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Mucosal Immune SystemMaintains Unresponsiveness of the Intestinal Apoptosis: One of the Mechanisms That

Phong T. Le, Susan Fisher and Liang QiaoPing Bu, Ali Keshavarzian, David D. Stone, Jianzhong Liu,

http://www.jimmunol.org/content/166/10/6399doi: 10.4049/jimmunol.166.10.6399

2001; 166:6399-6403; ;J Immunol 

Referenceshttp://www.jimmunol.org/content/166/10/6399.full#ref-list-1

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Print ISSN: 0022-1767 Online ISSN: 1550-6606. Immunologists All rights reserved.Copyright © 2001 by The American Association of1451 Rockville Pike, Suite 650, Rockville, MD 20852The American Association of Immunologists, Inc.,

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Apoptosis: One of the Mechanisms That MaintainsUnresponsiveness of the Intestinal Mucosal Immune System1

Ping Bu,2* Ali Keshavarzian,2§ David D. Stone,2* Jianzhong Liu,* Phong T. Le,‡ Susan Fisher,†

and Liang Qiao3*

Intestinal mucosa is constantly exposed to environmental Ags. Activation of lamina propria (LP) T cells by luminal Ags may leadto the production of inflammatory cytokines and subsequent mucosal inflammation and tissue damage. However, in normalcircumstances, LP T cells do not respond to antigenic stimulation. The mechanisms of this unresponsiveness in healthy subjectsare not fully understood. In this study, we found by in vivo analysis that, except for T cells in lymph nodules of the mucosa, 15%of LP T cells underwent apoptosis in normal individuals. In contrast, there was a marked reduction in apoptosis of LP T cells inpatients with inflammatory bowel disease (Crohn’s disease and ulcerative colitis) and those with specific colitis. Our findingssuggest that apoptosis might be a mechanism that turns off mucosal T cell responses to environmental Ags in healthy subjects, andresistance to apoptosis could be an important cause of mucosal immune dysregulation and tissue inflammation in colitis.TheJournal of Immunology,2001, 166: 6399–6403.

A lthough mucosal surfaces are exposed to many environ-mental Ags, the host’s immune system remains unre-sponsive to normal luminal Ags. The mechanisms un-

derlying that phenomenon are only partially understood. Oraladministration with soluble proteins in mice induces tolerance byinducing regulatory T cells in mucosal organized lymphoid tissues(1) and by anergizing or deleting Ag-specific T cells (2–6). Inhumans lamina propria (LP)4 T cells in normal individuals do notproliferate or produce T cell cytokines such as IL-2 and IFN-g inresponse to TCR stimulation (7–10). However, T cells do prolif-erate in mucosal organized lymphoid tissue such as Peyer’s patch,as determined in situ (10), suggesting that there are local environ-mental differences for T cell activation in organized lymphoid tis-sue and in diffuse lymphoid tissue such as LP. The Ag-specificresponse of LP T cells is down-regulated as a result of impairedsignal transduction through the TCR-CD3 complex (7, 8, 11, 12).The reduced response of LP T cells in normal individuals might beinduced by locally produced, small, nonprotein molecules withoxidative properties (13). Furthermore, LP macrophages do notprovide costimulation to T cells for proliferation in response toTCR stimulation (14). In inflamed intestinal mucosa, as in Crohn’sdisease, increased numbers of proliferating LP T cells have beendocumented (10, 15); proinflammatory cytokines, such as IL-1b,

IL-6, IL-15, IFN-g, and TNF-a, that are not expressed in normalmucosa are produced in the inflamed mucosa (10, 16), suggestingthat LP T cells and macrophages might have been activated as aresult of the loss of normal regulatory mechanisms.

