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Idiopathic inflammatory bowel disease (IBD) encompassesCrohn disease (CD) and ulcerative colitis (UC), which togetherconstitute the most common and serious chronic inflammatoryconditions of the bowel in man. The present view is that theseconditions arise as a result of the convergence of a geneticallydetermined susceptibility to inflammation and environmentaltriggers1. The genetic susceptibility is thought to be mainly inthe regulation of mucosal immune responses, and this is re-flected experimentally by the spontaneous development ofcolitis in transgenic animals deficient in essential immunoregu-latory molecules (review, ref. 2). The natural history of IBD ischaracterized by relapse and remission, and several factors areknown to trigger relapses, including infection, ingestion of non-steroidal anti-inflammatory drugs and changes in smokinghabits. Animal models of IBD have been useful in confirmingthese clinical observations, and have demonstrated, for exam-ple, the deleterious effect of non-steroidal anti-inflammatorydrugs on colitis3.

The effect of stress on IBD has long been a subject of contro-versy. Early studies favored a temporal relationship betweenstressful life events and clinical relapse4–6, and emphasized theimportance of monitoring and managing stress in the preven-tion and treatment of disease recurrence7– 9. However, other stud-ies have failed to demonstrate such a relationship, and havequestioned the roles of the psyche and stress in the natural his-tory of IBD (refs. 10–12). Nonetheless, the methodological diffi-culties involved in establishing a relationship betweenpsychological factors and disease activity are substantial, and ithas been acknowledged that the existing clinical literature is in-sufficient to exclude a relationship between stress and IBD (ref.13). Given the clinical implications of this issue, as well as recentinsights into the relationship of stress responses and chronic in-flammatory conditions14, the question regarding the role ofstress in IBD warrants further examination.

Certain observations in man and in animals support a link be-tween stress and the clinical expression of intestinal inflamma-tion. For example, ulcerative colitis was first described in

Bedouin Arabs after they had been moved to government hous-ing, and was attributed to the attendant social stress15. The de-velopment of colitis, which is sometimes complicated by thedevelopment of colonic cancer, has been observed in captiveSiamese gibbons and in cotton-top tamarins16–18, prompting theidea that the stress of captivity was involved in the clinical ex-pression of the disease.

Thus, we designed this study to investigate whether stress canreactivate quiescent colitis. We also sought to determine the pre-requisites for stress-induced reactivation, and whether it in-volves specific immune mechanisms and changes in colonicbarrier function. Our results show that stress does indeed reacti-vate colitis in mice that have recovered from acute colitis induced eight weeks previously by the hapten dinitrobenzene-sulfonic acid (DNBS). We show that the stress-induced reactiva-tion required lumenal exposure to a small dose of DNBS thatwhen given alone was insufficient to induce colitis. The reactiva-tion process was immune-dependent in that it could not be in-duced in athymic or SCID mice. Moreover, the susceptibility toreactivation by stress could be adoptively transferred to SCIDmice by CD4+ T cells taken from mice with active DNBS-inducedcolitis. Stress also reduced mucin production and increased per-meability in the colon, facilitating the entry of the hapten andother agents from the lumen. These results indicate the ability ofpsychological, immune and lumenal factors to converge and re-activate quiescent colitis, and have important implications forour conceptualization and management of IBD.

The induction and resolution of acute colitisIn Balb/c mice, intracolonic administration of 2 mg DNBS hadno effect on myeloperoxidase activity or on the appearance ofthe tissue (Fig. 1a and b). In contrast, 6 mg DNBS produced a se-vere colitis, characterized by extensive tissue damage and alarge acute inflammatory cell infiltrate (Fig. 1c), with an ulcerindex (UI) of 7.3 ± 1.5 units (U) and a significant increase inMPO activity, from 4.6 ± 1.4 to14.0 ± 1.9 U/g (P < 0.001). Thesechanges lasted several weeks, but the tissue appeared normal

The role of CD4+ lymphocytes in the susceptibility of mice tostress-induced reactivation of experimental colitis

B.S. QIU, B.A. VALLANCE, P.A. BLENNERHASSETT & S.M. COLLINS

Intestinal Diseases Research Unit, Division of Gastroenterology, Room 4W8, Department of Medicine,Faculty of Health Sciences, McMaster University. Digestive Diseases Program, Hamilton Health Sciences

Corporation, Hamilton, Ontario, Canada L8N 3Z5Correspondence should be addressed to S.M.C.; email: [email protected]

