Regulation of Intestinal Epithelial Cell Apoptosis and the Pathogenesis of Inflammatory Bowel Disorders

The intestinal epithelium is a highly dynamic tissue, characterized by a remarkable turnover rate and complete renewal of the entire cell population every 72 to 96 hours (1). Normal intestinal functions and morphology depend on a delicate balance in cell types and numbers from immature proliferating crypt cells to highly differentiated enterocytes that undergo apoptosis at the end of their life span. Morphologically and functionally, the intestinal epithelium can be separated into three different compartments: The basal crypt compartment comprises the immature stem cells with a high mitotic activity. Their daughter cells gradually differentiate into mature cell types (enterocytes, goblet cells, or enteroendocrine cells) during their migration along the crypt–villus axis (compartment of differentiating cells). After reaching the villus tip in the small bowel or crypt summit in the colon (compartment of mature, absorptive cells), these highly differentiated postmitotic cells die via apoptosis and are finally extruded into the gut lumen (2–5). Apoptosis (from Greek for “falling off” of leaves), the process associated with physiologic cell death, is the end-stage of migration and differentiation of intestinal epithelial cells along the crypt–villus axis. This programmed cell death is a counterpart to crypt-cell proliferation. A homeostatic balance between proliferation and apoptosis is essential to normal gut morphology and function (6). Exaggerated or accelerated cell dying via apoptosis results in a paucity of mature enterocytes leading to villus atrophy and epithelial destruction. However, excessive proliferation of immature crypt cells causes crypt hyperplasia, leading to loss of normal absorptive functions and potentially increased risk of tumorigenesis (7). Preferential differentiation of crypt cells rather than their self-renewal would result in a depleted stem-cell pool. An efficient and very closely regulated control system is a prerequisite, guaranteeing a functional steady state, and is the basis of normal intestinal functions. Proliferation, differentiation, and apoptosis of intestinal epithelial cells are tightly regulated by a variety of growth factors, cytokines, hormones, luminal nutrients, and mesenchymal structures. However, we only begin to understand the complex interplay of the various regulatory factors and their molecular mechanisms. The gut-associated lymphoid tissue constitutes an important source of cytokines and growth factors. The proximity between epithelial cells and immune cells, especially intraepithelial lymphocytes, allows an intensive bidirectional exchange between these two systems (8–10). Recent reports confirmed the necessity of normal mucosal T-cell function and cytokine production for physiologic growth and maturation of the intestine (11). Furthermore, intestinal epithelial cells themselves produce growth factors and cytokines that may act in an autocrine and a paracrine way (12–14). Thus, a complex network of enterocyte/enterocyte and lymphocyte–enterocyte interactions via cytokines and growth factors is essential for homeostasis of enterocyte number and function. Research during the past few years has shown that the homeostatic balance between proinflammatory and antiinflammatory factors is markedly disturbed in the intestinal mucosa of patients with immune-mediated bowel disorders, such as Crohn disease (CD), ulcerative colitis (UC), or celiac disease (15,16). Increasing evidence suggests that cytokines, released locally from infiltrating immune cells recruited to the sites of active inflammation, mediate the pathologically altered intestinal epithelial cell turnover and the high apoptosis rate observed under these inflammatory conditions (16). In this article, we give an overview of the role of intestinal epithelial cell apoptosis for homeostatic epithelial cell turnover. We will update our understanding of regulation and modulation of enterocyte apoptosis by cytokines, especially under inflammatory conditions. Finally, we discuss the implications of cytokine-induced intestinal epithelial cell apoptosis in the pathogenesis of inflammatory bowel disorders. REGULATING ENTEROCYTE APOPTOSIS UNDER PHYSIOLOGIC CONDISIONS Under normal conditions, apoptotic intestinal epithelial cells are mainly found at the villus tip / crypt summit, whereas in the basal crypt compartment, only slight apoptosis occurs in stem cells (5,17–20). Scant information exists on the induction of apoptosis after a mature enterocyte–colonocyte reaches the villus