Cell death by apoptosis regulates the lymphocyte population andterminates immune responses at sites of Ag exposure. Because LPis constantly exposed to environmental Ags, it is highly possiblethat lymphoid cells in LP are also regulated by apoptosis, i.e.,apoptosis might prevent the activation and clonal expansion oflymphocytes to unharmful environmental Ags. It has been shownthat isolated human LP T cells have a tendency to undergo apo-ptosis in vitro (17–19). In vitro, upon stimulation of LP T cellswith anti-CD2 Ab, the cells underwent apoptosis, which was me-diated via the CD95 pathway (17). Interestingly, CD2-mediatedapoptosis is reduced in patients with Crohn’s disease (19), and theresistance of T cells to apoptotic signals is associated with a higherratio of Bcl-2 to Bax (20). However, no study has yet shown thatLP T cells undergo apoptosis in vivo to confirm the physiologicsignificance of mucosal T cell apoptosis. In this study we deter-mined whether LP lymphocytes (T cells and plasma cells) under-went apoptosis in situ in normal mucosa. As a comparison we alsodetermined the apoptotic status of those cells in the inflamed mu-cosa of patients with colitis where lymphoid cells should have ahigher survival rate. We found that a significant amount of LPlymphocytes underwent apoptosis in normal mucosa and that ap-optosis was greatly reduced in mucosal inflammation.

Materials and MethodsSubjects

All subjects had endoscopic examination as part of their clinical evaluation.All patients with colitis had an established diagnosis of ulcerative colitis(UC), Crohn’s disease (CD), or specific colitis (infectious colitis, ischemiccolitis, and radiation colitis). The diagnosis of colitis was based on a stan-dard clinical, endoscopic, and histological criteria. All patients with colitiswere symptomatic and had endoscopically and histologically active dis-ease. Symptoms included abdominal pain (n5 6), diarrhea (n5 15),hematochezia (n5 10), and urgency (n5 8). Normal individuals (controls)had screening endoscopic procedure for colon cancer. None had gastroin-testinal symptoms, and all had normal endoscopic findings. Two pieces ofmucosa (;8 mg) were taken from areas of active inflammation (in colitispatients) or normal-appearing mucosa (in controls) in the sigmoid colon.

Departments of *Microbiology and Immunology,†Medicine, and‡Cell Biology, Neu-robiology and Anatomy, Stritch School of Medicine, Loyola University Chicago,Maywood, IL 60153; and§Division of Digestive Diseases, Rush University, Chicago,IL 60612

Received for publication October 25, 2000. Accepted for publication March 5, 2001.

The costs of publication of this article were defrayed in part by the payment of pagecharges. This article must therefore be hereby markedadvertisementin accordancewith 18 U.S.C. Section 1734 solely to indicate this fact.1 This work was supported in part by National Institutes of Health Grant CA81254 (toL.Q.).2 P.B., A.K., and D.D.S. contributed equally to this paper.3 Address correspondence and reprint requests to Dr. Liang Qiao, Department ofMicrobiology and Immunology, Stritch School of Medicine, Loyola University Med-ical Center, 2160 South First Avenue, Maywood, IL 60153. E-mail address:lqiao@luc.edu4 Abbreviations used in this paper: LP, lamina propria; AMCA, 7-amino-4-methyl-coumarin-3-acetic acid; CD, Crohn’s disease; DAB, 3,3-diaminobenzidine; IBD, in-flammatory bowel disease; UC, ulcerative colitis.

Copyright © 2001 by The American Association of Immunologists 0022-1767/01/$02.00

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Large fresh colonic specimens from normal mucosa were also obtainedfrom patients who were having colon cancer surgically removed. The mu-cosal tissues used were taken far away from the tumor and close to theresection margin and were macroscopically and microscopically normal.The biopsies and part of surgical specimens were immediately snap-frozenin liquid nitrogen and stored at280°C, and parts of the surgical specimenswere fixed in ice-cold methanol/PBS (6/1) solution before paraffin embed-ding for subsequent analysis. This study was approved by the institutionalreview board for safety of human subjects of Loyola University StritchSchool of Medicine and Rush University.

