Environmental stress–induced gastrointestinal permeability is mediated by endogenous glucocorticoids in the rat

GASTROENTEROLOGY 2 0 0 0 ; 1 1 9 : 1 0 1 9 - 1 0 2 8

Environmental Stress-Induced Gastrointestinal Permeability Is Mediated by Endogenous Glucocorticoids in the Rat JON B. MEDDINGS and M. G. SWAIN Gastrointestinal Research Group, University of Calgary, Alberta, Canada

Background &Aims: Abnormal presentation of luminal constituents to the mucosal immune system, caused by dysfunction of the intestinal epithelial barrier, is a candidate theory for the cause of Crohn's disease. Increased epithelial permeability is found in subgroups of patients at high risk for the development of Crohn's disease and has been found to precede disease recurrence. Clinical observations have suggested that disease recurrence can follow times of increased psychological stress, although the underlying mechanism remains obscure. We hypothesized that environmental stress increases gastrointestinal permeability, Methods: We evaluated site-specific gastrointestinal permeability after application of graded levels of stress in rats. Results: Increased epithelial permeability after stress was shown in all regions of the gastrointestinal tract and seemed to be mediated by adrenal corticosteroids. Stress-induced increases in epithelial permeability disappeared after adrenalectomy or pharmacologic blockade of glucocorticoid receptors. Dexamethasone treatment of control animals increased gastrointestinal permeability and mimicked the effects of stress. Conclusions: Psychological stress may increase gastrointestinal permeability, allowing luminal constituents access to the mucosal immune system. This provides a potential mechanism for the observation of stress-induced disease recurrence in Crohn's disease.

he relationship between psychological stress and inflammatory bowel disease (IBD) has a long history. It is clear that chronic disease may create stress for an individual; however, it also appears that exogenous stress can precipitate disease recurrence in patients with Crohn's disease. ~ 3 Furthermore, while anti-inflammatory therapy is the mainstay for disease therapy, specific measures, targeted at reducing stress, are of value. 4 This paradigm of stress-induced disease reactivation also seems to hold true in animal models of IBD. In animals in which recovery from trinitrobenzene sulfonic acidinduced colitis has been allowed to take place, reactivation of disease can be initiated by stress. 5 The mechanisms underlying these observations have not been clearly delineated. Evidence increasingly sug-

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gests that increased epithelial permeability plays an important role in the initiation and perpetuation of Crohn's disease, presumably by allowing abnormal presentation of luminal constituents to the mucosal immune system. The data in support of this are derived from both animal models and humans. In animals, bypassing the epithelial barrier and injecting luminal bacterial wall extracts directly into the submucosa initiates a chronic relapsing inflammatory syndrome similar to Crohn's disease. 6 Furthermore, simply decreasing epithelial barrier function in mice by altering intracellular adhesion molecule expression prompts the spontaneous generation of intestinal inflammation? Decreasing disease expression has not yet been shown by altering epithelial permeability, but altering luminal constituents can prevent or delay disease. This has been shown in genetically engineered animals at high risk of developing spontaneous inflammation resembling IBD. Housing these animals in a germfree environment dramatically reduces or delays the onset of disease. 8 Similar experiments are impossible to carry out in humans; however, there is solid, supportive evidence implicating increased epithelial permeability in the cause of Crohn's disease. The group at greatest risk of developing Crohn's disease are individuals who have a firstdegree relative with Crohn's disease. Multiple studies have now shown that within this group are a subgroup of individuals with increased epithelial permeability either initially or in response to an environmental challenge. 9-.3 It also seems that relapses in patients with Crohn's disease are preceded by an increase in small intestinal permeability. .4 This is of interest because nonsteroidal anti-inflammatory agents, which increase intestinal epithelial permeability, are also recognized to precipitate recurrences of Crohn's disease in some patients.15

Abbreviations used in this paper: FITC, fluorescein isothiocyanate; MPO, myeloperoxidase. © 2000 by the American Gastroenterological Association 0016-5085/00/$10.00 doi:10.1053/gast.2000.18152

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MEDDINGS ET AL.

