- Email: [email protected]
Introduction: apoptosis in the development and function of the immune system
Seminars in Immunology 15 (2003) 121–123
Editorial
Introduction: apoptosis in the development and function of the immune system The “tangled bank” concept from evolutionary biology suggests that complex environments favor organisms producing large numbers of highly divergent offspring, with the hopes that a few happen to have the characteristics that will favor their survival (the last paragraph of Darwin’s Origin of Species describes an “entangled bank,” hence the name of the theory). This idea, while intellectually satisfying, did not stand the tests of observation. However, in the development of the immune system, the tangled bank appears to be alive and well, and this is in part because most of the cells of the immune system die. Lymphocytes develop with a receptor generated by random processes capable of binding to antigens that may or may not ever appear in the body. For a while during their maturation these lymphocytes respond to the presence of their antigen by engaging a process that leads to their own deaths. If this does not occur, any subsequent engagement of the antigen can result in processes that orchestrate the immune response—proliferation, effector mechanisms, and long-term survival of some of the cells to create memory. This, of course, is the bare bones of clonal selection, the central tenet of modern immunology. It works because lymphocytes develop in huge excess of those that eventually survive to mediate immune responses. And this is needed because the problem of lymphocyte selection is so complex—a “tangled bank” of tissues that express tens of thousands of proteins that must be discriminated from those of the many invading organisms. Clonal selection, more or less in the current form, has been around since 1959. Since then, we have learned a few things about it. We know how antigen receptors on B and T lymphocytes are generated during development. We know a lot (but by no means everything) about how signals from antigen receptors lead to the cellular responses of proliferation and effector functions. But we know surprisingly little about how signals from the antigen receptor can lead to a cell’s death and how this decision to die is influenced by maturation. In a few cases we can explain the effect: for example, activation of T cells under some circumstances results in death via expression of Fas (CD95)-ligand, and this reacts with its death receptor to kill the cell. However, in most instances we do not know how the activation signal triggers cell death. 1044-5323/03/$ – see front matter © 2003 Published by Elsevier Science Ltd. doi:10.1016/S1044-5323(03)00028-9
Yet without cell death, especially antigen-specific cell death, the immune system cannot develop its critical features that allow it to function. This volume overviews several advances in our understanding of the initiation and control of cell death in cells of the immune system. The cell death discussed in this volume is apoptosis, the most common form of cell death in the body, and one that plays central roles in a wide variety of tissues and biological processes throughout the animals. Although it is technically defined by the morphology of the dying cell, the biochemical features that account for the features of the death have tended to replace this technical definition. But this is not without problems, since there is still much we do not know about the process. As apoptotic cells die, the chromatin condenses and the nucleus often fragments. These changes in nuclear morphology can be readily observed by the use of DNA dyes or by electron microscopy. The DNA of apoptosis is often digested between nucleosomes, so that when resolved by electrophoresis an oligonucleosomal ladder can be seen. Some assays for apoptosis are based on this DNA fragmentation, for example, assessment of sub-diploid propidium iodide staining of nuclei, or labeling of DNA ends by terminal deoxytransferase-catalyzed ligation of labeled nucleotides (the “sub-diploid peak” and “TUNEL” assays, respectively). Other assays for DNA fragmentation, whether qualitative or quantitative, often fail to give an indication of the number of cells that have undergone this effect. The plasma membrane of apoptotic cells frequently contorts into “blebs” that can break free, and the phospholipids of the bilayer scramble, destroying asymmetries in their distribution between inner and outer leaflets. One result of this scrambling is that phosphatidylserine (PS) in the inner leaflet of the plasma membrane now appears on the outer. There it can be detected, most commonly with labeled Annexin V, a PS-binding protein. All of these events appear to contribute to the phagocytosis of apoptotic cells and their rapid degradation. It is important to note, however, that these “markers” of apoptosis can occur by other means, and therefore none are definitive. How then, do we determine if a dead cell has undergone apoptosis? While arguments on this issue abound, I suspect that the answer is that it doesn’t matter. Our concern,
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Editorial