Determination of T cell apoptosis in situ

Detection of apoptotic cells in paraffin-embedded vs cryopreserved hu-man colonic mucosa.Paraffin sections (5mm) were deparaffinized in twochanges of xylene for 5 min each. Sections were hydrated in an alcoholgradient twice (100, 90, 75, and 50% for 3 min each) and incubated for 5min in tap water. Endogenous peroxidase was quenched by incubatingsections in 0.3% H2O2 in PBS for 10 min. Sections were washed twice for5 min each time in PBS and incubated in a humidified chamber for 1 h at37°C with TdT, 0.75 U/ml in TdT buffer (Life Technologies, Gaithersburg,MD) in solution with 100mM biotin-14-dCTP (Life Technologies). Afterwashing, sections were incubated for 30 min each time at room temperaturewith streptavidin-HRP diluted 1/300 (Amersham, Uppsala, Sweden). Sec-tions were developed with 3,3-diaminobenzidine (DAB; Sigma, St. Louis,MO), counterstained with methyl green, and dehydrated and mounted.Then they were analyzed by light microscopy. Cryopreserved sectionswere fixed in 1% formaldehyde in PBS (the PBS used throughout thisprocedure did not contain potassium), washed, blocked with 0.3% H2O2,and washed again. The frozen sections were then treated exactly as de-scribed for the paraffin-embedded tissues starting with the TdT incubationbefore being evaluated by light microscopy.Phenotype of apoptotic cells in situ.TdT end-labeling assay and immu-nofluorescence assay were used to determine the phenotypes of apoptoticmucosal cells (21). Human colonic sections (5mm) were fixed in 1% form-aldehyde for 15 min on ice. The sections were washed three times in PBS

(pH 7.4) for 5 min each time at 4°C to remove formaldehyde. Fixed colonicsections were incubated with 10ml of a solution containing 0.75 U/ml TdT(Life Technologies), 13TdT reaction buffer, and 100mM biotin-14-dCTP(Life Technologies) for 1 h at 37°C in humidified chambers. For negativecontrols, human colonic sections were incubated with a similar solutionwithout the TdT enzyme. To detect fragmented DNA with 39ends labeledwith biotinylated-dCTP, the sections were incubated for 60 min at roomtemperature with 40mg/ml streptavidin-7-amino-4-methylcoumarin-3-ace-tic acid (AMCA; Roche, Carpinteria, CA) in 43SSC staining buffer con-taining 0.1% Nonidet P-40 (v/v; Sigma) and 5% nonfat dry milk. Thesections were washed three times in cold PBS at 4°C between each incu-bation step. After incubation with streptavidin-AMCA solution, the sec-tions were incubated with mouse-anti-human CD3, CD38, or CD68 mAbsfor 60 min at room temperature. The mouse isotype of IgG1k (Sigma) wasused as a control. The FITC-conjugated anti-mouse IgG was used as thesecondary Ab. Immunofluorescence microscopy was performed with a mi-croscope (Leitz, Rockleigh, NJ) equipped with two different filters forFITC and AMCA (UV). The microphotographs were taken with either a35-mm camera (Nikon, Melville, NY) or a digital camera (Optronics, Go-leta, CA). The blue fluorescence representing apoptosis (AMCA) was con-verted to red using Magnafire, a digital imaging software associated withthe digital camera. This color conversion was performed for easy visual-ization of apoptotic cells against the dark background.