W i t h these d a t a we speculated t h a t stress-induced recurrences in Crohn's disease m i g h t involve a b n o r m a l l u m i n a l c o n s t i t u e n t delivery to the mucosal i m m u n e system secondary to stress-induced increases in gastrointestinal p e r m e a b i l i t y . Because m a n y o f the end organ effects o f stress are adrenally m e d i a t e d , we further specu l a t e d t h a t this m i g h t also be true of stress-induced alterations in g a s t r o i n t e s t i n a l p e r m e a b i l i t y . In vitro studies have s u g g e s t e d t h a t e n v i r o n m e n t a l stress can alter small intestinal t r a n s p o r t p r o p e r t i e s and barrier function in stress-susceptible strains o f rats via a cholinergic m e c h a n i s m ) 6,1v However, these observations have not been reported in vivo and no i n v e s t i g a t i o n of adrenal i n v o l v e m e n t has been performed. Therefore, the p u r p o s e of this s t u d y was to investigate w h e t h e r g r a d e d levels of stress could alter g a s t r o i n t e s t i n a l p e r m e a b i l i t y in a sitespecific m a n n e r and, if so, w h e t h e r the m e c h a n i s m s are m e d i a t e d by adrenal glucocorticoids.

Animals and Methods Animals Male Wistar rats were purchased (Charles River, St. Constant, Quebec, Canada) and housed in the University of Calgary Animal Care Center. Animals were allowed to acclimatize to the facility for 2 weeks unless otherwise noted and were handled daily by the same individuals who performed the experiments. On most days the animals were housed in gang cages, but during permeability testing they were placed in individual metabolic cages for urine collection. To acclimatize them to this, the daily handling included at least 2 days per week when they were placed in the metabolic cages and gavaged in a fashion identical to the permeability testing. In some experiments, animals underwent an adrenalectomy. This was performed under aseptic conditions with halothane anesthesia and a flank incision. Animals were allowed to recover for a week before experiments and were maintained with 0.9% saline for drinking water. Sham animals underwent an identical procedure, but the adrenal glands were simply manipulated and not removed. Experiments were approved by the University of Calgary Animal Care Committee.

Permeability Testing Animals were fasted for 4 hours and then gavaged with 1 g sucrose, 120 mg lactulose, 80 mg mannitol, and 60 mg sucralose in a 2-mL volume. Fasting was continued for 2 hours more, and then free access to water was allowed. Animals were placed in metabolic cages, and the urine passed over 24 hours after the gavage was collected into tubes containing 10% thymol as an antibacterial agent. These techniques have been validated previously. 18 Under these conditions, the urinary recovery of sucrose is a good marker for gastric permeability, and the lactulose-mannitol ratio for small intestinal permeability and the fractional excretion of sucralose reflect both

GASTROENTEROLOGYVol. 119, No. 4

small intestinal and colonic permeability. However, because these probes reach the colon within 4 hours, most of the sucralose absorption occurs within the colon. 18 Urine was assayed for the concentration of each probe by high-performance liquid chromatography techniques described by us previously38 For experiments using fluorescein isothiocyanate (FITC)labeled dextran (mol wt, 10,000), the compound was dissolved in sterile saline and given either intravenously or gavaged as noted in the text. Urine was collected as described, and the concentration of probe in the urine determined with an SLM4600 fluorometer (Urbana, IL) with excitation and emission wavelengths of 490 and 520 nm, respectively. Concentrations were calculated from a standard curve freshly prepared each day to bracket the concentrations observed in the urine. Over the range of concentrations studied, a linear relationship was observed between fluorescence and concentration. Stress Induction Two graded levels of stress were used in these studies. Modest stress was invoked by 3 hours of restraint in a plastic tube at room temperature. Animals were placed individually in plastic tubes where their movement was restricted, but they could reposition themselves with difficulty as previously described. .9 Permeability experiments were conducted over the subsequent 24 hours. More intense stress was applied by 20 minutes of swimming in room temperature water. For these experiments, animals were fitted with a flotation device, as described previously, 2° and allowed to swim without a solid support. Permeability experiments were conducted over the following 24 hours. Miscellaneous

