form that can activate the mitochondrial death pathway. (b) Other signals, such as the engagement of the TCR by peptide-MHC complexes on an APC, result in the activation of the mitochondrial death pathway. A key feature of this pathway is the release of AIF (apoptosis inducing factor) and cytochrome c from the inner mitochondrial membrane into the cytosol. Cytochrome c interacts with Apaf-1 and subsequently with procaspase 9 to form the active apoptosome. The apoptosome initiates the cleavage of procaspase 3, producing active caspase 3, which initiates the execution phase of apoptosis by proteolysis of substances whose cleavage commits the cell to apoptosis. [Adapted in part from S. H. Kaufmann and M. O. Hengartner, 2001. Trends Cell Biol. 11:526.]
C. elegans, where the death of cells was shown to be totally dependent upon the activity of a gene that encoded a cysteine protease with specificity for aspartic acid residues. We now know that in mammals there are at least 14 cysteine proteases or caspases, and all cell deaths require the activity of at least a subset of these molecules. We also know that essentially every cell in the body produces caspase proteins, suggesting that every cell has the potential to initiate its own death.
Cells protect themselves from apoptotic death under normal circumstances by keeping caspases in an inactive form within a cell. Upon reception of the appropriate death signal, certain caspases are activated by proteolytic cleavage and then activate other caspases in turn, leading to the activation of effector caspases.This catalytic cascade culminates in cell death. Although it is not well understood how caspase activation directly results in apoptotic death of the cell, presumably it is through the cleavage of critical targets necessary for cell survival.
T cells use two different pathways to activate caspases (Figure 10-19). In peripheral T cells, antigen stimulation results in proliferation of the stimulated T cell and production of several cytokines including IL-2. Upon activation, T cells increase the expression of two key cell-surface proteins involved in T-cell death, Fas and Fas ligand (FasL). When Fas binds its ligand, FasL, FADD (Fas-associated protein with death domain) is recruited and binds to Fas, followed by the recruitment of procaspase 8, an inactive form of caspase 8. The association of FADD with procaspase 8 results in the proteolytic cleavage of procaspase 8 to its active form; cas-pase 8 then initiates a proteolytic cascade that leads to the death of the cell.
Outside of the thymus, most of the TCR-mediated apop-tosis of mature T cells is mediated by the Fas pathway. Repeated or persistent stimulation of peripheral T cells results in the coexpression of both Fas and Fas ligand, followed by the apoptotic death of the cell. The Fas/FasL mediated death of T cells as a consequence of activation is called activation-induced cell death (AICD) and is a major homeostatic mechanism, regulating the size of the pool of T cells and removing T cells that repeatedly encounter self antigens.
The importance of Fas and FasL in the removal of activated T cells is underscored by lpr/lpr mice, a naturally occurring mutation that results in non-functional Fas. When T cells become activated in these mice, the Fas/FasL pathway is not operative; the T cells continue to proliferate, producing IL-2 and maintaining an activated state. These mice spontaneously develop autoimmune disease, have excessive numbers of T cells, and clearly demonstrate the consequences of a failure to delete activated T cells. An additional mutation, gld/gld, is also informative. These mice lack functional FasL and display much the same abnormalities found in the lpr/lpr mice. Recently, humans with defects in Fas have been reported. As expected, these individuals display characteristics of autoimmune disease. (See the Clinical Focus box.)
Fas and FasL are members of a family of related recep-tor/ligands including tumor necrosis factor (TNF) and its ligand, TNFR (tumor necrosis factor receptor). Like Fas and FasL, membrane-bound TNFR interacts with TNF to induce apoptosis. Also similar to Fas/FasL-induced apoptosis, TNF/TNFR-induced death is the result of the activation of caspase 8 followed by the activation of effector caspases such as caspase 3.
In addition to the activation of apoptosis through death receptor proteins like Fas and TNFR, T cells can die through other pathways that do not activate procaspase 8. For example, negative selection in the thymus induces the apoptotic death of developing T cells via a signaling pathway that originates at the TCR. We still do not completely understand why some signals through the TCR induce positive selection and others induce negative selection, but we know that the strength of the signal plays a critical role. A strong, negatively selecting signal induces a route to apoptosis in which mitochondria play a central role. In mitochondrially dependent apoptotic pathways, cytochrome c, which normally resides in the inner mitochondrial membrane, leaks into the cytosol. Cytochrome c binds to a protein known as Apaf-1 (apoptotic protease-activating factor-1) and undergoes an ATP-dependent conformational change and oligomeriza-tion. Binding of the oligomeric form of Apaf-1 to procaspase 9 results in its transformation to active caspase 9. The complex of cytochrome c/Apaf-1/caspase 9, called the apopto-some, proteolytically cleaves procaspase 3 generating active caspase 3, which initiates a cascade of reactions that kills the cell (Figure 10-19). Finally, mitochondria also release another molecule, AIF (apoptosis inducing factor), which plays a role in the induction of cell death.
Cell death induced by Fas/FasL is swift, with rapid activation of the caspase cascade leading to cell death in 2-4 hours. On the other hand, TCR-induced negative selection appears to be a more circuitous process, requiring the activation of several processes including mitochondrial membrane failure, the release of cytochrome c, and the formation of the apopto-some before caspases become involved. Consequently, TCR-mediated negative selection can take as long as 8-10 hours.
An important feature in the mitochondrially induced cell death pathway is the regulatory role played by Bcl-2 family members. Bcl-2 and Bcl-XL both reside in the mitochondrial membrane. These proteins are strong inhibitors of apop-tosis, and while it is not clear how they inhibit cell death, one hypothesis is that they somehow regulate the release of cy-tochrome c from the mitochondria. There are at least three groups of Bcl-2 family members. Group I members are anti-apoptotic and include Bcl-2 and Bcl-xL. Group II and Group III members are pro-apoptotic and include Bax and Bak in Group II and Bid and Bim in Group III. There is clear evidence that levels of anti-apoptotic Bcl-2 family members play an important role in regulating apoptosis in lymphocytes. Bcl-2 family members dimerize, and the anti-apoptotic group members may control apoptosis by dimerizing with pro-apoptotic members, blocking their activity. As indicated in Figure 10-19, cleavage of Bid, catalyzed by caspase 8 gen erated by the Fas pathway, can turn on the mitochondrial pathway. Thus signals initiated through Fas can also involve the mitochondrial death pathway.
While it is apparent there are several ways a lymphocyte can be signaled to die, all of these pathways to cell death converge upon the activation of caspases. This part of the cell-death pathway, the execution phase, is common to almost all death pathways known in both vertebrates and invertebrates, demonstrating that apoptosis is an ancient process that has been conserved throughout evolution.
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