Quantitation of apoptotic cells

At least two biopsies from each patient were used, and all of them wereexamined for the presence of apoptosis. The whole tissues of each biopsywere examined. To quantify the number of apoptotic cells, after preview ofthe section, different fields were photographed randomly, and the totalnumber of CD31, CD381, or CD681 cells and respective apoptotic cellswere counted by two investigators blinded to the sample groups. Four to 11fields/tissue were counted for each subject. Apoptosis was expressed as themean percentage of apoptotic CD31, CD381, or CD681 cells among all

FIGURE 1. Detection of apoptotic cells in cryo-preserved or Formalin-fixed and paraffin-embeddedhuman colonic tissues. Frozen tissues (aandc) andFormalin-fixed, paraffin-embedded tissues (bandd)were treated with TdT and biotin-dCTP, developedwith DAB, and counterstained with methyl green.Negative controls (candd) were incubated in solu-tion lacking TdT. Original magnification,3400.

FIGURE 2. Detection of ssDNA in cells from nor-mal human colonic tissue. Sections were stained using amAb against ssDNA followed by a biotinylated anti-mouse secondary Ab. The sections were incubated withExtravidin-peroxidase and developed with DAB andcounterstained. Brown cells in the lamina propria (a) arepositive for ssDNA (arrow). As a negative control (b) anisotype control Ab or PBS was substituted for anti-ssDNA or sections were treated with S1 nuclease beforeapplication of primary Ab. Original magnification,3400.

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CD31, CD381, or CD681 cells from all counted fields. Because of vari-able background staining at the margins of tissues, counting was consid-ered unreliable in those areas, and they were excluded from the analysis.

Detecting ssDNA in human colonic mucosa

We followed the procedure exactly as specified in the protocol accompa-nying Ab to ssDNA (Alexis Biochemicals, San Diego, CA). Briefly, mu-cosa was removed from resected colon of cancer patients and immediatelyfixed in ice-cold methanol/PBS (6/1) solution. The tissue and solution werereturned to220°C for 2 days. The fixed tissue was dehydrated in twochanges of absolute methanol and two changes of xylene and then embed-ded in paraffin. Fresh 4-mm sections were prepared before staining andheated at 56°C for 1–2 h. Sections were deparaffinized in two changes ofSafeclear (Fisher, Hanover Park, IL) and incubated in three changes ofmethanol/PBS (6/1) solution for 20 min each. Then they were rinsed withDulbecco’s PBS (the only PBS used throughout the procedure) and incu-bated for 5 min in PBS supplemented with 0.2% Triton X-100 and 5 mMMgCl2. Sections were then heated for exactly 6.5 min in a 99°C water bathfollowed by placement in ice-cold PBS for 10 min. As a negative control,sections were incubated with 100 U/ml S1 nuclease (Sigma) in acetatebuffer after ice-cold PBS wash, or an isotype control Ab (mouse IgM) or

PBS was substituted for the anti-ssDNA mAb. Endogenous peroxidase wasblocked using 3% H2O2 in PBS, and slides were treated with 0.1% BSA for30 min. After rinsing, anti-ssDNA mAb was applied to sections for 15 minand washed, and biotin-conjugated rat anti-mouse IgM (Zymed, San Fran-cisco, CA) was applied for another 15 min. After washing, ExtrAvidin-peroxidase (Sigma) was applied for 15 min. Sections were washed anddeveloped with DAB (Sigma).

Results and DiscussionTo determine whether LP lymphocytes undergo apoptosis in situ innormal intestinal mucosa, we initially used a TdT end-labelingassay to detect apoptotic cells in mucosal biopsies from normalindividuals. Many apoptotic cells were found in normal mucosaltissues. Considering that this much apoptosis has not been reportedin the literature, we determined whether our observation might bedue to the methods used. Previous studies that determined the ap-optosis of mucosal cells used formalin-fixed, paraffin-embedded

FIGURE 3. Double staining of various cellsurface markers and apoptosis in normal andinflamed frozen gut sections. Normal colonicsections (a–f) were stained with a mAb againstCD3 (a) to show T cells, against CD38 (c) toshow plasma cells, or against CD68 to showmacrophages (e). In each case, the same tissuesections were stained for apoptosis (b, d,andf). Apoptotic cells appear red. Arrows indicatedouble-positive cells in the same section. Gutsections from a patient with CD (g–l) weretreated in the same manner. Original magnifi-cation,3400.

