TY - JOUR
AU - Levine, Alan D.
AB - Summary Cells of the intestinal mucosa live in a harsh environment and therefore rely heavily on the highly regulated process of cell death, apoptosis, to maintain tissue integrity. Imbalance in the intracellular events that modulate apoptosis may contribute to the pathogenesis of inflammatory bowel disease. Apoptosis, Review, Intestinal epithelial cells, Lamina propria lymphocytes, Mucosa Introduction William Shakespeare may have said it best, “To be, or not to be, that is the question: Whether ’tis nobler in the mind, to suffer the slings and arrows of outrageous fortune; or, to take arms against a sea of troubles, and, by opposing, end them?” Cells of the gastrointestinal tract are continuously challenged “by a sea of troubles” in the form of toxins, mitogens, bacteria, viruses, and the antigens they yield. In response to these environmental insults a variety of complex and extremely well-regulated systems have developed in the wall of the gut to eliminate compromised cells and to minimize an inappropriate and extended immune response to nonpathogenic symbiotic microbiota. In particular, barrier epithelial cells and lamina propria and intraepithelial lymphocytes are two cell types of the intestinal mucosa that undergo extensive turnover. It is possible that the relatively rapid recycling of these cell populations may reduce the concentration of potentially damaged cells and eliminate the perpetuation of an immune response. Intestinal tissue homeostasis is maintained by a balance between cell proliferation and cell death, and a loss of this equilibrium, as has been proposed in a variety of conditions (Fig. 1), may be involved in disease pathogenesis (1,2). Cell proliferation, or mitosis, is positively regulated by growth factors and protooncogenes that stimulate cell cycle progression and negatively controlled by tumor suppressor genes, which inhibit the activity of protooncogenes (3,–5). Similarly, cell death is a highly regulated intracellular series of biochemical events called apoptosis, during which a cell activates its intrinsic suicide machinery that systematically dismantles the cell. This process generates small membrane-bound vesicles that are rapidly engulfed by neighboring cells or, in the specialized case of the intestinal epithelium, shed into the lumen. Fig. 1. Open in new tabDownload slide Diseases associated with the induction or inhibition of apoptosis (1,2). Regulation of cell death is essential for the development of all multicellular organisms (6). Normal cell death occurs in developing tissues, during amphibian and insect metamorphosis, during the defense against viral and bacterial infection, and the emergence of cancer (1, 7,8). Programmed cell death or apoptosis, referring to leaves dropping from trees in autumn, was originally described morphologically (9). Cells undergoing apoptosis are characterized by the shrinkage and condensation of their cytoplasm, increased mitochondrial permeability, chromatin condensation into caps at the edge of the nucleus, DNA fragmentation, and the appearance of plasma membrane blebs, often referred to as “apoptotic bodies” (10). Unlike necrosis, which is a passive process of cell death resulting in the rupture of the cell and initiation of inflammation, the events leading to an apoptotic cell are controlled through the expression of a number of evolutionarily conserved genes that function as activators, inhibitors, modulators, and effectors of the cell death process. Triggers of Apoptosis During embryogenesis, metamorphosis, maturation, and development, apoptosis appears to be a preprogrammed event that functions to eliminate lineagespecific unwanted cells to permit 1) sculpting structures (as in the formation of digits); 2) deleting unneeded structures (during the removal of subplate neurons); 3) controlling cell numbers (retraction of axons and deletion of neurons and oligodendrocytes); 4) eliminating abnormal, misplaced, nonfunctional, or harmful cells (thymocyte death during T lymphocyte differentiation); and 5) producing differentiated cells without organelles (skin keratinocytes and red blood cell enucleation and terminal differentiation) (7). These cell deaths are likely triggered by autonomous genetic programming (11), induced by death-activating signals, or stimulated by the deprivation of survival signals. Physiological mediators that have been reported to activate apoptotic death include the tumor necrosis factor (TNF) family of soluble and cell-bound proteins [Fas ligand, TRAIL, Apo3L (12,–14)], transforming growth factor (TGF)-β (15), glucocorticoids (16), calcium, and neurotransmitters such as dopamine, NMDA (N-methyl-D-aspartate), and glutamate. Deprivation of growth factors (17,18) or nutrients via antimetabolites (19) and loss of matrix attachment (20) or cell-cell contact (21) also initiate apoptosis. All of these stimuli represent biological processes employed by an organism to remodel tissue and maintain function. Paradoxically, these same signals may have opposing effects on growth, differentiation, or apoptosis between different cell types. Apoptosis is also initiated by harsh environmental insults, including heat shock, ionizing and ultraviolet radiation (22), viral infection (23), bacterial toxins, ischemia (24,25), oxidants (26), alcohol (27), and free radicals (28). The molecular mechanisms by which each of these varied insults activates the death pathway is not fully understood. Two cellular responses shared in common among these environmental triggers are the induction of the stress activation