in general, is to show (a) that a cell has died in a given circumstance, and (b) that this has occurred through an interesting molecular process (“interesting” is of course the operative word here). If one were to find a noxious treatment that caused cells to undergo all of the “apoptotic” changes we’ve mentioned, but without the participation of any cellular enzymes, I doubt that we would employ it to investigate physiologic cell death (examples of such effects exist, and indeed we do not use them as models for apoptosis; therefore I will not detail them here). In general, apoptosis proceeds through the action of caspases, cysteine proteases that cleave specific substrates, some of which then cause the changes we see as apoptosis. The subset of caspases that generally do this are called “executioner” caspases (caspases 3, 6, and 7 in mammals). These exist in living cells as inactive dimers, and it appears that they can only be activated by cleavage between what will be the large and small subunits of the active enzyme. For the most part, the enzymes responsible for activating the executioner caspases are also caspases, the “initiator” or “apical” caspases (caspases 8, 9, and 10 in humans, and probably caspase 2; rodents lack caspase 10). Unlike the executioner caspases, the initiator caspases exist in living cells as inactive monomers, and it appears that they can only be activated by enforced dimerization. Cleavage of these enzymes does not activate them. Caspase activation is an important part of the apoptotic process, and considerable controversy continues regarding the extent to which they are required for cell death. While targeted disruption of some caspases produces severe developmental defects, it is also apparent that cell death by apoptotic stimuli can proceed in the absence of caspases. Caspase inhibitors tend to be inefficient, and it is likely that many caspase “independent” effects in the literature are actually mediated by low level caspase activity. Others are similarly likely to be truly caspase independent, and with time we will hopefully know which is which. An indication that caspase activation has occurred in a cell can come with the detection of cleavage of known caspase substrates into predicted sizes. Antibodies to a variety of mammalian substrates are available, such as poly-ADP ribose polymerase (PARP), fodrin, gelsolin, D4-GDI, and the inhibitor of caspase-activated DNAse (iCAD), among others. Most of these are likely to be responsible for some of the effects of caspase activation in cells (however, the fact that a substrate is cleaved does not in any way indicate that its cleavage is important for the death process; in these cases there is additional evidence of involvement), and if in a particular cell death scenario several of these are found cut in appropriately sized fragments, it is likely that caspase activation has occurred. Caspase activity is also frequently detected through the use of synthetic substrates, however, while these are often advertised as “specific” for a particular caspase, they are far from definitive. This is, in part, because different caspases are present in cells in widely different amounts.
Similar problems persist for the use of “specific” synthetic inhibitors. An alternative that is often used to obtain evidence of involvement of a particular caspase is the cleavage of that caspase. As has already been noted, however, cleavage of an initiator caspase does not activate it, and therefore this cannot be taken as definitive evidence of its involvement. Another problem is that several caspases are also cleaved by calpain (at an irrelevant site that destroys the caspase) and fragments can appear to be of approximately the correct sizes to artifactually indicate that caspase-mediated cleavage has occurred. An evolving method to detect active caspases is the use of agents that bind to their active sites (if present, which would indicate that they are active enzymes) and can then be used to precipitate the molecule, which is then detected by immunoblot. This method has the potential to provide definitive evidence that a given caspase has been activated as part of the apoptotic process. Identification of activated initiator caspases is important because they serve to define distinct apoptotic pathways. As these new methods become available, there is hope that some of the confusion that surrounds this area will be resolved. The two best defined apoptotic pathways are the death receptor and mitochondrial pathways, sometimes called the extrinsic and intrinsic pathways, respectively. In these pathways, and presumably in other routes to apoptosis yet to be extensively characterized, the initiator caspases are activated by the binding of adapter molecules that enforce their dimerization. In the death receptor pathway, the adapter, FADD, forms part of the death inducing signaling complex (DISC) and then binds the initiator caspase 8 and/or 10, activating them. In the mitochondrial pathway, apoptotic signals induce the release of proteins from the intermembrane space of the mitochondria, including cytochrome c. The latter activates another adapter molecule, apoptotic protease activating factor-1 (APAF-1) that oligomerizes and recruits the initiator caspase 9 into an “apoptosome,” activating the caspase. In either case, the activated initiator caspases can cleave and activate executioner caspases that then orchestrate apoptosis. Permeabilization of the mitochondrial outer membrane is a critical event in apoptosis, as this not only releases cytochrome c and other proteins to the cytosol but also because it may commit the cell to death due to an ultimate failure of mitochondrial functions (even if caspases are not activated). This permeabilization event is mediated through the action of members of the Bcl-2 protein family. A subset of such proteins, called the Bcl-2 homology domain-3 (BH3) only proteins, appear to become activated through a variety of cellular stress pathways. Some of these are Bim, Bid, Bad, Bmf, Puma, and Noxa. At least some of these act on another subset, the multidomain or BH123 proteins, which include Bax and Bak, and act to produce the permeabilization of the mitochondrial outer membrane. Mice that lack Bax and Bak have a variety of apoptotic defects (resulting in profound