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mucosal tissues. Therefore, we compared frozen tissues with For-malin-fixed, paraffin-embedded tissues. We obtained normal mu-cosal tissues. One part was snap frozen, and another part was fixedwith Formalin and embedded in paraffin. Apoptosis was deter-mined in both parts by TdT end-labeling assay. As shown in Fig.1, more apoptotic cells in LP were detected in snap-frozen tissuethan in formalin-fixed and paraffin-embedded tissue from the samesubject. To further confirm the cell apoptosis in the mucosa, weused an assay to determine ssDNA, which has been shown to be aspecific method for detection of apoptotic cells (22, 23). As shownin Fig. 2, ssDNA-positive cells were found in LP of normal mu-cosa, thus confirming that LP cells underwent apoptosis in vivo.

To determine which cells underwent apoptosis in LP, we com-bined indirect immunofluorescence with a TdT end-labeling assay.Apoptotic cells were stained for T cell markers (CD3), plasma cellmarker (CD38), and macrophage marker (CD68). In the LP fromcontrol subjects (n5 7), a mean of 15% of LP T cells underwentapoptosis (Figs. 3 and 4). Both CD41 and CD81 T cells underwentapoptosis (data not shown). The apoptosis of LP T cells was foundin all seven of the healthy control subjects. In contrast, apoptotic Tcells were not found in solitary lymph nodules of control subjects(data not shown). Similarly, a mean of 20% of CD381 cells and16% of CD681 cells were positive in the TdT end-labeling assay(Fig. 3). We used CD38 as a marker for plasma cells because of thehigh background when we initially used anti-human IgA and IgGto detect plasma cells. Although CD381 cells are most likelyplasma cells, CD38 can also be expressed by activated T cells.Therefore, the percentage of plasma cells might be an overestima-tion. We found that CD681 cells were positive in the TdT end-labeling assay, possibly because macrophages are deleted to con-trol the immune responses to normal Ags, as macrophages arepotentially important APCs. However, we could not exclude thepossibility that those macrophages were positive in the TdT end-labeling assay because they engulfed the apoptotic bodies/cells.That possibility will be investigated in future studies. It should alsobe noted that the apoptotic cells may not be evenly distributedalong the colon length. Therefore, the mean percentages of apo-ptotic cells are just an estimate and are subject to sample error.Nonetheless, the data clearly showed that a significant proportionof T cells and plasma cells underwent apoptosis in vivo in LP ofnormal colonic mucosa.

It has been shown that mucosal T cells have a tendency to un-dergo apoptosis in vitro (17). Here we demonstrated that LP T

cells, but not T cells in mucosal organized lymphoid tissue, un-derwent apoptosis in vivo. This finding suggests that T cells in theorganized lymphoid tissue are able to respond to antigenic stimu-lation. Our findings are consistent with previous studies in miceshowing that T cells in Peyer’s patches can proliferate and producecytokines upon Ag stimulation (24, 25). There are at least twopossible explanations for our observed difference in T cells fromLP and the organized lymphoid tissues. The first possibility is thatT cells that are activated in the organized lymphoid tissue prefer-entially home to LP where the T cells encounter normal environ-mental Ags and undergo apoptosis upon stimulation with an over-whelming Ag load. The second possibility is that the LP is anenvironment where reactive T cells are induced to undergo apo-ptosis so that they are not able to respond to antigenic stimulation(environment-driven apoptosis). Similarly, the apoptosis of plasmacells could be explained by two possibilities, i.e., Ag or environ-ment-driven. To directly address whether apoptosis is caused byantigenic stimulation, an animal model is needed; however, theapoptosis of LP lymphoid cells has been detected only in humans,not in mice or rabbits (L. Qiao, unpublished observation).