Experiments

In some experiments, animals received a subcutaneous injection of 0.1 mg/kg dexamethasone and permeability was assessed over the subsequent 24 hours. In others, 10 mg/kg of the glucocorticoid antagonist RU-486 (generously donated by Roussel, UCAF, France) was administered intraperitoneally 1 and 12 hours immediately before the application of stress. RU-486 is a glucocorticoid antagonist, and we have previously shown that this dosing regimen blocks endogenous glucocorticoid activity. 21,z2 Plasma corticosterone concentrations were determined by a sensitive radioimmunoassay (ICN, Costa Mesa, CA), as described previously, t9 Macroscopic scoring of damage, histologic assessment, and tissue myeloperoxidase (MPO) determinations were performed 24 hours after stress or dexamethasone administration. Statistical Analysis All comparisons between groups were performed using analysis of variance and a Tukey test as a post hoc analysis. Calculations were performed on a minicomputer using the software Prism (Graphpad Inc., San Diego, CA). A probability of <0.05 was considered significant.

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Animal Acclimatization Transport of animals is a stressful situation, and we took advantage of this to sequentially follow gastrointestinal permeability after arrival at the Animal Care Center. These data are presented in a standard format illustrating the fractional excretion of sucrose (gastric permeability; Figure 1A), lactulose-mannitol ratio (small intestinal permeability; Figure 1B), and fractional excretion of sucralose (small intestinal and colonic permeability; Figure 1C). The x-axis refers to the time after arrival in days. A significant decrease in gastrointestinal permeability was observed during the acclimatization period. This seemed to reach a nadir in all regions of the gastrointestinal tract by 1 week; however, to be safe we selected a 2-week acclimatization period for all subsequent experiments.

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Stress-Induced Permeability Alterations Stress is only one of several potential explanations for the data shown in Figure 1. To test whether stress altered gastrointestinal permeability, the experiments illustrated in Figure 2 were performed. After acclimatization, baseline permeabilities were determined and animals were then subjected to either restraint or swimming stress with permeability measured immediately thereafter. One week later, a third permeability determination was performed. Restraint stress provoked significant increases only in gastric permeability (Figure 2A). However, swimming stress not only provoked a significantly greater increase in gastric permeability but also significant increases in the lactulose-mannitol ratio and fractional excretion of sucralose (Figure 2B and C). These increases in gastrointestinal permeability were not associated with visible evidence of gastrointestinal damage. Careful visual and microscopic examination of the entire gastrointestinal mucosa was carried out following both levels of stress in 5 animals. The only abnormality observed was mild erythema of the gastric mucosa in a few animals (data not shown). No ulcers were evident. In all cases, gastrointestinal permeability had returned to basal levels 1 week after either stress. Because sucralose can be absorbed across either the small or large intestinal epithelium, it was not immediately evident if the increase in fractional sucralose excretion illustrated in Figure 2C was secondary to the increased small intestinal permeability shown by the lactulose-mannitol ratio in Figure 2B. This important point is clarified by the lactulose-sucralose ratios presented in Figure 2D. As previously described, e3 this ratio is a sensitive marker of where permeability alterations occur. W i t h swimming stress, a significant decrease in this ratio was observed, which implies that the fractional

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excretion of sucralose had increased to a greater extent than was attributable to the increase in the fractional excretion of lactulose. Because the only functional difference between these probes is the site of permeation, these

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GASTROENTEROLOGY Vol. 119, No. 4

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data imply that colonic permeability significantly increased in addition to small intestinal permeability. Figures 1 and 2 show increased gastrointestinal permeability on arrival to the animal care facility or after graded levels of stress. Because adrenal glucocorticoids are potent mediators of the stress response, these differing degrees of stress should exhibit differences in plasma corticosterone concentrations. These data are presented in Figure 3. For comparison, Figure 3A illustrates gastric permeability in animals on arrival, after acclimatization for 2 weeks, or after graded degrees of stress. Figure 3B shows the corresponding plasma corticosterone concentrations. Clearly, the increases in gastric permeability were paralleled by increases in plasma corticosterone concentration.

