Enterocyte apoptosis and TPN-associated intestinal mucosal atrophy: a view of the chicken or the egg?

October 2003

ENTEROCYTE APOPTOSIS AND TPNASSOCIATED INTESTINAL MUCOSAL ATROPHY: A VIEW OF THE CHICKEN OR THE EGG? Yang H, Fan Y, Teitelbaum DH (Department of Surgery, University of Michigan Medical School and CS Mott Children’s Hospital, Ann Arbor, Michigan). Intraepithelial lymphocytederived interferon-gamma evokes enterocyte apoptosis with parenteral nutrition in mice. Am J Physiol Gastrointest Liver Physiol 2003;284:G629 –G637. The homeostatic balance between the rate of cell production and cell death is especially pertinent in the intestine because the rates of enterocyte turnover are extremely high. Total parenteral nutrition (TPN) is associated with the development of intestinal mucosal atrophy. This atrophy might possibly play a role in attenuating the intestinal barrier to bacteria and bacterial products, thus contributing toward an increased risk for sepsis and/or liver injury in parenterally fed patients. It is therefore clinically relevant to more fully understand how TPN perturbs the vital balance of enterocyte turnover to promote intestinal mucosal atrophy. In addition to reduced enterocyte proliferation, TPN is associated with elevated rates of enterocyte apoptosis (J Nutr 2002;132:2010 –2014). In a recent report, Teitelbaum et al. provide an important new insight into the pathogenesis of intestinal mucosal atrophy by exploring a potential mechanism for the TPN-associated accelerated enterocyte apoptosis (Am J Physiol Gastrointest Liver Physiol 2003;284:G629 –G637). In this study, they expand on their prior observations of increased interferon gamma (IFN-␥) messenger RNA expression within intraepithelial lymphocytes (IEL) harvested from the intestine of mice receiving TPN (J Surg Res 1998;79:91–96). They specifically test the hypothesis that the increased IEL expression of IFN-␥ activates enterocyte apoptosis. In addition, they explore the notion that the apoptosis activation by IFN-␥ occurs via the Fas/FasL system. In the first series of experiments, TPN or crystalloid solution was intravenously infused to mice for 7 days. In the control group, standard laboratory chow and water were provided ad libitum. The animals in both groups were estimated to have received similar calories. Mucosal atrophy was confirmed by a 32% decrease in the jejunal villus height from the animals receiving TPN. In addition, TPN was associated with a 45% reduction in rates of enterocyte proliferation and a 3-fold increase in enterocyte apoptosis. Next, IEL expression of IFN-␥ protein was measured by enzyme-linked immunosorbent assay and found to be 3.5-fold higher in the TPN group. Using flow cytometry, the investigators further characterized the IEL subpopulations and found that TPN significantly increased the IFN-␥ expression within CD8⫹ and CD3⫹, but not CD4⫹, cells. Finally, whereas Fas expression was found to be unchanged, the expression of FasL in the TPN animals was found to be significantly elevated. To