We hypothesize that if apoptosis is important in controlling theresponses of mucosal lymphoid cells to normal luminal Ags, thenthe apoptosis would be reduced in an inflammatory microenviron-ment where, in most cases, lymphocytes are activated. To test thishypothesis, we determined apoptosis of lymphoid cells in the pa-tients with colitis. Apoptosis of T cells, plasma cells, and macro-phages in LP was markedly reduced in all tissue samples from thepatients with colitis (Figs. 3 and 4). This marked decrease in ap-optosis did not appear to be due to anti-inflammatory medication,because all patients had reduced apoptosis regardless of type oftherapy. In addition, there was no correlation between disease ac-tivity or disease extent and degree of apoptosis because most dis-eased mucosa had very low levels of T cell apoptosis. Our dataalso showed that reduced T cell apoptosis was not specific for UC,CD, or specific colitis and was seen in the inflamed colon regard-less of the etiology. Hence, reduced apoptosis may be a factor inthe immune dysregulation seen in IBD, but it may not play a pri-mary etiological role. The data from these patients contrasted withresults from murine colitis models, where increased T cell apopto-sis was found in inflamed mucosa (26, 27). This finding might bedue to the fact that there are few apoptotic LP T cells in normalmouse mucosa.

The apoptosis of LP T cells could occur through CD95 and/orother receptors. It has been shown that most LP T cells isolatedfrom uninvolved areas of resected colon carcinoma expressedCD95, as analyzed by flow cytometry (18). When LP cells werestimulated by CD2, the T cells also underwent apoptosis, whichcould be blocked by anti-CD95 Ab (17). However, unstimulatedLP T cells underwent apoptosis in vitro, which could not beblocked by an inhibitory anti-CD95 Ab (17). Thus, the apoptosisof unstimulated LP T cells might be induced by an uncharacterizedpathway, as shown in the in vitro experiment (17). It also has beenshown that T cells isolated from areas of inflammation in CD, UC,and other inflammatory states were resistant to CD2-induced ap-optosis, and that resistance was accompanied by elevated Bcl-2levels (19). T cells grown from CD lesions were resistant to CD95-or NO-mediated apoptosis, with similarly increased Bcl-2:Bax ra-tios (20). Those data suggest that CD95 plays a role in inducingapoptosis in LP T cells; however, other receptors or pathways bywhich apoptosis is induced also might be involved.

It appears that the reduced apoptosis in IBD is a consequence ofinflammation rather than a cause of IBD, since it was also noted inspecific colitis. Thus, reduced apoptosis could be due to proin-flammatory cytokines that are abundantly present in the inflamed

FIGURE 4. Percentage of lamina propria apoptotic CD31, CD381, andCD681 cells in mucosa from normal individuals (norm) and from patientswith UC, CD, and specific colitis (SC). The bars represent SDs of the meanpercentage of apoptosis in each group of patients (the number of patientsper group is shown on thetop of bars). The mean percentage of apoptosisin control patients (norm) is significantly different from that of the otherthree groups, withp 5 0.0018 for CD31 cells andp 5 0.0001 for CD381

cells and for CD681 cells. Differences among the groups were comparedusing ANOVA. Duncan’s multiple range test was used for pairwise com-parisons to maintain the overalla level at 0.05.

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mucosa. Many proinflammatory cytokines, such as IL-1, IL-6,IL-2, IFN-g, and IL-15, are expressed in IBD (16, 28). In partic-ular, IL-15 might have an important effect in counteracting apo-ptosis of LP cells, because it was shown that IL-15 was producedby macrophages in inflamed mucosa of patients with IBD (28).Bcl-2 expression can be induced as a result of signaling via thecommong-chain of the IL-2 receptor, which serves as a signalingcomponent of the receptor for IL-2, IL-4, IL-7, and IL-15. Thus,IL-15 might greatly enhance the expression of Bcl-2, which furtherenhances resistance of LP T cells against induction of apoptosis.