Idiopathic inflammatory bowel disease is a chronic relapsing condition. The role of stress in caus-ing relapses of inflammatory bowel disease remains controversial. We now show that colitis in-duced in mice by dinitrobenzenesulfonic acid (DNBS) resolves by 6 weeks, but can subsequentlybe reactivated by stress plus a sub-threshold dose of DNBS, but not by DNBS alone. Stress re-duced colonic mucin and increased colon permeability. Susceptibility to reactivation by stress re-quired CD4+ lymphocytes and could be adoptively transferred. We conclude that stressreactivates experimental colitis by facilitating entry of luminal contents that activate previouslysensitized CD4 cells in the colon.

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(Fig. 1d) by 6 weeks after DNBS (UI, 0.4 ± 0.06 U; MPO activity5.7 ± 1.1 and 4.6 ± 1.4 U/g, P > 0.05). A similar profile was seenin C57Bl/6 mice (data not shown).

The effect of stress on quiescent colitis in Balb/c miceThese studies used mice that had recovered from DNBS colitis6–8 weeks previously. The combination of acoustic and re-straint stress resulted in a significant increase in plasma corti-costerone concentrations, from 106 ± 6 to 277 ± 30 ng/ml (P <0.001). In Balb/c mice, stress plus ethanol (vehicle) had no ef-fect on the microscopic appearance of the colon (Fig. 2a), anddid not significantly increase either the UI or MPO activity: UI,0.2 ± 0.05 and 0.1 ± 0.05 U (P > 0.05); MPO activity, 7.4 ± 1.3and 6.1 ± 0.7 U/g (P > 0.05). As stated above, 2 mg DNBS alonehad no effect on the colon, but when combined with stress, itcaused significant colonic damage (Fig. 2b). The UI increasedfrom 0.4 ± 0.02 to 6.2 ± 0.8 U (P < 0.001) and MPO activity in-creased from 5.7 ± 1.1 to 12.0 ± 1.4 U/g (P < 0.01).

DNBS also caused a transient acute colitis in congenitallyathymic mice, which recovered fully by 8 weeks (data notshown). However, the combination of stress and 2 mg DNBS at8 weeks caused only minor reactivation of the colitis; the UI in-creased from 0.4 ± 0.04 to only 1.0 ± 0.8 U (P > 0.05) and therewas no substantial change in either microscopic appearance ofthe colon (Fig. 2c) or in MPO activity (5.8 ± 1.2 to 6.7 ± 1.6 U/g).These results indicate that T lymphocytes are important for thefull expression of the stress-induced reaction of colitis.

The effect of stress on colitis in CD8 and CD4 knockout miceThe combination of stress and 2 mg DNBS caused significant tis-sue damage (UI from 0.4 ± 0.4 to 5.4 ± 1.3 U; P < 0.01) and MPOactivity (from 6.0 ± 1.4 to 14.0 ± 2.1 U/g; P < 0.05) at 8 weeksafter recovery from acute colitis induced by 6 mg DNBS inC57Bl/6 mice. A similar profile was found in CD8 knockoutmice (UI, 6.0 ± 1.4 U; MPO activity, 15.0 ± 2.5 U/g; P < 0.05compared with respective controls). In contrast, in CD4-defi-cient mice that had recovered from DNBS-induced acute colitis8 weeks before, stress plus 2 mg DNBS failed to induce anychange in the microscopic appearance of the tissue (Fig. 2d), UI(0.4 ± 0.04 U) or MPO activity (6.0 ± 1.4 U/g). These results indi-cate that CD4+ cells are essential for the reactivation the colitis.

Adoptive transfer of the susceptibility to stress using CD4+ cellsThe combination of stress and 2 mg DNBS failed to cause tissuedamage (UI, 0.6 ± 0.08 U) or an increase in MPO activity (4.2 ±0.7 U/g) in naive mice with severe combined immune deficiency(SCID)(Fig. 3). Similarly, this protocol failed to induce colitis inSCID mice that had received CD4-depleted cells from Balb/cmice with acute colitis (Fig. 3c). In contrast, SCID mice that hadreceived a CD4-enriched cell population that contained morethan 95% CD4+ cells developed a substantial increase in UI andMPO activity (Fig. 3a and b); this was reflected by microscopyshowing tissue damage and an acute inflammatory infiltrate(Fig. 3d). These results identify CD4+ cells as being prerequisitefor the expression of colitis reactivated by stress and hapten.