Editorial
developmental problems) and their mitochondria fail to release cytochrome c when the cells are stressed. The pro-apoptotic effects of these members of the Bcl-2 family are antagonized by another subset of these proteins, which are anti-apoptotic, and include Bcl-2, Bcl-xL, Bcl-w, Mcl-1, and A1/Bfl. While the regulation of apoptosis occurs largely through the actions of the Bcl-2 family proteins, other molecular mechanisms of control on this process also play important roles. Mitochondrial membrane permeabilization is also affected by the function of AKT/PKB, and while not fully understood, this may be independent of the effects of the Bcl-2 proteins. Caspases are inhibited by inhibitor of apoptosis proteins (IAPs) and this effect appears to be controlled by molecules released from the mitochondria, such as Smac/DIABLO and Omi/Htra2. Proteins that control the death receptor signaling pathway are also known, including a protein often referred to as c-FLIP, although its precise role in regulating apoptosis continues to be debated.
123
As you peruse the reviews in this volume, it is useful to bear in mind that the process of apoptosis is almost certainly more complex than we know, due in part to the inherent complexity of the systems and to the limitations of our methods to probe them. Nevertheless, the importance of apoptosis and its regulation is clearly central to many immunologic phenomena, and any differences in our viewpoints may be taken as starting points for what may be exciting new avenues of research. Clearly, we have much to learn. Douglas R. Green Division of Cellular Immunology La Jolla Institute for Allergy and Immunology 10355 Science Centre Dr., San Diego, CA, 92121 USA Fax: +1-858-558-3594 E-mail address: [email protected] (D.R. Green)
Editorial
Introduction: apoptosis in the development and function of the immune system The “tangled bank” concept from evolutionary biology suggests that complex environments favor organisms producing large numbers of highly divergent offspring, with the hopes that a few happen to have the characteristics that will favor their survival (the last paragraph of Darwin’s Origin of Species describes an “entangled bank,” hence the name of the theory). This idea, while intellectually satisfying, did not stand the tests of observation. However, in the development of the immune system, the tangled bank appears to be alive and well, and this is in part because most of the cells of the immune system die. Lymphocytes develop with a receptor generated by random processes capable of binding to antigens that may or may not ever appear in the body. For a while during their maturation these lymphocytes respond to the presence of their antigen by engaging a process that leads to their own deaths. If this does not occur, any subsequent engagement of the antigen can result in processes that orchestrate the immune response—proliferation, effector mechanisms, and long-term survival of some of the cells to create memory. This, of course, is the bare bones of clonal selection, the central tenet of modern immunology. It works because lymphocytes develop in huge excess of those that eventually survive to mediate immune responses. And this is needed because the problem of lymphocyte selection is so complex—a “tangled bank” of tissues that express tens of thousands of proteins that must be discriminated from those of the many invading organisms. Clonal selection, more or less in the current form, has been around since 1959. Since then, we have learned a few things about it. We know how antigen receptors on B and T lymphocytes are generated during development. We know a lot (but by no means everything) about how signals from antigen receptors lead to the cellular responses of proliferation and effector functions. But we know surprisingly little about how signals from the antigen receptor can lead to a cell’s death and how this decision to die is influenced by maturation. In a few cases we can explain the effect: for example, activation of T cells under some circumstances results in death via expression of Fas (CD95)-ligand, and this reacts with its death receptor to kill the cell. However, in most instances we do not know how the activation signal triggers cell death. 1044-5323/03/$ – see front matter © 2003 Published by Elsevier Science Ltd. doi:10.1016/S1044-5323(03)00028-9