tip / crypt summit. A current hypothesis is that factors implicated in the maturation and differentiation process of intestinal epithelial cells also activate the ultimate cell-death machinery. Transforming growth factor β (TGF-β), implicated in enterocyte maturation (21), has been proposed as one factor with such properties. In vitro, gastric and colonic epithelial cells of cancer origin rapidly underwent apoptosis when stimulated with TGF-β, supporting this hypothesis (22,23). Analysis of mRNA expression and protein concentration of TGF-β along the crypt–villus axis revealed a region-specific pattern, with a gradual increase from crypt cells to maximal expression in villus enterocytes (24). Based on this pattern of distribution and additional in vitro analyses, it was suggested that TGF-β arrests enterocyte growth when sufficient numbers of maturing enterocytes leave the basal proliferative compartment. It is tempting to speculate about the physiologic role of TGF-β, aside from its antiproliferative properties, in triggering apoptosis of mature enterocytes. However, further factors and their molecular mechanisms that couple immature crypt-cell proliferation with mature intestinal epithelial-cell apoptosis remain unknown. During the past few years, the molecular mechanisms of apoptosis have become clearer. Apoptotic cell death, in contrast with necrosis, does not provoke an inflammatory reaction in the surrounding tissue (Fig. 1). Thus, the cell membrane remains intact, avoiding leakage of potentially inflammatogenic cytoplasmic enzymes and proteins, such as proteases, ribonuclease, and so forth. However, apoptotic cells are characterized by the loss of their cell membrane asymmetry, with the appearance of phosphatidyl serine, normally located at the inner leaflet of the plasma membrane, on the outer surface (25). This externalization of phosphatidyl serine, triggered by a specific flipase, is an important signal for macrophages or neighboring epithelial cells to eliminate these early apoptotic cells by phagocytosis—a process that takes only a few hours. A further characteristic of apoptosis is the nuclear condensation and DNA fragmentation by internucleosomal cleavage, leading to the appearance of fragments of 180 base pairs and their multiples, resulting in a characteristic DNA-ladder with electrophoresis (26). This degradation of DNA is the basis for the formation of so-called apoptotic bodies (Fig. 2). Understanding the molecular mechanisms underlying these morphologic changes and the intracellular apoptotic machinery is a field of active investigation.FIG. 1.: Apoptotic versus necrotic cell death: Apoptosis is an energy-dependent silent form of cell death that does not create an inflammatory reaction in the surrounding tissue, in contrast to necrosis. During apoptosis, the cell membrane remains intact for a long time. However, at the onset of apoptosis, the cell membrane characteristically looses its asymmetry with exposure of phosphatidyl serine (PS) on the outer leaflet of the plasma membrane. Further morphologic characteristics of apoptosis include nuclear condensation and fragmentation leading to the formation of apoptotic bodies. During necrosis, the energy-depleted cell swells until the membrane ruptures and the whole cytoplasmic content leaks into the surrounding tissue, leading to an inflammatory reaction.FIG. 2.: Morphologic characteristics of apoptotic enterocytes: After staining with the DNA-dye HOECST 33342, nuclear condensation, fragmentation of the DNA, and formation of apoptotic bodies (arrow) can be observed in apoptotic human intestinal epithelial cells. In contrast, normal enterocytes (left side) display an ovaloid, regular nucleus.To date, several different apoptosis pathways are known. One important signaling cascade is the caspase cascade, activated by the formation of a death inducing signaling complex (27). This death-inducing signaling complex is a complex of activated death receptor, such as Fas or p55 tumor necrosis factor (TNF) receptor, an adapter protein called Fas-associated death domain and caspase-8. After a death-inducing signaling complex is formed, caspase-8 is activated, allowing it to propagate the apoptotic signal. Caspases are constitutively present in the cytosol as zymogens, single-chain proenzymes (28). In response to various pro-apoptotic stimuli, specific caspases are activated to become fully functional proteases, first by a proteolytic cleavage to divide the single chain into a large and a small subunit and a second cleavage to remove the prodomain (N-terminal). The subunits assemble into tetramers with two active