Effect of stress on colonic mucin productionColonic mucin production decreased by 35.6% in mice afterstress in the absence of previous colitis or administration of asubthreshold dose of the hapten (from 12.5 ± 1.2 to 8.05 ± 1.2dpm/g protein; P < 0.01). At 6 weeks after colitis induction,mucin production in these mice was not significantly differentfrom that of control mice (12.5 ± 1.2 compared with 10.2 ± 1.5dpm/g protein; P > 0.05). However, the subsequent applicationof stress resulted in a 30% reduction in mucin production (from10.2 ± 1.5 to 7.0 ± 0.9 dpm/g protein; P < 0.05).

Effect of stress on colon permeabilityThere was a small but statistically insignificant increase in per-meability in mice that had recovered from colitis 6 weeks previ-ously (Fig. 4). Although stress for 5 days did not increase colonicpermeability to 3H-mannitol in control mice, it caused a signifi-cant increase in permeability in those mice that had DNBS colitis6 weeks previously.

DiscussionThe results of this study indicate that stress reactivates quiescentcolitis and that the susceptibility to stress requires CD4+ lympho-cytes as well as a subclinical lumenal challenge of the sensitizinghapten. The immunological prerequisite for reactivation bystress was demonstrated by the absence of reactivation in con-

Fig. 1 Acute colitis induced by DNBS in Balb/c mice. The distal colon inBalb/c mice in the absence of DNBS (a); 3 d after 2 mg DNBS (b); 3 d after6 mg DNBS (c); and 42 d after 6 mg DNBS (d).

Fig. 2 The effect of stress on the colon. The distal colon of Balb/c micethat had recovered 8 weeks previously from DNBS colitis, and after 5 d ofcombination of restraint and acoustic stress. a, Stress alone had no effect onthe colon. b–d, Stress followed by a single intracolonic administration of 2mg DNBS caused reactivation of colitis in Balb/c mice (b); however, thiscombination had no effect in athymic mice (c) or in CD4-deficient mice (d)that had recovered from colitis 8 weeks previously.

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Fig. 3 The effect of stress on the colon of SCID micethat received T cells from mice with colitis. Naive SCIDmice received either CD4-depleted cells or CD4-enrichedcells before stress plus 2 mg intracolonic DNBS. a and b,Stress plus DNBS had no effect on the ulcer index (a) orMPO activity (b) in mice that received CD4-depleted cells(CD4–). In contrast, stress plus DNBS caused a significantincrease (*, P < 0.05) in both indices in mice that receivedCD4-enriched cells from mice with colitis (CD4+). c andd, The results in a and b were reflected in the histologicalappearance of the colon, which resembled control tissuein mice that had received CD4-depleted cells (c), in con-trast to the presence of acute inflammation and ulcera-tion after stress plus DNBS in mice that had receivedCD4-enriched cells (d).

Fig. 4 The effect ofstress on colonic per-meability. Colonic seg-ments were perfusedwith 3H-mannitol for 4h, and recovery of uri-nary 3H-mannitol indpm/cm (mean ± 1s.e.m.; n = 5) was mea-sured in control micewithout colitis (greybars) and from mice 6weeks after DNBS induction of colitis (filled bars), in the presence or absenceof stress (below graph). Stress caused a significant increase in colonic perme-ability above control in mice that had previous colitis: *, P < 0.05.

genitally athymic and in SCID mice. The essen-tial role of CD4 lymphocytes was shown by theabsence of reactivation in CD4-deficient miceand by the demonstration of stress-induced reac-tivation of colitis in naive SCID mice after thetransfer of CD4+ cells from mice with previouscolitis. The transfer of these cells alone was insuf-ficient to induce colitis, which also required thecombination of stress and local challenge with asubclinical dose of hapten. This finding raisedthe question of whether stress reduces the protective barrier inthe colon, thereby facilitating entry by the hapten and other lu-minal factors such as bacterial antigen.

These results build on previous results in rats, in which stressalone rekindled the inflammatory process induced 6 weeks be-fore by the hapten trinitrobenzene sulfonic19. However, this pro-duced only a small increase in MPO activity that was notaccompanied by macroscopic or histological evidence of colitis.Thus, our study here represents the first demonstration, to ourknowledge, of a stress-induced reactivation of colitis, accompa-nied by the classical changes of mucosal inflammation and ul-ceration. The difference in results between these studies mayreflect the fact that stress was not accompanied by luminal expo-sure to the hapten in the rat study19.