Permeability in Adrenalectomized Animals To determine whether adrenally mediated mechanisms played a role in these observations, these experiments were repeated in animals after either surgical adrenalectomy or a sham procedure. These data are presented in Figures 4 and 5. N o increase in gastrointestinal permeability was observed in adrenalectomized animals (Figure 4). This was not secondary to the surgical procedure per se because sham-operated animals exhibited

Figure 2. Gastrointestinal permeability after stress. (A-C) The same format as in Figure 1. However, the animals had all been acclimatized as described in Results. Permeability was determined either at baseline or after either restraint or swimming. One week after the stress episode, permeability was determined again (Post Stress). Significant increases in permeability were observed with stress (see text for details). (D) Lactulose-sucralose ratio for the same animals. A decrease in this ratio is observed when coIonic permeability increases to a greater extent than small intestinal permeability. * P < 0 . 0 5 vs. baseline; * * P < 0 . 0 5 vs. both baseline and restraint stress.

the same permeability responses to stress observed in the control animals (Figure 5).

Pharmacologic Blockade of Glucocorticoid Effects Adrenal mediation of the stress response could be secondary to either cortical or medullary hormones. To differentiate these possibilities, control animals, after acclimatization, were treated with either RU-486 (10 mg/kg intraperitoneally 12 hours and 1 hour before stress) or vehicle. One hour after the last dose of RU-486, they were exposed to a single swimming stress provocation, and permeability was measured immediately after this and compared with their pretreatment results. These data are presented in Figure 6. Animals treated with vehicle had the same increases in gastric (Figure 6A), small intestinal (Figure 6B), and colonic permeability (Figure 6C) elicited in control animals (Figure 2). However, animals receiving RU-486 had no increase in permeability at any site and appeared identical to the adrenalectomized animals.

Effect of Dexamethasone Figure 7 illustrates gastrointestinal permeability after a single injection of either saline or dexamethasone

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permeability alterations in adrenalectomized animals. The format for this figure is identical to Figure 2 with the exception that the lactulose-sucralose ratio is not shown. No increase in permeability after stress was observed in these animals.

infiltration. N o differences were observed in the stomach; however, there was a significant reduction in M P O content of the small intestine after either dexamethasone or swimming (6.8 + 0.8 vs. 3.6 -+ 0.7 and 4.3 + 0.4 m U M P O / m g tissue for controls, swimming, and dexamethasone treatment, respectively; P < 0.05). A similar re-

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GASTROENTEROLOGY Vol. 119, No. 4

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duction was also observed in the colon (1.8 --- 0.6 vs. 0.8 +-_ 0.1 and 1.1 + 0.1 m U M P O / m g tissue for controls, swimming, and dexamethasone, respectively; P < 0.05).

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Swimming Figure 6. Glucocorticoid receptor blockade. Acclimatized animals were studied before (11) and after (D) swimming stress, Animals received either RU-486 or vehicle before swimming as described. Permeability measurements are presented in the same format as in Figure 4. Significant increases are apparent in vehicle-treated animals but not in those receiving RU-486. * P < 0.05 vs. baseline determination.

examined the uptake of FITC-labeled dextran (average mol wt, 10,000). W h e n given intravenously, 90% 11% (n = 10 animals) of the probe was collected in the

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Dexamethasone

Figure 7. Effect of dexamethasone on permeability. Acclimatized animals were studied either before or after dexamethasone or vehicle administration as described in the text. Permeability data are in the same format as in Figure 4. Significant increases in permeability were apparent after dexamethasone but not saline. * P < 0.05 vs. baseline.

urine over 24 hours (data not shown). Figure 8 shows data obtained in 10 animals when the probe was given orally and urine collected for 24 hours either before or after a standard swimming stress. There is a clear dou-