sites (28). This proteolytic activation of caspases occurs in a cascadelike fashion, whereby these proteases serve as one another's substrates or autocatalytically as their own substrates, thereby allowing efficient propagation and enhancement of pro-apoptotic stimuli. In a final step, several key substrates, such as structure proteins (i.e., Lamin A), repair enzymes (i.e., polyadenosine-diphosphate-ribosyl polymerase) or executioner enzymes (i.e., DNA-fragmentation factor) are proteolytically cleaved (29–31), resulting in definitive apoptotic cell death (Fig. 3). This intracellular apoptosis machinery is fully functional in intestinal epithelial cells, as we recently demonstrated (32). In Caco-2 cells, caspase-3–mediated degradation of polyadenosine-diphosphate-ribosyl polymerase is critical for apoptosis (33). Furthermore, we showed in nontransformed human intestinal epithelial cells, that caspase-3 and ICE (interleukin-1 converting enzyme) caspases are the main executioner caspases in response to death-receptor–induced apoptosis (Ruemmele et al., unpublished data).FIG. 3.: Schematic overview of the intracytoplasmic signaling-cascade of the death receptors p55 TNF-R and Fas. The receptor's cytoplasmic death domain (DD) binds intracellular adapter molecules, also characterized by a DD, such as TRADD (TNF-R associated death domain) or FADD (Fas-associated death domain). TRADD serves as an adapter platform for various other molecules. FADD is necessary to trigger apoptosis in response to the p55 TNF-R and Fas via a death-inducing signaling complex (DISC), resulting in activation of caspase-8 and the classic caspase cascade. Caspase-3 serves as the main executionary caspase in intestinal epithelial cells. The bcl-2 gene family (bcl-2, bak, bax) regulates a cell's threshold at which it undergoes apoptosis after specific stimulation.Recent studies by Grossman et al. (34) showed that intestinal epithelial cells react with activation of their intracellular apoptosis-signaling machinery on loss of physiologic contact with the basal membrane. In this experimental model, the caspase cascade was activated within 30 minutes of detachment from the basal membrane. Another important regulatory pathway of apoptosis involves the proteins of the bcl-2 gene family. Except for bax, they are all integral membrane proteins in the mitochondria, the endoplasmic reticulum, or the nuclear envelope (35). These proteins form heterodimers of an anti-and pro-apoptotic member, thereby titrating each other's function. Bcl-2 and bcl-xl potently inhibit apoptosis, whereas bax, bak, and bcl-xs are important pro-apoptotic molecules (36). The ratio between anti-and pro-apoptotic members defines a cell's threshold to undergo apoptosis on specific stimuli. Bax acts directly on mitochondria by binding to the permeability transition pore complex, leading to increased mitochondrial membrane permeability (37). Three-dimensional analysis suggested that some bcl-2 family members form ion channels upon homodimerization (38–40), potentially changing the function of mitochondria, critical to generating energy. These pores may produce leakage of the outer membrane, allowing the exit of factors that activate the intracytoplasmatic caspase cascade. In fact, translocation of cytochrome c from the mitochondria into the cytosol was recently found to induce apoptosis through the formation of a so-called apoptosome (a complex of cytochrome c, caspase-9 and Apaf-1). In the colon, high bak expression positively correlates with apoptosis (41). However, in the basal crypt compartment, bcl-2-expression, a major inhibitor of apoptosis, predominated (42). Recently, we showed in the Caco-2 cell model that neo-expression of bak is critical to activating the caspase cascade in response to butyrate-induced apoptosis. On inhibition of protein synthesis in this in vitro model, no apoptotic response was observed, confirming the regulatory function of these molecules in enterocyte/colonocyte apoptosis (33). Important differences in the pattern of bcl-2 family expression have been observed between the small and large bowel (43). This difference has given rise to speculations for the higher frequency of carcinoma in the colon compared with the small bowel. Little is known about regulation of the expression of bcl-2 family proteins in the gut. INTESTINAL EPITHELIAL CELL AND LYMPHOCYTE APOPTOSIS IN INFLAMMATORY BOWEL DISEASE The homeostatic, physiologic intestinal epithelial cell turnover is markedly disturbed in the acutely inflamed intestinal mucosa of patients with immune-mediated bowel disorders (6). Macroscopically, the intestinal mucosa is