In summary, we demonstrated that LP T cells underwent apo-ptosis in normal mucosa, which might have an important effects inmaintaining the unresponsiveness of LP T cells to normal luminalAgs and the homeostasis of mucosal lymphoid tissue. The level ofLP-cell apoptosis is greatly reduced in the inflamed colonic mu-cosa, which may contribute to the mucosal immune dysregulationnoted in IBD.

AcknowledgmentsWe thank Dr. Joseph Losurdo for collecting samples.

References1. Weiner, H. L. 1997. Oral tolerance: immune mechanisms and treatment of au-

toimmune diseases.Immunol. Today 18:335.2. Chen, Y., J.-I. Inobe, V. K. Kuchroo, and H. L. Weiner. 1995. Peripheral deletion

of antigen-reactive T cells in oral tolerance.Nature 367:177.3. Marth, T., W. Strober, and B. L. Kelsall. 1996. High dose oral tolerance in

ovalbumin TcR-transgenic mice: systemic neutralisation of interleukin 12 aug-ments TGFb secretion and T cell apoptosis.J. Immunol. 157:2348.

4. Kyburz, D., P. Aichele, D. E. Speiser, G. Hengartner, R. M. Zinkernagel, andH. Pircher. 1993. T cell immunity after a viral infection versus T cell toleranceinduced by soluble viral peptides.Eur. J. Immunol. 23:1956.

5. Migita, K., K. Eguchi, Y. Kawabe, T. Tsukada, Y. Ichinose, and S. Nagataki.1995. Defective TCR-medicated signaling in anergic T cells.J. Immunol. 155:5083.

6. Whitacre, C. C., I. E. Gienapp, C. G. A. Orosz, and D. M. Bitar. 1991. Oraltolerance in experimental autoimmune encephalitis. III. Evidence for clonal an-ergy.J. Immunol. 147:2155.

7. Qiao, L., G. Schurmann, M. Betzler, and S. C. Meuer. 1991. Activation andsignaling status of human lamina propria T lymphocytes.Gastroenterology 101:1529.

8. Targan, S. R., R. L. Deem, M. Liu, S. Wang, and A. Nel. 1995. Definition oflamina propria T cell responsive state: enhanced cytokine responsiveness of Tcells stimulated through the CD2 pathway.J. Immunol. 154:664.

9. Boirivant, M., I. Fuss, C. Fiocchi, J. S. Klein, S. A. Strong, and W. Strober. 1996.Hypo-proliferative human lamina propria T cells retain the capacity to secretelymphokines when stimulated via the CD2/CD28 accessory signaling pathways.Proc. Assoc. Am. Physicians 108:55.

10. Autschbach, F., G. Schurmann, L. Qiao, G. Merz, R. Wallich, and S. Meuer.1995. Cytokine messenger RNA expression and proliferation status of intestinal

mononuclear cells in noninflamed gut and Crohn’s disease.Virchows Arch. 426:51.

11. DeMaria, R., S. Fais, M. Silvestri, L. Frati, F. Pallone, A. Santoni, and R. Testi.1993. Continuous in vivo activation and transient hyporesponsiveness to TCR/CD3 triggering of human gut lamina propria T lymphocytes.Eur. J. Immunol.23:3104.

12. Qiao, L., G. Schurmann, M. Betzler, and S. C. Meuer. 1991. Down-regulation ofprotein kinase C activation in human lamina propria T lymphocytes: influence ofintestinal mucosa on T cell reactivity.Eur. J. Immunol. 21:2385.

13. Qiao, L., G. Schurmann, F. Autschbach, R. Wallich, and S. C. Meuer. 1993.Human intestinal mucosa alters T cell reactivities.Gastroenterology 105:814.

14. Qiao, L., J. Braunstein, M. Golling, G. Schurmann, F. Autschbach, P. Moller, andS. Meuer. 1996. Differential regulation of human T cell responsiveness by mu-cosal versus blood monocytes.Eur. J. Immunol. 26:922.