Discussion These data provide convincing support for the hypothesis that environmental stress can increase gastrointestinal permeability. Swimming stress constitutes a more profound form of stress than does restraint, 24 and as illustrated in Figure 3, there was a significantly increased adrenal glucocorticoid response to this degree of stress. Not only was there a dose-response relationship between the degree of stress and plasma corticosterone concentration, but also with the elicited permeability increase (Figures 2 and 4). Furthermore, the stress-induced increase in permeability was transient, disappearing by 7 days. All segments of the gastrointestinal tract were involved in this response, but the magnitude of the effect was greatest in the stomach and least in the small intestine (Figure 2). Stress-induced gastric ulcers have been reported previously, but have usually been associated with cold water immersion restraint in stress-sensitive animal strains. The animals in this study showed only occasional, mild gastric erythema. W e observed no statistical increase in damage score in any treated animal or histologic abnormalities in random samples. Furthermore, from a biochemical perspective, there was no evidence of tissue damage as reported by MPO content. There was a significant reduction of this parameter in the small intestine and colon of animals after either dexamethasone treatment or swimming stress. Therefore,

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these stress-induced permeability alterations reflected a subvisual abnormality as shown in previous studies, iv Perhaps most interesting of all, these effects were mediated by glucocorticoid secretion. Adrenalectomy completely abolished these stress responses, as did blockade of glucocorticoid receptors (Figures 4 and 6). Furthermore, administering a pharmacologic dose of a glucocorticoid reproduced the end organ effect (Figure 7). The doses used were certainly lower than those given to patients with Crohn's disease; however, a clear effect on permeability was observed. These results differ from those observed by Saunders et al. 16 who examined small intestinal permeability after restraint stress. In this study, atropine pretreatment, given in vivo, abolished the stress-induced alterations in small intestinal permeability. However, the measurements were performed in vitro using an Ussing chamber approach. Whether removing the intestine from extrinsic innervation, blood flow, adrenal glands, or washing the tissue accounts for the differences is unclear. What does seem clear is that in the intact animal, corticosteroids are integral in this stress-induced response. Administration of glucocorticoids to rats has been shown to reduce biliary immunoglobulin A secretion and increase bacterial adherence to the intestinal mucosa. 2~ In regions of the gastrointestinal tract with high concentrations of luminal bacteria, such as the cecum, this is associated with decreased electrical resistance of the epithelium. 25 In other studies, pathogenic bacterial adherence to epithelial cells has been associated with a series of intracellular events culminating in myosin light-chain phosphorylation, loosening of tight junctions, and increased epithelial permeability. 26y Whether these events are involved in the responses observed in this study are unclear; however, the time course of the stress response within the upper gastrointestinal tract was extremely rapid. We have previously shown that sucrose leaves the stomach within an hour of administration. 18 Despite this, increases in sucrose permeability were observed immediately after a 20-minute swimming stress or the administration of dexamethasone. Lactulose and mannitol are present within the small intestine for only 3 - 4 hours, yet similar increases in small intestinal permeability were seen within this time frame. However, it is conceivable that the alterations in colonic permeability, which took place over 24 hours, may have been secondary to alterations in luminal flora. It was also apparent that stress-induced increases in epithelial permeability are applicable to both small and larger molecules. Probe molecules over the size range of several hundred to 10,000 molecular weight were exam-