pathway and damage to chromosomal DNA. In many of these insults, cells within the central area of damage appear to die rapidly of necrosis. Perimeter cells lie just outside the central core of destruction, possibly representing those cells that were compromised but not killed by the insult. These cells die over a more protracted time period through the process of apoptosis. Activation of these pathways at times leads to induction of p53, which in turn can initiate apoptosis (29), and thus provides a direct link between cell cycle progression and cell death. In particular, the p53 tumor suppressor gene, an inducer of the cell cycle inhibitor p21, is the most frequently mutated gene in human tumors (30), and reintroduction of an active p53 transgene into transformed cells can induce apoptosis (31,32). Consistent with a role for apoptosis in tumorigenesis, oncogenes can be either pro- or antiapoptotic, and the latter family can inhibit apoptosis induced by the former (33,34). Caspases: Effectors of Apoptosis A hallmark attribute of apoptosis is death from within, mediated by proteolytic dismantling of the cell. Proteolytic cleavage of specific enzymatic and structural targets is mediated by a family of cysteine-dependent aspartate specific proteases (caspases). Currently 11 human caspases have been identified (Fig. 2), all of which share a high degree of homology in the carboxy-terminal catalytic domain. Caspases are synthesized as proenzymes (zymogens) that usually exhibit little activity relative to the cleaved, activated proteases. Activation of caspases, by other caspases or autoproteolysis, involves the proteolytic cleavage between two domains resulting in the removal of an amino-terminal prodomain, a linker region, and the assembly of ˜20 kDa and ˜10 kDa subunits into an active enzyme. Based on substrate specificity and their likely biological function, caspases are divided into three major groups (Fig. 2): 1) cytokine activators involved primarily in inflammation; 2) initiators of apoptosis; and 3) executioners of apoptosis (35,–37). Within the longer amino-terminal regions of the initiator caspases are two distinct protein-protein interaction structural motifs, the death effector domain (DED), and the caspase recruitment domain (CARD), which loosely associate these proteases with the extrinsic (i.e., TNF-α, Fas ligand, etc.) or intrinsic (DNA damage, stress pathway, etc.) triggers of apoptosis discussed earlier. Fig. 2. Open in new tabDownload slide Enzymatic and structural homology among inflammatory, initiator, and executioner caspases. In a nonapoptotic and resting inflammatory cell, caspases exist as zymogens, inactive proteases in their full length form, which consists of an amino-terminal peptide of varying length and a catalytic domain. Caspases are activated by proteolytic cleavage at conserved aspartic acid residues within the catalytic domain, yielding the large and small subunits, as indicated in the upper part of this figure. Both of these subunits are essential for the proteolytic activity and substrate specificity of this protease family. An additional short linker peptide, which appears to have no biological function, is released during the activation step. The amino acid sequence within the large and small units of the catalytic domain is highly conserved. The amino-terminal peptide, depicted in the lower half of Fig. 2, varies in length from 22 amino acids in caspase 6 to greater than 200 amino acids in caspase 8. The amino-terminal peptides of the inflammatory and initiator caspases contain conserved sequence motifs, indicated as CARD (caspase recruitment domain) and DED (death effector domains). The DED domains on procaspases 8 and 10 participate in protein-protein interactions that recruit these proenzymes to the intracellular domain of the death receptors. CARD domains are believed to contribute to the assembly of activator complexes in both inflammatory and apoptotic cells. The amino-terminal peptide of procaspase 9 is not removed subsequent to its activation. The substrate specificity of each caspase is indicated in the right column. W, tryptophan; L, leucine; E, glutamic acid; H, histidine; D, aspartic acid; I, isoleucine; V, valine; X, any amino acid. Initiator Caspases of the Extrinsic Triggers of Apoptosis When ligands for the cell surface death receptors, Fas, TNFR1, DR3, DR4, and DR5 (14), are engaged, these receptors form homotrimeric complexes that recruit adapter proteins to the inner leaflet of the plasma membrane through like-like interactions between death domains (DD). A death-inducing signaling complex (DISC) forms between the cytoplasmic domain of the death receptor and the DD of such proteins as FADD, TRADD, RAIDD, RIP, and MADD (38). Whereas the DD of these proteins is required for association with the death receptors, the amino terminus of FADD also contains a DED, which is critical for recruiting the DED containing initiator proteases procaspase 8 and/or procaspase 10. Procaspase 8 can be proteolytically activated by oligomerization following its recruitment to the DISC (39). While caspase 2 does not contain a DED, it is often grouped with the initiator caspases since it can bind to the TNF receptor via RIP and RAIDD. The exact biological functions of caspase 2 and caspase 10 as initiators of apoptosis are not fully defined. Initiator Caspases of the Intrinsic Triggers of Apoptosis The second trigger of apoptosis is an intracellular death signal mediated by a cytoplasmic complex that contains Apaf-1 (apoptotic protease activating factor-1) and holocytochrome