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determine whether enterocytes (expressing Fas) were more vulnerable to apoptosis induced by IEL-derived FasL expression, enterocytes were isolated from TPN and control mice and incubated with an anti-Fas antibody. The engagement of Fas by this antibody resulted in higher rates of enterocyte apoptosis from the TPN mice versus the control group. In the next series of experiments, TPN was administered to IFN-␥ null and wild-type mice. In the absence of IFN-␥ expression, villus heights were taller and TPN did not activate enterocyte apoptosis to the same degree as in the control group. Further, the expected induction of FasL expression by TPN within IELs of the IFN-␥ null mice did not occur. In addition, the induction of apoptosis by anti-Fas antibody in isolated enterocytes derived from the IFN-␥ null mice was higher when compared with controls. In the final experiments, TPN was given to mice deficient in either Fas (lpr) or FasL (gld) expression. Rates of apoptosis in these 2 different mice strains were significantly lower when compared with the wild-type mice. Further, villus heights were greater in both strains when compared with the control mice given TPN. Comment. These elegant studies provide important mechanistic insight into the pathogenesis of increased enterocyte apoptosis and mucosal atrophy that occurs in association with TPN. The experimental techniques are sophisticated and well designed, and make a strong case for INF-␥ and enterocyte death receptor engagement (particularly Fas and IEL-derived FasL) as primary mediators of apoptosis activation. Several points should be considered in the interpretation of the data from this report. In comparison with wild-type mice, TPN was associated with reduced enterocyte apoptosis in each of the strains of mice deficient in the expression of IFN-␥, Fas, or FasL. However, rates of apoptosis were not completely normalized when compared with control animals not receiving TPN. This would suggest (as the investigators appropriately discuss) that perhaps other death receptor–mediated pathways such as tumor necrosis factor may be involved to direct enterocyte apoptosis. What was not mentioned is the role for the intrinsic (non– death receptor) pathway in this model of increased enterocyte apoptosis. In particular, the execution of apoptosis may be significantly affected by alterations in the expression of various bcl-2 family members. Another experimental paradigm for increased enterocyte apoptosis is the adaptation response of the remnant bowel to massive intestinal resection (J Gastrointest Surg 1998;2:44 – 49). Under these conditions, the expression of the proapoptotic gene bax is increased simultaneously with a reduced expression of the antiapoptosis gene bcl-w (J Gastrointest Surg 2000;4:93–100). Further, the activation of enterocyte apoptosis after intestinal resection is prevented in bax-null mice (Surgery 2000;128:165–170). Therefore, it may be informative to monitor altered expression profiles of the various bcl-2 homologues during parenteral nutrition in this model. In addition to local proapoptotic factors within the intestine, the genesis of TPN-associated mucosal atrophy may be caused by reduced secretion of multiple enterotrophic endocrine and exocrine-derived peptides and growth factors that are up-regulated in response to oral feeding. Thus, it should be considered that the absence of a luminal or endocrine prosurvival growth factor or peptide (as opposed to the local production of a proapoptotic ligand as proposed in this study) may be more decisive in the genesis of atrophy. Even without endocrine/exocrine secretion, enteral nutrition alone probably ac-

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counts, at least in part, for the decreasing gradient of intestinal mucosal thickness that occurs from the duodenum to the terminal ileum. Along these lines, the authors compared mice that were parenterally fed with mice that were fed the same calories enterally. It is therefore impossible to determine whether the reported findings were caused by lack of trophic luminal nutrient, diminished trophic endocrine/exocrine secretion because of no oral feeding, or even the parenteral nutrition solution. An additional experimental group of animals receiving intravenous crystalloid solution, but no oral intake, might have been useful. While beyond the scope of the current study, which focuses on the regulation of apoptosis, consideration must be afforded to the effect that TPN has on rates of enterocyte proliferation. Because the investigators showed significantly reduced rates of proliferation in normal mice receiving TPN, it is unclear as to the magnitude that this perturbation undoubtedly contributes toward the development of intestinal mucosal atrophy. Similarly, altered rates of enterocyte proliferation might have accounted for the intestinal phenotype observed in the mice deficient in IFN-␥, Fas, or FasL expression. It would be interesting to compare rates of enterocyte proliferation in these mice with the wild-type mice receiving TPN. Finally, it is presently unclear as to whether the changes in inflammation and/or apoptosis described in the present report are the cause or effect of the intestinal mucosal atrophy. The investigators imply that these alterations are fundamental toward the development of mucosal atrophy. However, the animals were studied only after the atrophy had already taken place. The atrophic mucosa would predictably be more susceptible to the translocation of bacteria and/or bacterial products from the intestinal lumen. As such, it would not be surprising that extrinsic death receptor activation is involved in enterocyte apoptosis once the atrophy had already developed. Future studies at earlier time points to link the timing of IFN-␥, Fas, or FasL expression, increased apoptosis, and the development of mucosal atrophy will be critical to elucidate more fully the pathogenesis of intestinal mucosal atrophy associated with parenteral nutrition. BRAD W. WARNER, M.D.