15. Qiao, L., M. Golloing, G. Schurmann, F. Autschbach, and S. C. Meuer. 1994. Tcell receptor repertoire and functional behavior of lamina propria T lymphocytesin inflammatory bowel diseases.Clin. Exp. Immunol. 97:303.

16. Fiocchi, C. 1998. Inflammatory bowel disease: etiology and pathogenesis.Gas-troenterology 115:182.

17. Boirivant, M., R. Pica, R. DeMaria, R. Testi, F. Pallone, and W. Strober. 1996.Stimulated human lamina propria T cells manifest enhanced Fas-mediated apo-ptosis.J. Clin. Invest. 98:2616.

18. DeMaria, R., M. Boirivant, M. G. Cifone, P. Roncaioli, M. Hahne, J. Tschopp,F. Pallone, A. Santoni, and R. Testi. 1996. Functional expression of Fas and Fasligand on human gut lamina propria T lymphocytes.J. Clin. Invest. 97:316.

19. Boirivant, M., M. Marini, G. D. Felice, A. M. Pronio, C. Montesani, R. Tersigni,and W. Stober. 1999. Lamina propria T cells in Crohn’s disease and other gas-trointestinal inflammation show defective CD2 pathway-induced apoptosis.Gas-troenterology 116:557.

20. Ina, K., J. Itoh, K. Fukushima, K. Kusugami, T. Yamaguchi, K. Kyokane,A. Imada, D. G. Binion, A. Musso, G. A. West, et al. 1999. Resistance of Crohn’sdisease T cells to multiple apoptotic signals is associated with a Bcl-2/Bax mu-cosal imbalance.J. Immunol. 163:1081.

21. Le, P. T., H. T. Maecker, and J. E. Cook. 1995. In situ detection and character-ization of apoptotic thymocytes in human thymus.J. Immunol. 154:4371.

22. Frankfurt, O. S., J. Robb, E. V. Sugarbaker, and L. Villa. 1996. Monoclonalantibody to single-stranded DNA is a specific and sensitive cellular marker ofapoptosis.Exp. Cell Res. 226:387.

23. Frankfurt, O. S., J. A. Robb, E. V. Sugarbaker, and L. Villa. 1996. Apoptosis inhuman breast and gastrointestinal carcinomas: detection in histological sectionswith monoclonal antibody to single-stranded DNA.Anticancer Res. 16:1979.

24. Xu-Amano, J., W. K. Aicher, T. Taguchi, H. Kiyono, and J. R. McGhee. 1992.Selective induction of Th2 cells in murine Peyer’s patches by oral immunization.Int. Immunol. 4:433.

25. Taguchi, T., J. R. McGhee, R. L. Coffman, K. W. Beagley, J. G. Eldridge,K. Takatsu, and H. Kiyono. 1990. Analysis of Th1 and Th2 cells in murinegut-associated tissues: frequencies of CD41 and CD81 T cells that secrete IFN-gand IL-5.J. Immunol. 145:68.

26. Bregenholt, S., J. Reimann, and M. H. Claesson. 1998. Proliferation and apopto-sis of lamina propria CD41 T cells from scid mice with inflammatory boweldisease.Eur. J. Immunol. 28:365.

27. Bonhagen, K., S. Thoma, P. Bland, S. Bregenholt, A. Rudolphi, M. H. Claesson,and J. Reimann. 1996. Cytotoxic reactivity of gut lamina propria CD41 ab Tcells In SCID mice with colitis.Eur. J. Immunol. 26:3074.

28. Liu, Z., K. Geboes, S. Colpaert, G. R. D’Haens, P. Rutgeerts, and J. L. Ceuppens.2000. IL-15 is highly expressed in inflammatory bowel disease and regulateslocal T cell-dependent cytokine production.J. Immunol. 164:3608.

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