Yet without cell death, especially antigen-specific cell death, the immune system cannot develop its critical features that allow it to function. This volume overviews several advances in our understanding of the initiation and control of cell death in cells of the immune system. The cell death discussed in this volume is apoptosis, the most common form of cell death in the body, and one that plays central roles in a wide variety of tissues and biological processes throughout the animals. Although it is technically defined by the morphology of the dying cell, the biochemical features that account for the features of the death have tended to replace this technical definition. But this is not without problems, since there is still much we do not know about the process. As apoptotic cells die, the chromatin condenses and the nucleus often fragments. These changes in nuclear morphology can be readily observed by the use of DNA dyes or by electron microscopy. The DNA of apoptosis is often digested between nucleosomes, so that when resolved by electrophoresis an oligonucleosomal ladder can be seen. Some assays for apoptosis are based on this DNA fragmentation, for example, assessment of sub-diploid propidium iodide staining of nuclei, or labeling of DNA ends by terminal deoxytransferase-catalyzed ligation of labeled nucleotides (the “sub-diploid peak” and “TUNEL” assays, respectively). Other assays for DNA fragmentation, whether qualitative or quantitative, often fail to give an indication of the number of cells that have undergone this effect. The plasma membrane of apoptotic cells frequently contorts into “blebs” that can break free, and the phospholipids of the bilayer scramble, destroying asymmetries in their distribution between inner and outer leaflets. One result of this scrambling is that phosphatidylserine (PS) in the inner leaflet of the plasma membrane now appears on the outer. There it can be detected, most commonly with labeled Annexin V, a PS-binding protein. All of these events appear to contribute to the phagocytosis of apoptotic cells and their rapid degradation. It is important to note, however, that these “markers” of apoptosis can occur by other means, and therefore none are definitive. How then, do we determine if a dead cell has undergone apoptosis? While arguments on this issue abound, I suspect that the answer is that it doesn’t matter. Our concern,
122
Editorial
in general, is to show (a) that a cell has died in a given circumstance, and (b) that this has occurred through an interesting molecular process (“interesting” is of course the operative word here). If one were to find a noxious treatment that caused cells to undergo all of the “apoptotic” changes we’ve mentioned, but without the participation of any cellular enzymes, I doubt that we would employ it to investigate physiologic cell death (examples of such effects exist, and indeed we do not use them as models for apoptosis; therefore I will not detail them here). In general, apoptosis proceeds through the action of caspases, cysteine proteases that cleave specific substrates, some of which then cause the changes we see as apoptosis. The subset of caspases that generally do this are called “executioner” caspases (caspases 3, 6, and 7 in mammals). These exist in living cells as inactive dimers, and it appears that they can only be activated by cleavage between what will be the large and small subunits of the active enzyme. For the most part, the enzymes responsible for activating the executioner caspases are also caspases, the “initiator” or “apical” caspases (caspases 8, 9, and 10 in humans, and probably caspase 2; rodents lack caspase 10). Unlike the executioner caspases, the initiator caspases exist in living cells as inactive monomers, and it appears that they can only be activated by enforced dimerization. Cleavage of these enzymes does not activate them. Caspase activation is an important part of the apoptotic process, and considerable controversy continues regarding the extent to which they are required for cell death. While targeted disruption of some caspases produces severe developmental defects, it is also apparent that cell death by apoptotic stimuli can proceed in the absence of caspases. Caspase inhibitors tend to be inefficient, and it is likely that many caspase “independent” effects in the literature are actually mediated by low level caspase activity. Others are similarly likely to be truly caspase independent, and with time we will hopefully know which is which. An indication that caspase activation has occurred in a cell can come with the detection of cleavage of known caspase substrates into predicted sizes. Antibodies to a variety of mammalian substrates are available, such as poly-ADP ribose polymerase (PARP), fodrin, gelsolin, D4-GDI, and the inhibitor of caspase-activated DNAse (iCAD), among others. Most of these are likely to be responsible for some of the effects of caspase activation in cells (however, the fact that a substrate is cleaved does not in any way indicate that its cleavage is important for the death process; in these cases there is additional evidence of involvement), and if in a particular cell death scenario several of these are found cut in appropriately sized fragments, it is likely that caspase activation has occurred. Caspase activity is also frequently detected through the use of synthetic substrates, however, while these are often advertised as “specific” for a particular caspase, they are far from definitive. This is, in part, because different caspases are present in cells in widely different amounts.