c. In the presence of dATP or ATP, Apaf-1 binds to and activates procaspase 9, which in turn activates the executioner caspase 3 (40). Biochemical analysis of this reaction reveals that Apaf-1 hydrolyzes dATP/ATP, which promotes the binding of cytochrome c and the subsequent recruitment and autocatalytic activation of procaspase 9 (41). Apaf-1 is a 130 kDa protein with an amino-terminal domain shared in common with the amino-terminal domain of some caspases, followed by a CARD domain. The carboxy-terminal region shares homology with a death-promoting protein (CED-4) from Caenorhabditis elegans, which contains Walker's A and B boxes capable of nucleotide binding and 12-13 WD40 repeats, a motif mediating protein-protein interaction (42). The apoptosis promoting activity of cytochrome c is independent of its redox potential, and its release from the mitochondria into the cytosol corresponds to the observed damage to mitochondria in cells undergoing apoptosis (43). The requirement to form an Apaf-1/ cytochrome c complex may help to avoid spontaneous apoptosis caused by the occasional release of cytochrome c intothe cytoplasm, and may nucleate the activation of procaspase 9 by enhancing its aggregation, leading to autoproteolysis. The role of the CARD domain is less clear since it is found on both proinflammatory caspases 1, 4, 5, and 13 and the initiator caspases 2 and 9. The assignment of caspases 2, 8, 9, and 10 as initiator caspases is based not only on their association with the triggers of apoptosis, as described above. These initiator proteases can all undergo autoproteolysis since their specific cleavage recognition site is found within their own sequence. Furthermore, initiator caspase recognition sites are contained in the executioner caspases 3, 6, and 7, suggesting that the proteolytic cascade proceeds sequentially by autoproteolysis and activation of initiator caspases, which in turn cleave and activate executioner caspases. In addition, since executioner caspases do not contain their own cleavage sites, this also implies that the executioner caspases are incapable of self activation. Consistent with this distinction in function among these caspases, the amino-terminal peptide of caspases 3, 6, and 7 is small, with no known biological function (35,–37). The difference in biological functions for caspases is an evolving and, most likely, overlapping field of investigation, which is currently based on molecular genetic homology, substrate specificity, and preferences for natural intracellular substrates. Since all caspases and granzyme B (see immunology section below) require an aspartate in the P1, of the substrate, the distinction among known caspases is based largely on changes in the P4 amino acid in the target cleavage site. As further information on intracellular localization and cell type expression of caspases is obtained, and when highly specific protease inhibitors become available, a more refined attribution of biological relevance for each protease will be forthcoming. Inhibitors of Apoptosis In light of the extremely rapid and global proteolytic destruction brought upon a cell by caspase activation, not surprisingly three distinct pathways exist to keep this ubiquitous cascade of proteolysis in check: 1) caspase inhibitors; 2) inactive caspase homologues; and 3) decoy death receptors. Caspase Inhibitors The inhibitor of apoptosis (IAP) family of proteins was first described in baculoviruses, where they suppress the death response of the host cell to a viral infection. Five human homologues of these viral inhibitors of apoptosis have been identified, c-IAP-1, c-IAP-2, XIAP, NAIP, and survivin, that inhibit activated caspases, prevent caspase activation, and block apoptosis (44). These proteins contain one [survivin (45)], two or three copies of a 70 amino acid BIR (baculovirus IAP repeat) motif (Fig. 3), which is required for their caspase inhibitory and antiapoptotic activity (46,–48). In addition to BIR motifs, some IAPs (c-IAP-1, c-IAP-2, XIAP) contain carboxy-terminal RING (really interesting new gene) finger zinc-binding domain (47), whose function is unclear (48). Furthermore, c-IAP-1 and c-IAP-2 contain a CARD domain (49), which may implicate these IAP as direct inhibitors of procaspase 9 (a CARD containing zymogen) activation by the Apaf-1/cytochrome c complex. Fig. 3. Open in new tabDownload slide Domain structure of inhibitors of apoptosis proteins (IAP). Shared sequence motifs indicate the location of the BIR (baculoviral IAP repeat) and Ring (Really Interesting New Gene) domains conserved among this family of caspase protease inhibitors. Amino acid length is noted to the right of each protein. Currently there is limited understanding of the structure and molecular mechanism of IAP activity. Since exogenous expression of IAPs can suppress apoptosis initiated by a variety of triggers, two possible models have been proposed. First, mammalian IAPs, especially XIAP, prevent apoptosis by directly inhibiting the active site of the executioner caspases 3 and 7, but not caspase 1, 6, 8, or 10 (50,51). Second, IAP also inhibit apoptosis by blocking the activation of procaspase 9, by binding directly to the zymogen form of this protease (52,53). The dual activities of IAP demonstrate that they can effectively block both the extrinsic (TNF-α, Fas ligand, etc.) and intrinsic (Apaf-1/cytochrome c) pathways of cell death. If a cell is engaged via its death receptor, the presence of an IAP can inhibit the activity of executioner caspases and thereby terminate the apoptotic cascade before the cell