characterized by an inflammatory reaction along with erosions or ulcerations (44). In UC and celiac disease, histologic analyses revealed a dramatically increased number of dying intestinal epithelial cells at the site of acute inflammation, characterized by an inflammatory infiltrate in the form of activated lymphocytes, neutrophils, and monocytes (17,18,45). However, crypt elongation is also frequently encountered in inflammatory bowel disease (IBD) tissue, indicating a compensatory, hyperproliferative state (6). Further analysis of the underlying mechanisms revealed that intestinal epithelial cells die preferentially by apoptosis in immune-mediated bowel disorders. The physiologic pattern of apoptotic cells, normally restricted to the villus tip / crypt summit, is completely disturbed, with a great number of apoptotic cells along the entire crypt–villus axis (17,45). This abnormal increase in premature death of differentiating enterocytes by apoptosis, combined with the excessive proliferation of immature crypt cells, leads to functional loss of normal absorptive capacity along with inappropriate secretion of intestinal fluid and electrolytes, typical of immature crypt epithelial cells (46). For the patient, this manifests clinically in malabsorption and diarrhea, along with abdominal cramps, excessive gas, and bloating. Recent research suggests that in patients with CD, regulation of apoptosis in T cells is defective, whereas enhanced intestinal epithelial-cell apoptosis was a predominant feature in UC (47–49). Isolated lamina propria lymphocytes from patients with CD were highly resistant to Fas-induced as well as CD2-mediated apoptosis (47,48), whereas T cells from healthy controls and patients with UC were highly susceptible to apoptosis. Lamina propria lymphocytes from patients with CD displayed an anti-apoptotic feature of the blc rheostat with clear up-regulation of anti-apoptotic bcl-2 and bcl-xl, protecting these cells from apoptosis (47). Recently, Atreya et al. (50) confirmed these findings of T-cell resistance to apoptosis in patients with CD and in experimental animal models of colitis. They discovered that the interleukin-6 trans-signaling pathway (a complex of IL-6 and soluble IL-6 receptor activating the STAT-3 signaling pathway) was an important regulatory mechanism protecting these cells from apoptosis. Blockade of this pathway in these colitis animal models was effective in reducing the inflammatory reaction. Therefore, a new approach for treating CD was proposed by inhibiting this IL-6 trans-signaling pathway to restore normal lamina propria T-cell apoptosis. CYTOKINE REGULATION OF INTESTINAL EPITHELIAL CELL APOPTOSIS Alterations in the local production of cytokines in IBD (15,16) are crucial to inducing morphologic changes in the intestinal mucosa. Among the huge array of cytokines and other inflammatory mediators (IL-1, IL-6, IL-8, monocyte chemoattractant protein-1, RANTES, etc.) studied in various animal models and tissues of patients with IBD, there is increasing evidence that TNFα and interferon γ (IFNγ) act as key mediators of these inflammatory disorders (51–54). Tissue levels and the expression/secretion of TNFα and IFNγ were significantly higher in zones of acute inflammation compared with non-IBD tissues (51,53). Recent reports of the successful induction of remission in otherwise treatment-resistant patients with CD using chimeric anti-TNFα antibodies (55,56) further support the role of TNFα as a critical inflammatory mediator. In some patients, a single injection of anti-TNFα antibodies resulted in prompt clinical improvement with normalization of disease activity index scores. Our understanding of the underlying molecular mechanisms of how TNFα contributes to intestinal inflammation and damage of the intestinal epithelium remains limited. Research in our laboratory showed that at the mRNA and protein level, normal human intestinal epithelial cells express p55 and p75 TNF-R under physiologic conditions (Ruemmele et al., submitted). Functional studies show that TNFα, especially in combination with IFNγ, on nontransformed human or intestinal epithelial cell turnover intestinal epithelial cell and intestinal epithelial cell (Ruemmele et al., submitted). IFNγ no with cytokines apoptosis in a with than apoptotic cells after hours in in vitro Analysis of the underlying molecular mechanisms showed that the caspase cascade is directly activated in response to of the main executioner caspase-3 with a highly specific the pro-apoptotic of TNFα, indicating the necessity of a functional in enterocytes (32). In the results were the pro-apoptotic of