Similar problems persist for the use of “specific” synthetic inhibitors. An alternative that is often used to obtain evidence of involvement of a particular caspase is the cleavage of that caspase. As has already been noted, however, cleavage of an initiator caspase does not activate it, and therefore this cannot be taken as definitive evidence of its involvement. Another problem is that several caspases are also cleaved by calpain (at an irrelevant site that destroys the caspase) and fragments can appear to be of approximately the correct sizes to artifactually indicate that caspase-mediated cleavage has occurred. An evolving method to detect active caspases is the use of agents that bind to their active sites (if present, which would indicate that they are active enzymes) and can then be used to precipitate the molecule, which is then detected by immunoblot. This method has the potential to provide definitive evidence that a given caspase has been activated as part of the apoptotic process. Identification of activated initiator caspases is important because they serve to define distinct apoptotic pathways. As these new methods become available, there is hope that some of the confusion that surrounds this area will be resolved. The two best defined apoptotic pathways are the death receptor and mitochondrial pathways, sometimes called the extrinsic and intrinsic pathways, respectively. In these pathways, and presumably in other routes to apoptosis yet to be extensively characterized, the initiator caspases are activated by the binding of adapter molecules that enforce their dimerization. In the death receptor pathway, the adapter, FADD, forms part of the death inducing signaling complex (DISC) and then binds the initiator caspase 8 and/or 10, activating them. In the mitochondrial pathway, apoptotic signals induce the release of proteins from the intermembrane space of the mitochondria, including cytochrome c. The latter activates another adapter molecule, apoptotic protease activating factor-1 (APAF-1) that oligomerizes and recruits the initiator caspase 9 into an “apoptosome,” activating the caspase. In either case, the activated initiator caspases can cleave and activate executioner caspases that then orchestrate apoptosis. Permeabilization of the mitochondrial outer membrane is a critical event in apoptosis, as this not only releases cytochrome c and other proteins to the cytosol but also because it may commit the cell to death due to an ultimate failure of mitochondrial functions (even if caspases are not activated). This permeabilization event is mediated through the action of members of the Bcl-2 protein family. A subset of such proteins, called the Bcl-2 homology domain-3 (BH3) only proteins, appear to become activated through a variety of cellular stress pathways. Some of these are Bim, Bid, Bad, Bmf, Puma, and Noxa. At least some of these act on another subset, the multidomain or BH123 proteins, which include Bax and Bak, and act to produce the permeabilization of the mitochondrial outer membrane. Mice that lack Bax and Bak have a variety of apoptotic defects (resulting in profound
Editorial
developmental problems) and their mitochondria fail to release cytochrome c when the cells are stressed. The pro-apoptotic effects of these members of the Bcl-2 family are antagonized by another subset of these proteins, which are anti-apoptotic, and include Bcl-2, Bcl-xL, Bcl-w, Mcl-1, and A1/Bfl. While the regulation of apoptosis occurs largely through the actions of the Bcl-2 family proteins, other molecular mechanisms of control on this process also play important roles. Mitochondrial membrane permeabilization is also affected by the function of AKT/PKB, and while not fully understood, this may be independent of the effects of the Bcl-2 proteins. Caspases are inhibited by inhibitor of apoptosis proteins (IAPs) and this effect appears to be controlled by molecules released from the mitochondria, such as Smac/DIABLO and Omi/Htra2. Proteins that control the death receptor signaling pathway are also known, including a protein often referred to as c-FLIP, although its precise role in regulating apoptosis continues to be debated.
123
As you peruse the reviews in this volume, it is useful to bear in mind that the process of apoptosis is almost certainly more complex than we know, due in part to the inherent complexity of the systems and to the limitations of our methods to probe them. Nevertheless, the importance of apoptosis and its regulation is clearly central to many immunologic phenomena, and any differences in our viewpoints may be taken as starting points for what may be exciting new avenues of research. Clearly, we have much to learn. Douglas R. Green Division of Cellular Immunology La Jolla Institute for Allergy and Immunology 10355 Science Centre Dr., San Diego, CA, 92121 USA Fax: +1-858-558-3594 E-mail address: [email protected] (D.R. Green)