is proteolytically dismantled. Activation of cell death through the intrinsic pathway is prevented by IAPs at two levels: 1) IAPs, possibly through their CARD domain, may prevent the binding of procaspase 9 to the Apaf-1/ cytochrome c complex and block the activation of the protease; and 2) similar to the extrinsic pathway, IAPs directly inhibit executioner caspases (44). The expression and function of IAP in a variety of cellular and animal models implicate these proteins in disease processes. For example, survivin protein levels are decreased in a transformed cell that is resistant to apoptosis (45), and inhibition of survivin translation with an antisense oligonucleotide induces apoptosis and inhibits cell proliferation in a tumor cell (54). The gene for neuronal apoptosis inhibitory protein (NAIP) is partially deleted in individuals with spinal muscular atrophy (55), and increased expression of NAIP delays apoptosis in cerebellar granule neurons and reduces ischemic damage in the rat hippocampus (56,57). Nonproteolytic Caspase Homologues Mammalian cells also express an inhibitor of death receptor mediated apoptosis by preventing procaspase recruitment to the DISC complex. cFLIP [cellular FADD-like ICE inhibitory protein (58,–62)] is an inactive homologue of caspases 8 and 10 that contains two amino-terminal DED domains. The caspase 8-like domain is mutated in several critical residues in the catalytic active site and the aspartate binding site. cFLIP acts as a competitive inhibitor, binding to FADD, procaspase 8, and procaspase 10 via its DED, and blocking the activation of these zymogens at the DISC that forms after a death receptor has been engaged (61,63). Not surprisingly, ectopic expression of cFLIP inhibits apoptosis induced by death receptors, but not the intrinsic pathway stimulated by staurosporine or UV irradiation (63). Decoy Death Receptors Apoptosis induced by TRAIL or Fas can be specifically inhibited through the expression of decoy receptors. DcRl and DcR2, specific for TRAIL, and DcR3, specific for Fas ligand, prevent apoptosis by competing with the appropriate death receptors (DR4, DR5, Fas) for the ligand (64,–66). The TRAIL decoy receptors (DcR1 and DcR2) are unable to stimulate cell death because they lack a DD or express a truncated DD in the cytoplasmic tail, respectively. It is likely that the ratio in cell surface levels of the death receptor to the decoy receptor modulates a cell's sensitivity to TRAIL or Fas-mediated apoptosis. Therefore, differential expression of each receptor class in normal versus tumor cells may impact the ability of the immune tumor surveillance system to function adequately (14). Bcl-2 Family of Proteins: Modulators of Apoptosis Superimposed on the direct action of caspases, caspase inhibitors, and decoy receptors in effecting cell death is a complex system of proteins that appear to predominantly regulate the integrity of the mitochondria as a means for modulating apoptosis. The Bcl-2 family of proteins includes both proapoptotic and antiapoptotic members that share a similar modular distribution of homologous domains, known as BH (Bcl-2 homology) (Fig. 4). Bcl-2 proteins are located on the cytoplasmic surface of various organelles, including the mitochondria, endoplasmic reticulum, and nucleus. The proapoptotic members appear to initiate cell killing by augmenting the release of cytochrome c from the mitochondria (67). Three theories are currently under investigation to provide a biochemical mechanism for Bcl-2 protein effects on cytochrome release: 1) permeability transition pore (PTP) (68); 2) ion channel (69); and 3) BH3 dimerization (70). Fig. 4. Open in new tabDownload slide Conserved structural homologies among antiapoptotic and proapoptotic human proteins of the Bcl-2 family. Only human Bcl-2 family proteins are shown, grouped by the presence and absence of the modular BH (Bcl-2 homology) sequence motifs, BH1, BH2, BH3, and BH 4, and the transmembrane domain (TM). The range in amino acid length is indicated to the right of each protein classification. Permeability Transition Pore Most members of the Bcl-2 family contain a carboxyterminal hydrophobic transmembrane domain, which may enable the protein to integrate into the outer mitochondrial membrane and form a PTP that leads to a loss of membrane potential and cytochrome c release observed in an apoptotic cell (71,–73). Consistent with this model, cyclosporin A, an inhibitor of PTP, blocks Baxmediated cell death (74). Bax is proposed to translocate to the outer membrane of the mitochondria where it interacts with the voltage-dependent anion channel (VDAC, also called porin) to form pores (75). Other reports suggest that Bax also associates with inner membrane proteins such as adenine nucleotide transporter (76) and proteins located at the intersection of the outer and inner membranes, such as hexokinase and adenylate kinase (77). However, Bax association with these inner membrane proteins appears to occur late in apoptosis, which is observed in the absence of membrane depolarization (71,72). With these seemingly conflicting findings it is currently safe to state that how Bcl-2 proteins truly destabilize the mitochondrial membrane is unknown. Ion Channel When the three dimensional structure of Bcl-xL was solved by nuclear magnetic resonance (NMR), it revealed a hydrophobic helical hairpin flanked by a pair of amphipathic helices, similar to those seen in diptheria toxin, which forms ion channels after insertion into lipid bilayers (78). In agreement with the pro- and antiapoptotic activity of Bax