Our results indicate that the reactivation of colitis is unlikelyto reflect an altered hypothalamic–pituitary response to stress inanimals that had previous colitis. As in the rat study19, plasmacorticosterone response to stress was normal in mice with previ-ous colitis. These measurements were made because of previouswork demonstrating the role corticotropin-releasing hormone incolonic responses to stress20,21 and of the postulated relationshipbetween the impaired hypothalamic secretion of corticotropin-

releasing hormone in response to stress22 and the promotion ofinflammation in peripheral organs23. Our finding is consistentwith a recent study that questions the role of CRF in the abilityof stress to enhance the inflammatory response to subsequentinjury in the colon24.

The results of this study emphasize the importance of CD4lymphocytes in the expression of hapten-induced colitis25,26.Although CD4 cells transferred from mice with colitis migrate tothe colon of naive recipient mice26, this in itself was insufficientto induce colitis in the presence or absence of a subthresholddose of the hapten. Our results demonstrate the essential role ofstress in the reactivation of colitis in this model, and provide in-sights into the underlying mechanism. We have shown thatstress reduces the colonic barrier to luminal contents by decreas-ing mucus production and increasing colonic permeability.These changes facilitate the access of the hapten and other lumi-nal factors such as bacterial antigens, to the colonic milieu insufficient concentration to activate pre-sensitized CD4 cells26,27

and reactivate colitis. Essential to this interpretation are thedemonstrations that neither the hapten nor the ethanol vehiclealone could reactivate colitis in the absence of stress.

Our finding that stress increases the permeability of the gut isin keeping with previous results28. However, the stress-induceddecrease in colonic mucin production differs from results show-ing a stress-induced increase in mucin production in ratcolon29,30. This apparent discrepancy may reflect the fact that thestress-induced decrease in mucin production in our study was inmice recovering from colitis, whereas the previous studies in ratswere done on healthy colon. It is possible that remodeling afterinflammation may alter the neural control of gut function.

Our study emphasizes the importance of the nervous systemin modulating intestinal inflammation. Recent studies havedrawn attention to this neuro–immune axis in the context ofcolitis in both non-human animals and man. For example, theseverity and duration of experimental colitis in mice may bemodulated by manipulating the bioavailability of the sensoryneurotransmitter substance P (ref. 31). In man, the importance

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of neural pathways has been demonstrated in a report showingthat spinal stimulation, used to control pain, induced relapses ofulcerative colitis32.

The results of our study have important implications for themanagement of patients with IBD. The demonstration of a rolefor stress in reactivating colitis endorses the need to incorporatestress management into advice given to patients with IBD (ref. 8).Moreover, the demonstration of an immunological prerequisitefor the reactivation by stress supports the current trend towardsaggressive treatment of IBD patients with immunomodulatoryagents, rather than the more traditional style of relying almostexclusively on anti-inflammatory drugs for active disease. Finally,our results broaden the conceptual framework within which thepathogenesis of IBD must be viewed. This revised frameworkshould incorporate behavioral as well as immunological and localgut influences in the expression of active IBD.

MethodsAnimals. Balb/c mice were purchased from Harlan Sprague Dawley(Indianapolis, Indiana); athymic Balb/c mice, from NCI (Frederick,Maryland); C57BL/6 mice, from Taconic (Germantown, New York); andCB.17 SCID mice (congenic with Balb/c mice) were obtained from a colonyat McMaster University (Hamilton, Ontario, Canada). The original colony ofCD4-deficient (ref 33) and CD8-deficient (ref. 34) mice (back-crossed ontoa C57BL/6 background) was obtained from T.W. Mak (University ofToronto, Canada) and bred at McMaster University (Hamilton, Ontario,Canada). Only male mice 6–10 weeks old were used in this study, and allmice were housed in specific pathogen-free conditions. All studies were ap-proved by the Animal Utilization Committee of McMaster University.

Induction of colitis. Colitis was induced by intracolonic administration ofdinitrobenzenesulfonic acid (DNBS; ICN, Aurora, Ohio), which is compara-ble to the more widely used trinitrobenzenesulfonic acid35. A stock solutionof DNBS was made by dissolving 60 mg of the hapten in 0.5 ml of 100%ethanol, to which 0.5 ml of water was added. Mice anaesthetized with 2%enflurane were injected in the distal 4 cm of the colon with 100 µl of this so-lution, containing 6 mg of DNBS, using a 1 ml tuberculin syringe (BecktonDickinson, Franklin Lakes, New Jersey) and PE90 tubing (Clay Adams,Parsippany, New Jersey). Mice were given 8% sucrose in 0.2% saline to pre-vent dehydration during the first week after DNBS administration.