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ined in this study; a similar pattern was apparent for all. In terms of intestinal inflammation, it is not clear what size of molecule may be important in disease initiation. Clearly, molecules the size of traditional antigens may be involved. However, it is important to recognize that much smaller molecules can both initiate and exacerbate intestinal inflammation. Trinitrobenzene sulfonic acid, a small molecule, is frequently used as a model of hapteninduced intestinal inflammation in animals and can initiate an inflammatory reaction when given in the presence of a barrier breaker such as ethanol. In both animal models of disease and in humans, very small bacterial products such as the formylated tripeptide f-met-leu-phe (fmlp, present in the colon and distal small intestine) can induce inflammation and play an important role in the disease process. Therefore, increased epithelial permeability over the size range of monosaccharides and disaccharides may be extremely relevant for the pathogenesis of human IBD. However, regardless of how important the smaller size range is eventually shown to be, the data presented show that stress-induced increases in permeability are apparent over a size range of several hundred to at least 10,000. This would allow many differently sized luminal molecules access to the submucosa during stress that normally would be excluded. The implications of these observations for disease are intriguing. Patients with Crohn's disease have been reported to undergo disease reactivation after environmental stress. 3,28 However, in humans, a causal relationship does not always seem present when studied prospectively. 29'3° This raises difficult questions regarding the veracity of the previous observations or whether only specific subsets of patients undergo stress-induced disease recurrences. The observation that certain parameters of IBD in animal models of colitis can be reactivated by stress suggests that a mechanism by which this can occur does exist. 5 Stress may mediate effects on intestinal inflammatory conditions by modulating the neuroimmune system within the gut. Previous work has shown that this does occur. Reductions in mucosal sympathetic neurotransmitters and interleukin l b messenger R N A have been shown in the colon of stressed animals after previous colitis. 5 However, stress may also directly affect the initiation of inflammation. The mechanisms that underlie initiation of inflammation in IBD have not been identified with certainty. However, evidence increasingly suggests that increased mucosal permeability may allow abnormal delivery of luminal constituents to the mucosal immune system and initiate an inflammatory response.

October 2000

Bypassing the epithelial barrier and introducing luminal constituents directly into the bowel wall can initiate an inflammatory process with many of the features found in Crohn's disease. 6 Furthermore, chronically increasing epithelial permeability, by introducing a dominant negative N-cadherin mutant gene and inducing it specifically within the small intestine, initiates IBD. v,31 This, and other work in animals, support the hypothesis that one mechanism leading to chronic intestinal inflammation is abrogation of intestinal barrier function. Whether a similar mechanism underlies Crohn's disease in humans is unclear, but there is evidence to support such a contention. Individuals at the highest risk of developing Crohn's disease, first-degree relatives of patients, contain a subgroup with abnormally high small intestinal permeability, m,~,13 There also seems to be a subgroup who are abnormally responsive to exogenous factors that can increase epithelial permeability. 32 Finally, evidence clearly shows that inducing remission of Crohn's disease is associated with a reduction of epithelial permeability. 33 The cause-and-effect relationship of this is unclear because increased permeability is observed with inflammatory disease per se. However, during remission of Crohn's disease, the presence of increased small intestinal permeability can predict disease recurrence. ~4 These data support a link between increased intestinal permeability, luminal constituent delivery to the mucosal immune system, and inflammatory disease activity. Our data show that environmental stress increases gastrointestinal permeability, thereby suggesting a plausible mechanism for stress-induced disease reactivation. The same mechanism could be invoked for the relationship between nonsteroidal anti-inflammatory drugs (NSAIDs) and reactivation of Crohn's disease, 15 because these agents increase permeability to a degree similar to the abnormalities reported in this study. 18 Also, not all patients with Crohn's disease report that NSAIDs worsen their disease. In some ways this is analogous to the stress situation, because not all studies have been able to show this relationship. However, in the case of NSAIDs it has been reported that a subgroup of individuals have abnormally increased permeability responses to an NSAID. 13 The effects of stress on human gastrointestinal permeability have not been investigated, and the possibility that a subgroup of patients have an abnormally increased permeability response to stress cannot be excluded. However, if these results in rats are similar to those in humans, we would predict that stress increases human gastrointestinal permeability transiently and to a degree