and Bcl-xL, when inserted into planar lipid bilayers and vesicles, these proteins formed anion-selective and cation-specific channels (79,80). In addition, the channels formed by Bax and Bcl-2 have other distinct characteristics including conductance, voltage dependence, and rectification (81). While Bax alone can destabilize lipid membranes and induce porosity at submolar concentrations, Bcl-xL, can neither interact with Bax directly nor protect against its destabilizing effects (82). The ability to distinguish between pro- and antiapoptotic Bcl-2 proteins by analyzing ion channel function agrees with the demonstration that Bcl-2 and Bcl-xL can protect against Bax-induced mitochondria damage, cytochrome c release (83,84), and cell death (69,82). An alternate interpretation of these reports is that pro- and antiapoptotic Bcl-2 proteins may compete for the same target protein on the outer membrane of the mitochondria, such as porin, or that the relative ratios of pro- and antiapoptotic Bcl-2 proteins may regulate the flow of ions (85). An additional antiapoptotic activity attributed to Bcl-2 and Bcl-xL is their ability to bind Apaf-1 and prevent procaspase 9 activation (86,–88). BH3 Dimerization Not all Bcl-2 proteins, such as Bid and Bad, express a transmembrane domain, therefore they appear to lack the potential of forming a channel or pore. Instead Bid and Bad are cleaved during apoptosis by caspase 8, and a carboxy-terminal BH3 domain is released. This BH3- containing fragment translocates from the cytosol to the mitochondria where it triggers cytochrome c release by selectively permeabilizing the outer membrane (89,90) perhaps by interacting with Bax (91). While the mechanism by which the BH3 domain induces mitochondrial permeability is unknown, mutagenesis studies in the BH3 domain demonstrate a correlation between loss of Bid binding to other Bcl-2 proteins with an inability to stimulate cytochrome c release (92). Cleavage of Bid by caspase 8 may be a mechanism by which death receptorinitiated apoptosis can be amplified by stimulating the Apaf-1/cytochrome c/procaspase 9 pathway. In support of this model, the death of cells undergoing death receptor-mediated apoptosis can be abrogated by caspase 8 inhibitors, by blocking direct caspase 8 activation of executioner caspases and by obstructing the indirect pathway of caspase 8 activation of Bid-mediated apoptosis (89,90). In contrast, while the antiapoptotic proteins Bcl-2 and Bcl-xL can inhibit Bid-mediated cytochrome c release, they cannot prevent cell death initiated by the death receptors due to the direct activation of executioner caspases by caspase 8 (93). Cell Proliferation and Cell Death in the Intestinal Epithelium The intestinal epithelium undergoes enormous cellular turnover, yet tissue integrity is maintained by the delicate balance between rapid proliferation of stem cells in the base of the crypt and exfoliation of terminally differentiated senescent cells at the luminal surface. It is possible that the ongoing renewal of the epithelium minimizes the accumulation of deleterious mutations that could promote epithelial cell transformation by their continuous exposure to the plethora of luminal bacteria, toxins, and mitogens. Indeed the unfortunate reality that colon cancer is among the neoplasias with the highest incidence in the world may reflect the tremendous burden placed upon this tissue. However, despite the presumed similarity in epithelial cell proliferation in the small and large intestine, the discrepancy in the rate of adenocarcinomas between the two tissues suggests that differences in environmental and cellular mechanisms must contribute to the neoplastic event. In addition, differences in the susceptibility to apoptosis between these tissues has been proposed as one explanation for these clinical observations (94). It has been proposed that between 1 and 72 intestinal epithelial stem cells exist in each crypt (95), and that the proliferative zone consists of a hierarchy of totipotent and pluripotent stem cells (96). The totipotent stem cell might give rise to two distinct pluripotent cell populations: one that undergoes downward migration differentiating into a Paneth cell and another that yields the three upward migrating columnar, goblet, and enteroendocrine cell types (97). It is the latter three cells that experience true “terminal” differentiation at the luminal surface. Two growth factors that differentially regulate the proliferation and death of intestinal epithelial cells are transforming growth factor (TGF)-α and TGF-β. TGF-α stimulates proliferation while TGF-β is a potent cell cycle inhibitor. When both growth factors are added together in cell culture, the antiproliferative activity of TGF-β predominates over the growth promoting activity of TGF-α (98). Paradoxically, the expression of TGF-α is highest in the nonproliferative villus, while production of TGF-β, although controversial, is highest in the crypt (99,–103). The constitutive expression of TGF-β and its dominant effect on epithelial cell proliferation has led to the proposal that remodeling of the epithelium may be regulated in part by the growth inhibitory activity of TGF-β (97). Furthermore, since TGF-β modulates the expression of protein constituents of the basement membrane, and production of TGF-β is regulated by the composition of the extracellular matrix, it is likely that changes in the extracellular matrix play a dynamic role in regulating cell proliferation and death in the epithelium (104). Apoptosis in the Epithelium Two sites of apoptosis are