Stress and hapten challenge. In preliminary studies, we evaluated re-straint stress36 and sonic stress37 separately and in combination, and foundthat larger and more predictable responses were obtained with the com-bined stressors. In the final protocol, during the weeks 7–8 after colitis in-duction, each mouse was placed in a 50-ml polypropylene conicalcentrifuge tube (Sarstedt) modified with 10 drilled holes of 5 mm in diame-ter to permit penetration of sound. A sonicating water bath, placed 1 footaway, was used to generate noise at 25 decibels for 2 h. This was repeatedtwice daily for 5 successive days. The mice were challenged with a single in-tracolonic administration of 0.1 ml of 2 mg DNBS in 50% ethanol immedi-ately after the final stress session. The mice were killed by cervicaldislocation 3 d after DNBS administration.

Corticosterone assay. Blood was collected immediately after the last stresssession by cardiac puncture, and plasma cortisol levels were measured byradioimmunoassay38.

Assessment of the severity of colitis. The colon was removed, opened bylongitudinal incision and macroscopic damage was immediately assessedusing a dissecting microscope. Microscopic damage was assessed after thefixation of tissues in 10% formalin followed by hematoxylin and eosin stain-ing. Damage (ulcer index; UI) was assessed quantitatively in arbitrary unitsusing published scoring systems39. Myeloperoxidase (MPO) activity wasused to assess the acute inflammatory infiltrate as described40. MPO activitywas expressed in units/mg of tissue, where 1 unit corresponds to the activ-ity required to degrade 1 mmol hydrogen peroxide in 1 min at 24 °C.

Lymphocyte purification and transfer experiments. T cells were collectedfrom mesenteric lymph nodes of Balb/c mice killed on day 10 after colitis in-duction, as described41. Cells were then counted, and then incubated for 20min at 4 °C with magnetic beads CD4 (Miltenyi Biotec, Sunnyvale,California) labeled with rat monoclonal antibody against mouse CD4 (cloneGK1.5). The cells were then passed through a Minimacs column (VS type)previously washed with column buffer (phosphate buffered saline, 2 mMEDTA, 0.5% BSA, pH 7.2) and chilled to 4 °C. The unbound cells were al-lowed to pass through the column, which was then washed three timeswith 3 ml column buffer. The column was then removed from the magnetand the purified CD4+ cells were eluted with 5 ml column buffer. The iso-lated cells were then counted and analyzed for viability, which was found tobe greater than 95%, using Trypan blue exclusion. Flow cytometric analysisshowed the purity of CD4+ cells to be more than 90%, whereas the CD4-de-pleted fraction contained only 0.4% CD4+ cells. In the adoptive transfer ex-periments, 1.5 × 106 CD4-depleted cells or 1.5 × 106 positively selected CD4cells were injected into the tail veins of recipient naive SCID mice 4–6 weeksbefore the subsequent application of stress.

Colonic mucin assay. Colonic mucosal explants were used to assess spon-taneous mucin release as described28. To confirm that 3H-glucosamine wasincorporated into mucin, the trichloroacetic acid precipitate was assessedby density gradient ultracentrifugation as described42.

Colonic permeability to mannitol. Mice were anesthetized by intraperi-toneal injection of sodium pentobarbitol (0.6 mg/mouse). A laparotomywas done and the renal artery was ligated. The colon was mobilized and a3- to 4-cm loop was created. 3H-mannitol (10 µCi; 30 Ci/mmol; NEN) wasinjected into the inferior vena cava before closure of the abdominal incision.Mice were killed 4 h later and urinary 3H-mannitol excretion was measuredas described43.

Statistical analysis. Results shown are mean values ± 1 standard error of themean (s.e.m.) from a minimum of four experiments. Differences betweenmore than two mean values were analyzed by ANOVA, and P values < 0.05were considered statistically significant.

AcknowlegmentsThis study was supported by grants to SMC from the Medical Research Councilof Canada, and from a Research Initiative Award from the CanadianAssociation of Gastroenterology and Astra Canada.

RECEIVED 22 JULY; ACCEPTED 16 AUGUST 1999

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