STRESS AND GASTROINTESTINAL PERMEABILITY 1027

similar to that observed with the administration of NSAIDs. In conclusion, environmental stress induces a transient increase in gastrointestinal permeability similar in magnitude to that observed with NSAID administration. The mechanism involves mediation by glucocorticoids because adrenalectomy or blockade of glucocorticoid receptors prevents this response and it can be reproduced by the administration of glucocorticoids. This provides a plausible mechanism for the involvement of stress in the reactivation of IBD, in particular Crohn's disease. References 1. Porcelli P, Zaka S, Centonze S, Sisto G. Psychological distress and levels of disease activity in inflammatory bowel disease. Ital J Gastroenterol 1994;26:111-115. 2. Greene BR, Blanchard EB, Wan CK. Long-term monitoring of psychosocial stress and symptomatology in inflammatory bowel disease. 8ehav Res Ther 1994;32:217-226. 3. Garrett VD, Brantley PJ, Jones GN, McKnight GT. The relation between daily stress and Crohn's disease. J Behav Med 1991; 14:87-96. 4. Milne B, Joachim G, Niedhardt J. A stress management programme for inflammatory bowel disease patients. J Adv Nurs 1986;11:561-567. 5. Collins SM, McHugh K, Jacobson K, Khan I, Riddell R, Murase K, Weingarten HP. Previous inflammation alters the response of the rat colon to stress. Gastroenterology 1 9 9 6 ; 1 1 1 : 1 5 0 9 - 1 5 1 5 . 6. Yamada T, Sartor RB, Marshall S, Specian RD, Grisham MB. Mucosal injury and inflammation in a model of chronic granulomatous colitis in rats. Gastroenterology 1 9 9 3 ; 1 0 4 : 7 5 9 - 7 7 1 . 7. Hermiston ML, Gordon Jl. Inflammatory bowel disease and adenomas in mice expressing a dominant negative N-cadherin. Science 1995;270:1203-1207. 8. Rath HC, Herfarth HH, Ikeda JS, Grenther WB, Hamm TE, Jr., Balish E, Taurog JD, Hammer RE, Wilson KH, Sartor RB. Normal luminal bacteria, especially bacteroides species, mediate chronic colitis, gastritis, and arthritis in HLA- 827/human ~2 microglobulin transgenic rats. J Clin Invest 1996;98:945-953. 9. Hollander D, Vadheim CM, Brettholz E, Petersen GM, Delahunty T, Rotter Jl. Increased intestinal permeability in patients with Crohn's disease and their relatives. Ann Intern Med 1986;105: 883-885. 10. May GR, Sutherland LR, Meddings JB. Is small intestinal permeability really increased in relatives of patients with Crohn's disease? Gastroenterology 1993;104:1627-1632. 11. Yacyshyn BR, Meddings JB. CD45RO expression on circulating CD19 + B cells in Crohn's disease correlates with intestinal permeability. Gastroenterology 1995;108:132-137. 12. Peeters M, Geypens B, Claus D, Nevens H, Ghoos Y, Verbeke G, Baert F, Vermeire S, Vlietinck R, Rutgeerts P. Clustering of increased small intestinal permeability in families with Crohn's disease. Gastroenterology 1 9 9 7 ; 1 1 3 : 8 0 2 - 8 0 7 . 13. Hilsden RJ, Meddings JB, Sutherland LR. Intestinal permeability changes in response to acetylsalicylic acid in relatives of patients with Crohn's disease. Gastroenterology 1 9 9 6 ; 1 1 0 : 1 3 9 5 - 1 4 0 3 . 14. Wyatt J, Vogelsang H, HQbl W, Waldh6er T, Lochs H. Intestinal permeability and the prediction of relapse in Crohn's disease. Lancet 1 9 9 3 ; 3 4 1 : 1 4 3 7 - 1 4 3 9 . 15. Kaufmann HJ, Taubin HL. Nonsteroidal anti-inflammatory drugs activate quiescent inflammatory bowel disease. Ann Intern Med 1987;107:513-516. 16. Saunders PR, Hanssen NPM, Perdue MH. Cholinergic nerves