observed in the normal intestinal epithelium: one within the crypt and the other at the luminal surface (105). Detection of an apoptotic cell in the lower third of the crypt is a rare event, observed once every fifth crypt (106), which is not surprising since the apoptotic bodies that form are rapidly phagocytosed by neighboring cells. The presence of spontaneous apoptosis in the crypt, in close proximity to the stem cells, may serve to regulate the rate of differentiation of Paneth, goblet, columnar, and enteroendocrine cells within the epithelium (106,107). These differentiated epithelial cells ultimately endure the same apoptotic fate by migrating upward along the basement membrane and at the villus tip are shed into the lumen as apoptotic cells. The integrity of the epithelium is maintained by neighboring epithelial cells that extend cellular processes underneath the exfoliating cell, forming tight junctions and desmosomes (108). It has been suggested, but not confirmed, that subbasement membrane macrophages extend cytosolic processes beneath the exfoliating epithelial cell and push the dying cell into the lumen (109,110). Initial characterization of the exfoliation process reported DNA fragmentation, a hallmark of apoptosis, at the villus tip using the TdT-mediated dUTP- biotin nick end labeling (TUNEL) technique (111), but some controversy concerning these data remains (107). A potential resolution has recently been proposed (20). Exfoliating cells at the luminal surface exhibit very subtle morphological features of apoptosis (112), while whole cell bodies with condensed chromatin, microvilli, and autophagocytic vacuoles in the cytoplasm are located in the intestinal lumen (113). Surface epithelial cells are shed from the mucosa and thereby detached from the extracellular matrix. While in the gut lumen these cells undergo a process of apoptosis known as detachment-induced cell death (DICD), thus preventing ectopic growth of IEC after exfoliation (104). Apoptosis plays a pivotal role in diseases of the epithelium, the most clinically important and widespread being colorectal cancer. In the gastrointestinal tract, carcinogenesis of the epithelium has been proposed to progress through a series of gene mutations (114), many of which culminate in a loss of apoptotic death. In normal intestinal tissue the proapoptotic Bcl-2 family member, Bak, appears to be highly expressed in epithelial cells (115) with a limited role for the antiapoptotic Bcl-2 protein itself. In contrast, colorectal tumors exhibit an increased expression of Bcl-2 and corresponding decreased expression of Bax, another proapoptotic family member (116,117). These changes in the balance between proand antiapoptotic Bcl-2 proteins are accompanied by a resistance to apoptosis in the developing neoplasia (118,119). Similarly, mutation and expression of the tumor suppressor gene p53 are inversely related to the expression of Bcl-2. Loss of p53 function in adenocarcinomas, particularly after DNA damage, results in genomic instability (120), defective apoptosis (121), and decreased survival (122,123). Furthermore, overexpression of a wild-type p53 cDNA induces apoptosis in a human colonic adenocarcinoma (124). Finally, many of the anecdotal effects of diet on the incidence and progression of colorectal cancer may have a mechanistic explanation in their impact on apoptosis of adenocarcinomas. For example, a high fiber diet, a controversial protector against colon cancer, stimulates increased luminal bacterial fermentation of the short-chain fatty acid sodium butyrate that promotes epithelial cell apoptosis (19,125,126). Many other environmental and clinical insults induce apoptosis in the intestinal epithelium. Nonsteroidal antiinflammatory drugs (NSAIDs) induce damage to the intestinal epithelium (127), which is accompanied by increased apoptosis. Chemoprotective NSAIDs, such as sulindac (128,129), induce apoptosis in colon cancer cells in vitro (130,131) and modulate crypt cell proliferation and apoptosis in a rat model of colon carcinogenesis in vivo (132). Activation of an immune or inflammatory response also initiates apoptosis in the epithelium. For example, apoptosis is increased in graft versus host disease (133), or after invasive bacteria bind to epithelial cells in vitro (134) or crypt cells in vivo (135). The proinflammatory cytokine interferon (IFN)-γ appears to prime adenocarcinoma cell lines for TNF-α and Fas-induced apoptosis (136,–139). Adenocarcinoma cells are often characterized by decreased expression of Fas (140,141) and are thus resistant to immune-mediated apoptosis. Colon cancer cells may additionally escape immune tumor surveillance by mounting a Fas counterattack, by expressing the Fas ligand, which enables them to kill T lymphocytes (142). Finally, the gastrointestinal epithelium is particularly sensitive to apoptosis induced by chemotherapeutic agents and ionizing radiation (143), yet the sites of spontaneous and induced apoptosis are distinct between the large and small intestine (144). Since the frequency and location of apoptotic cells differ between the small bowel and colon, an intriguing theory has been described to explain the higher incidence of cancer in the colon (94). Apoptosis in the ileum is found within the region of the stem cell, while in the large intestine the apoptotic target zone is well above the stem cell region. Therefore, it was suggested that in the small intestine highly proliferative mutated or transformed cells will be rapidly deleted within this apoptotic zone. In contrast, a damaged stem cell that