Reply. Despite the well-appreciated observation that administration of TPN results in increased epithelial cell apoptosis, little is known of the cause of this process. As was pointed out, our study investigated only one potential mechanism by which epithelial cell apoptosis may be mediated. Clearly the intrinsic pathway for apoptosis may also have relevance. We have recently addressed this in a preliminary report by examining the expression of the bcl-2 family member (J Ped Surg 2003;38:92–96). In this study, we found that the antiapoptotic protein bcl-2 significantly declined with TPN administration, suggesting that the loss of its expression may also contribute to the observed increase in epithelial cell apoptosis. The expression of a multitude of endocrine and exocrine factors has been shown to be disturbed with administration of TPN. As mentioned, TPN results in numerous changes in both phenotype and function of intraepithelial lymphocytes (Dig Dis 2002;47:1147–1157). We have recently noted that the observed alterations in intraepithelial lymphocytes do not occur if mice receiving TPN are also permitted to take in an enteral diet (unpublished observation). This suggests that key factors, via enteral intake, mediate such changes, rather than through the action of the TPN solution itself. The decline in epithelial cell proliferation that we observed is more than likely caused by a multitude of factors. Interestingly, we have shown that ␥␦ T-cell receptor IEL expression of keratinocyte growth factor (KGF) is markedly diminished with TPN administration.

Additionally, the exogenous administration of human recombinant KGF (rHuKGF; Amgen Corp., Thousand Oaks, CA) results in a prevention of this decline in epithelial cell proliferation (JPEN J Parenter Enteral Nutr 2002;26:333–341). This suggests that such an agent may have a potentially beneficial action with the administration of TPN. DANIEL H. TEITELBAUM, M.D. HUA YANG, M.D., PH.D.

PROPHYLACTIC PANCREATIC DUCT STENTING: A PANACEA? Fazel A, Quadri A, Catalano MF, Meyerson SM, Geenen JE (St. Luke’s Hospital, Milwaukee, Wisconsin). Does a pancreatic duct stent prevent post-ERCP pancreatitis? A prospective randomized study. Gastrointest Endosc 2003;57:291–294. Acute pancreatitis is the most common complication of endoscopic retrograde cholangiopancreatography (ERCP). Although there exists a risk of pancreatitis regardless of the underlying pathology or indication for the procedure, patients with sphincter of Oddi dysfunction (SOD) and those with papilla that are difficult to cannulate are at exceptionally high risk. Such patients are also more likely than others to develop severe pancreatitis. Fazel et al. report their experience with pancreatic duct stenting to reduce the risk of post-ERCP pancreatitis in high-risk patients. Over a 6-year period, patients with difficult cannulation at ERCP, or who had undergone sphincter of Oddi manometry (SOM) or endoscopic sphincterotomy were prospectively randomized to either short-term placement of a pancreatic duct stent or no stent on completion of ERCP. Eight percent of the patients had sphincterotomy alone as the risk factor. Exclusion criteria included acute or chronic pancreatitis at the time of the procedure and failure of cannulation of the pancreatic duct. Seventy-six patients at high risk for post-ERCP pancreatitis were enrolled and randomized. Two patients in the stent group were excluded because cannulation of the pancreatic duct deep enough to allow stent placement was not achieved. Thirty-six patients underwent stent placement and 38 patients were assigned to the control group without a pancreatic duct stent. Post-ERCP pancreatitis occurred in 2 (5%) of the patients in the stent group and 10 (28%) of the patients in the control group (P ⬍ 0.05). Mean duration of hospitalization was also shorter in the stent group at 2.5 days versus 5.5 days (P ⬍ 0.20). Both cases of pancreatitis in the stent group were mild, whereas half of the cases of pancreatitis in the control group were moderate or severe. The investigators conclude that a pancreatic duct stent reduces the frequency and severity of post-ERCP pancreatitis and recommend a pancreatic duct stent be placed after ERCP in patients at high risk for procedure-related pancreatitis. Comment. The end result of pancreatitis regardless of cause including post-ERCP pancreatitis involves the activation of trypsinogen to trypsin. Trypsin in turn activates other digestive enzymes including