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mediate stress-induced intestina! transport abnormalities in Wistar-Kyoto rats, Am J Physiol Gastrointest Liver Physiol 1997; 273:G486-G490. Saunders PR, Kosecka U, McKay DM, Perdue MH. Acute stressore stimulate ion secretion and increase epithelial permeability in rat intestine. Am J Physiol Gastrointest Liver Physiol 1994; 267:G794-G799. Meddings JB, Gibbons I. Discrimination of site-specific alterations in gastrointestinal permeability in the rat. Gastroenterology 1998;114:83-92. Swain MG, Maric M. Defective corticotropin-releasing hormone mediated neuroendocrine and behavioral responses in cholestatic rats: implications for chotestatic liver disease-related sickness behaviors. Hepatology 1 9 9 5 ; 2 2 : 1 5 6 0 - 1 5 6 4 . Swain MG, Maric M. Improvement in cholestasis-associated fatigue with a serotonin receptor agonist using a novel rat model of fatigue assessment. Hepatology 1997;25:291-294. Tjandra K, Kubes P, Rioux K, Swain MG. Endogenous glucocorticolds inhibit neutrophil recruitment to inflammatory sites in cholestatic rats. Am J Physiol 1996;270:G821-G825. Swain MG, Maric M. Prevention of immune-mediated arthritis in cholestatic rats: involvement of endogenous glucocorticoids. Gastroenterology 1 9 9 4 ; 1 0 7 : 1 4 6 9 - 1 4 7 4 . Goda T, Takase S. Dietary carbohydrate and fat independently modulate disaccharidase activities in rat jejunum. J Nutr 1994; 124:2233-2239. Glavin GB, Pare WP, Sandbak T, Bakke H, Murison R. Restraint stress in biomedical research: an update. Neurosci Biobehav Rev 1994;18:223-249. Spitz J, Hecht G, Taveras M, Aoys E, Alverdy J. The effect of dexamethasone administration on rat intestinal permeability: the role of bacterial adherence. Gastroenterology 1 9 9 4 ; 1 0 6 : 3 5 - 4 1 . Spitz J, Yuhan R, Koutsouris A, Blatt C, AIverdy J, Hecht G. Enteropathogenic Escherichia coil adherence to intestinal epithelial monolayere diminishes barrier function. Am J Physiol Gastrointest Liver Physiol 1995;268:G374-G379.

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27. Yuhan R, Koutsouris A, Savkovic SD, Hecht G. Enteropathogenic Escherichia coil-induced myosin light chain phosphorylation alters intestinal epithelial permeability. Gastroenterology 1997; 113:1873-1882. 28. Duffy LC, Zielezny MA, Marshall JR, Byers TE, Weiser MM, Phillips JF, Calkins BM, Ogra PL, Graham S. Relevance of major stress events as an indicator of disease activity prevalence in inflammatory bowel disease. Behav Med 1991;17:101-110. 29. North CS, Alpers DH, Helzer JE, Spitznagel EL, Clouse RE. Do life events or depression exacerbate inflammatory bowel disease? A prospective study. Ann Intern Med 1991;114:381-386. 30. von Wietersheim J, Kohler T, Feiereis H. Relapse-precipitating life events and feelings in patients with inflammatory bowel disease. Psychother Psychosom 1992;58:103-112. 31. Hermiston ML, Gordon Jl. in vivo analysis of cadherin function in the mouse intestinal epithelium: essential roles in adhesion, maintenance of differentiation, and regulation of programmed cell death. J Cell Biol 1 9 9 5 ; 1 2 9 : 4 8 9 - 5 0 6 . 32. Hancock DL, Coupar IM. Functional characterization of the adenosine receptor mediating inhibition of intestinal secretion. Br J Pharmacol 1995;114:152-156. 33. Sanderson IR, Boulton P, Menzies I, Walker-Smith JA. Improvement of abnormal lactulose/rhamnose permeability in active Crohn's disease of the small bowel by an elemental diet. Gut 1987;28:1073-1076. Received October 22, 1999. Accepted May 10, 2000. Address requests for reprints to: Jon Meddings, M.D., 1705 Health Sciences Center, 3330 Hospital Drive N.W., Calgary, Alberta, Canada T2N 4N1. e-mail: [email protected]; fax: (403) 220-8747. Supported by generous grants from the Medical Research Council of Canada and Searle Canada Inc. Dr. Meddings is an Alberta Heritage Foundation Medical Senior Scholar, and Dr. Swain is a Medical Research Council Medical Scholar and an AHFMR Clinical investigator. The authors thank Kim Tran, Tai Le, and lan Gibbons for their technical expertise in the performance of these studies.