acquires an oncogenic mutation in the large bowel will be able to freely replicate in the crypt before it migrates toward the lumen and encounters the apoptotic zone higher in the colonic crypt, thereby escaping deletion. Apoptosis Regulates Immune Responses Mice that carry spontaneous recessive homozygous mutations in either of two alleles, lpr (lymphoproliferation) and gld (generalized lymphoproliferative disease), develop lymphadenopathy and splenomegaly, associated with the accumulation of CD4- (helper phenotype) CD8- (cytolytic phenotype) T lymphocytes. In addition to the T-cell defect, B cells in these mutant mice accumulate, leading to elevated serum immunoglobulin levels, with a similar increase in antisingle strand and double strand DNA antibodies (145). Genetic and molecular characterization and reconstitution of the lpr and gld mutations demonstrated that the unregulated immunological response was due to loss of function mutations in the Fas and Fas ligand genes, respectively (146,147). The full characterization of these mice revealed that Fas-mediated apoptosis plays a critical role in the regulation of T-cell activation and the deletion of activated and autoreactive T and B lymphocytes (148). These results along with many other experimental models underscored the fundamental importance of apoptosis in maintaining immune homeostasis (149,150). Furthermore, defective apoptosis is believed to contribute to the development of autoimmune diseases (151), such as congenital lymphoproliferative syndrome, asthma, chronic atopic dermatitis, and rheumatoid arthritis (152,–155). In addition, alterations in the balanced expression of proapoptotic and antiapoptotic Bcl-2 family members in T lymphocytes is observed in multiple sclerosis, aging, infectious mononucleosis, and cancer (156,–159). Apoptosis is particularly important in regulating T lymphocytes, which die under a variety of conditions. Greater than 95% of thymocytes that immigrate into the thymus die of apoptosis, after failing to successfully navigate the demands of positive (incorrect specificity of their T-cell antigen receptor) or negative selection (autoreactive T-cell receptor) (13,160). An additional opportunity to eliminate autoreactive T cells by apoptosis occurs in the periphery when they encounter self antigens (160). During an immune response T cells recognize foreign antigenic peptides in the context of the major histocompatibility complex and are stimulated to proliferate, mature, and differentiate into effector cells. This process of clonal expansion is, however, self limited and the fully activated T cell is removed from circulation and tissue by Fas-mediated apoptosis, referred to as activation-induced cell death (AICD) (150,161,162). The cytolytic activity of natural killer (NK) cells and cytotoxic T lymphocytes (CTL), predominantly of the CD8+ phenotype, is realized by inducing apoptosis in the target cell. Two mechanisms, both of which ultimately stimulate caspase-mediated dismantling of the target cell, have been described. Antigen-specific activation of CTLs after engagement of the T-cell receptor by foreign peptide in association with proteins of the major histocompatibility complex induces the expression of Fas ligand (12). Fas ligand engages Fas on the target cell and causes apoptosis (161). Similarly, engagement of the T-cell receptor in CTLs stimulates the degranulation of T cells, leading to the release of perforins and granzymes. Perforins are believed to form pores in the plasma membrane of the target cell through which granzymes enter. Granzyme B is a serine protease with specificity for aspartic acid that proteolytically activates caspase 3, among other proteases, thus initiating cell execution by apoptosis (163,164). There appears to be little cross-talk between these two mechanisms of cytotoxicity. CTLs isolated from perforin-deficient mice kill target cells via the Fas pathway (165,166), and CTLs from gld mice and target cells from lpr mice can lyse or be lysed through the release of perforin and granzyme (167,–169). CD8+ CTLs and NK cells use both the perforin/granzyme and Fas ligand/Fas pathways, while CD4+ CTLs preferentially use the Fas system (13). Since CTLs deficient in both functional perforin and Fas ligand show residual cytotoxic activity (170), other death receptors may also participate, albeit to a much lesser extent, in target cell killing during an immune response. Apoptosis specifically regulates inflammation in the eye and testis, whose immune responses are suppressed by a process identified as immune privilege. While activated leukocytes can enter these tissues, they are rapidly killed by Fas-mediated apoptosis. Constitutive expression of Fas ligand was detected in corneal epithelium and endothelium, iris, and cilliary cells of the eye and the Sertoli cells of the testis (171,172). In addition, immune privilege in these tissues is lost in gld mice (171,172), providing further support for the critical role played by the Fas system in regulating immune reactivity. Apoptosis of Mucosal Lymphocytes: Impact on Inflammatory Bowel Disease While naive T cells in general do not express Fas and are thus resistant to Fas-mediated apoptosis, memory T cells, the predominant cell in the normal intestinal mucosa, express high levels of Fas (150). After engagement of the Fas receptor, not unexpectedly, normal intestinal lamina propria T cells (LPT) rapidly undergo apoptosis (173,174), which is in stark contrast to the observed resistance of LPTs derived from the mucosa of patients with Crohn's disease (CD) (175). In