TNFα on intestinal epithelial cells These are of in vitro analyses with cells, which not the physiologic Based on from nontransformed cell such as human intestinal epithelial cells, highly susceptible to apoptosis, one speculate that anti-TNFα acts not only on the between cells themselves also directly intestinal epithelial cells. In the past few years, other members of the family were shown to induce apoptosis on specific One such receptor is the Fas receptor, on all cells, whereas the specific the Fas is mainly by epithelial cells constitutively express Fas (Fig. colon we observed a difference in the of Fas with a signal on mature epithelial cells compared with immature crypt cells, in with other reports Recently, et al. that the Fas mechanism is potentially in the pathogenesis of lymphocytes Fas are recruited to the zones of acute In fact, on with an rapidly underwent apoptosis. The apoptotic response to specific of Fas in normal colonic is markedly increased in the of the proinflammatory cytokines IFNγ or we showed by in apoptotic cells were all along the after with Fas and IFNγ One of the underlying molecular mechanisms of this increased to Fas-induced apoptosis is cytokine-induced up-regulation of Fas. mechanisms are the up-regulation of expression of caspases These may be important in the pathogenesis of In with our in vitro increased Fas expression was in the inflamed tissue of patients with IBD Therefore, we speculate that IFNγ or TNFα, by the infiltrating cells, by this to intestinal epithelial cell of Fas expression in intestinal epithelial cells by inflammatory expression of Fas on human intestinal epithelial cells was and by after specific of Fas. expression was significantly after with interferon The combination of interferon γ and showed a with an up-regulation of than The signal of membrane staining also et al. mechanism of intestinal epithelial cell apoptosis is by the threshold for apoptosis on the bcl-2 gene family is a major of apoptosis. Therefore, inflammatory factors may the balance between anti-apoptotic members of this bcl-2 family in enterocytes. In fact, et al. recently demonstrated in a human colon cancer cell that TNFα and IFNγ apoptosis by anti-apoptotic bcl-2 expression with pro-apoptotic These that inflammatory cytokines may on the intracellular level, the threshold at which intestinal epithelial cells undergo apoptosis on specific AND In apoptosis of intestinal epithelial cells is an important regulatory mechanism that homeostasis of this highly dynamic Apoptosis is a to proliferation of intestinal epithelial cells. This mechanism to a distribution and postmitotic absorptive intestinal epithelial cells as to normal intestinal functions. specific intestinal epithelial cells activate their intracellular apoptosis machinery to programmed cell The physiologic pattern of apoptosis in intestinal epithelial cells and in lamina propria T cells to be markedly disturbed under immune-mediated conditions. Increasing evidence that T-cell resistance to apoptosis is a main feature of Crohn disease, whereas intestinal epithelial cell apoptosis is in members of the are important and of apoptosis in intestinal epithelial cells under such inflammatory conditions. Recent in vitro and in research confirmed that TNFα and Fas to the up-regulation of enterocyte apoptosis. These factors, from activated cells, activate specific death which are directly or to the intracellular caspase cascade, for apoptosis. exists between different members of the which each on enterocytes. These and further understanding the disease mechanisms that to intestinal inflammatory about the pathogenesis of immune-mediated bowel disorders remain A understanding of the physiologic and regulation of enterocyte turnover and especially apoptosis and the underlying signaling pathways will to the of for a of of this research have resulted in the clinical of anti-TNFα antibodies to patients with otherwise treatment-resistant This may active in the intestinal resulting in of the inflammatory cascade. Another hypothesis the of anti-TNFα antibodies is the of activated lymphocytes by inducing apoptosis. these on cells, anti-TNFα also may directly act at the of intestinal epithelial cells. Another be to directly with the turnover rate of intestinal epithelial cells. of the apoptosis rate with antibodies to or of executioner caspases by be a for patients with with these recently apoptosis in the injection of a caspase inhibitor significantly the rate of apoptotic in this animal However, further and intensive research is necessary to the and clinical of these at the of intestinal epithelial cells in