fact, LPTs derived from inflamed CD mucosa are not only resistant to Fasmediated cell death, but also apoptosis induced by engaging the CD2 receptor, nitric oxide, and growth factor deprivation (175,176). The reported decrease in susceptibility to apoptosis for LPTs derived from inflamed CD mucosa was not observed for LPTs derived from ulcerative colitis (UC) patients (175). This finding demonstrates that differences in apoptotic responses in mucosal T cells cannot be attributed solely to intestinal inflammation, but are likely an intrinsic property of the CD T cell. A confirmation of this hypothesis reported that decreased Bcl-2 levels were observed in lamina propria mononuclear cells from UC, but not CD patients (175), which was in agreement with an earlier report comparing only CD with controls (176). Furthermore, lower Bax protein levels and an increase in the ratio of Bcl-xL/Bax proteins in LPTs from CD, but not normal and UC LPTs were described (177). Together these results suggest that the resistance of LPTs from the CD mucosa to apoptosis may be attributed to a relative increase in the balance of Bcl-2 proteins in favor of an antiapoptotic phenotype. One possible function for the high level of Fas expression on normal LPTs (178) and mRNA for Fas ligand in Paneth cells (179) is to limit clonal expansion and the development of self-reactive T cells (149,180,181). It is believed that the T cell in the healthy intestinal mucosa is long lived, possibly to provide protection against the continuous antigenic challenge present in the gut (182). Since normal LPTs express high levels of Fas and are susceptible to apoptosis, other mechanisms must exist in the local mucosa to regulate their rate of turnover (183). Furthermore, the decreased susceptibility of LPTs to apoptosis in CD suggests that these regulatory pathways have been disrupted and that increased T-cell residency in the mucosa may contribute to the chronicity of the inflammatory response. These differences between the two forms of inflammatory bowel disease (IBD) are further supported by the increased loss of epithelium due to apoptosis in UC (184,185) and the proposed role of Fas ligand-bearing mucosal T cells mediating this process in UC, but not CD lesions (186,187). An imbalance in the regulation of apoptosis in many other cell types of the intestinal mucosa has also been implicated in chronic intestinal inflammation. The accumulation of neutrophils (PMNs) in the mucosa in IBD may be due to a reduction in the degree of PMN apoptosis. PMNs cultured in conditioned media from mucosal explants of patients with active IBD exhibited reduced apoptosis (188). Elevated levels of granulocyte colony stimulating factor (G-CSF) and granulocyte-macrophage colony stimulating factor (GM-CSF) were detected in IBD tissue, and the resistance of neutrophils to apoptosis appeared to be mediated by G-CSF (188). Markers for cells undergoing apoptosis were detected on twice as many colonic macrophages analyzed in situ and isolated from inflamed mucosal tissue than on macrophages from control tissue (189). Similarly, elevated expression of mRNA for granzyme B was colocalized with increased numbers of apoptotic cells in focal lesions of the lamina propria from CD, but not UC patients (190). Unfortunately, these current data paint a paradoxical picture of the role of apoptosis in IBD, demonstrating both increased and decreased cell death in the inflamed intestine. Over the past 5 years there has been an explosion in the number of mouse models of IBD, many of which have provided new insights into chronic intestinal inflammatory disease pathogenesis. Yet, consistent with the contradictory effect of apoptosis in human IBD, seemingly conflicting results have also been reported on the rate of cell death in the inflamed murine lamina propria. When CD4+ T cells are transferred into SCID (severe combined immunodeficiency) mice, a rapid increase in both cell proliferation and apoptosis is observed in the inflamed colonic mucosa (191). It is unclear which of these responses dominates during inflammation. In the TNBS (trinitrobenzene sulfonic acid)-induced model of colitis, treatment with a neutralizing antibody to interleukin (IL)-12 not only blocks disease progression, but also increases the number of apoptotic (TUNEL+) lamina propria cells, a process that appears to be Fas mediated (192). Where will the Future Lead US? The exquisite detail in the activation pathways that regulate cell death, as described in the first four sections of this review, demonstrate that molecular genetic and biochemical characterizations of apoptosis are making huge advances toward our understanding the machinery of cell death. In contrast, knowledge on the pathology of dysfunctional apoptosis, especially in the IBD, lags far behind. Is UC due to excessive apoptosis of the epithelium mediated by inflammatory cells? Can CD be attributed to decreased susceptibility of mucosal T cells to apoptosis leading to prolonged inflammatory responses? These simple solutions to incredibly complex diseases most likely disguise a far more intricate web of cellular, immunological, and neurological responses that develop in the chronically inflamed gut. 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TI - Apoptosis: Implications for Inflammatory Bowel Disease
JF - Inflammatory Bowel Diseases
DO - 10.1097/00054725-200008000-00006
DA - 2000-08-01
UR - https://www.deepdyve.com/lp/oxford-university-press/apoptosis-implications-for-inflammatory-bowel-disease-uurgbNW26k
SP - 191
EP - 205
VL - 6
IS - 3
DP - DeepDyve
ER -