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The Journal of Immunology, 1999, 163: 4033-4040.
Copyright © 1999 by The American Association of Immunologists

Protection from Radiation-Induced Colitis Requires MHC Class II Antigen Expression by Cells of Hemopoietic Origin

Samuel Marguerat1,*, H. Robson MacDonald*, Jean-Pierre Kraehenbuhl{dagger} and Joost P. M. van Meerwijk2,*

* Ludwig Institute for Cancer Research, Lausanne Branch, University of Lausanne, Epalinges, Switzerland; and {dagger} Swiss Institute for Experimental Cancer Research (ISREC), Epalinges, Switzerland


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Ulcerative colitis, an inflammatory bowel disease, is believed to result from a breakdown of dominant tolerance mechanisms that normally control intestinal immunity. Although CD4+ T lymphocyte subpopulations and expression of MHC class II molecules have been shown to play a role in the pathogenesis of the disease, the nature of the responsible mechanisms remains unclear. In this paper we describe a novel mouse model for inflammatory bowel disease, radiation-induced colitis, that occurs with complete penetrance 6–8 wk postinduction. A combination of high dose gamma-irradiation and lack of MHC class II expression on cells of hemopoietic origin results in development of colitis in C57BL/6 mice. Because of its versatility (due to susceptibility of mice of the widely genetically manipulated C57BL/6 background), high reproducibility, and 100% penetrance, radiation-induced colitis will be a useful mouse model for colitis and a significant tool to study dominant immunological tolerance mechanisms. Moreover, our data imply that tolerization to enteric Ags requires MHC class II mediated presentation by APC of hemopoietic origin.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
Ulcerative colitis and Crohn’s disease are two human chronic diseases characterized by metaplasia and inflammation of the gastrointestinal tract and resulting atrophy. Their etiology remains unclear, but an involvement of the immune system is now strongly assumed. Several murine models show pathology similar to that of these human diseases. Mice deficient in TCR{alpha} or -ß (1), IL-2 (2), IL-10 (3), MHC class II (1), or TGF-ß (4), spontaneously develop colitis-like symptoms. Colitis can also be induced in SCID mice by transfer of congenic CD45RBhigh T cells. In the latter model, cotransfer of CD45RBlow lymphocytes blocks disease development (5, 6, 7). Dextran sulfate sodium feeding (8, 9) as well as intracolonic injection of the haptenizing agent 2,4,6-trinitrobenzene sulfonic acid (TNBS).3 TNBS (10) also induce murine colitis. TNBS-induced colitis can be prevented by orally tolerizing animals with haptenized colon proteins before rectal TNBS administration (11). Ab-blocking experiments have indicated that immunoregulation in the SCID and TNBS systems depends on TGFß activity (11, 12). Also, it was recently suggested that IL-10 plays a role in protection from colitis (13).

Mombaerts et al. (1) have described development of colitis in MHC class II deficient mice (MHC II°) between 4 and 6 mo of age. MHC class II mediated protection from immunopathology has also been reported for a variety of autoimmune disorders: Development of diabetes in the nonobese diabetic (NOD) mouse (which does not express I-E molecules) is inhibited by expression of I-E transgenes (14), and also some I-A alleles have been shown to confer resistance to this disease (15, 16). Similar observations have been reported for the rodent model for rheumatoid arthritis, collagen-induced arthritis (CIA) (17), the multiple sclerosis model experimental autoimmune encephalomyelitis (18), myastenia gravis-susceptible B10 mice (19), and systemic lupus erythematosis in autoimmune BXSB mice (20).

The mechanism(s) responsible for MHC class II-mediated protection from (auto)immune disorders remain unknown. Expression of protective MHC class II molecules has been hypothesized to lead to thymic tolerization of autoreactive T lymphocytes, and experimental data in favor of (21, 22) but also contesting this model (16, 23, 24) have been reported. Alternatively, positive selection of protective T lymphocytes may be mediated by these molecules (25). CD4-expressing subpopulations have indeed been reported to confer protection from diabetes in transgenic I-Ad-expressing NOD mice (26), from neonatal thymectomy-induced autoimmunity (27) and also from colitis induced in SCID mice (6, 7). Moreover, it has been proposed that protective MHC class II molecules may capture and thereby deviate determinants that in the context of other MHC class II molecules are recognized by autospecific T cells (28). Also, a diversion of an autoaggressive Th1 to a benign Th2 phenotype of autospecific T lymphocytes can be envisaged.

The nature of the ligand(s) involved in colitis remains unknown. Although the fact that inflammatory bowel disease in TCR{alpha} knockout (KO) mice is accompanied by a variety of autoantibodies may reflect an autoimmune nature of this disease (29, 30), a breakdown of tolerance toward intestinal flora in experimental colitis has also been reported (31, 32). Moreover, germ-free IL-2 KO and TCR{alpha} KO mice do not develop colitis, suggesting a role for enteric flora in the etiology of this disease (33, 34). It has become clear that, in contrast to most autoimmune diseases, T lymphocytes are not required for the development of colitis (1), although they can induce it (6). Although TCR{alpha} KO mice spontaneously develop colitis, recombinase-activating gene-1 (RAG-1)-deficient mice do not (1), suggesting that B lymphocytes play a role in its etiology. Therefore, a cognate interaction between B or T lymphocytes and (foreign) antigenic determinants seems to elicit inflammation, which under normal conditions is controlled by regulatory T cells. However, colitis can be induced in SCID mice by dextran sulfate sodium feeding, suggesting that, at least in this model, B and T cells are not required (35, 36, 37).

As mentioned above, mice deficient in MHC class II expression develop colitis at 4–6 mo of age (1). Therefore, expression of MHC class II molecules somehow protects mice from the development of this disease. Because these molecules are known to play a major role in development (38), peripheral survival (39, 40), and activation of CD4+ T lymphocytes (41), we wished to investigate the mechanism(s) by which MHC class II molecules protect mice from colitis. In this paper we report that irradiation hemopoietic chimeras that lack MHC class II expression on bone marrow-derived cells but express these molecules on radioresistant cells invariably develop colitis ~8 wk postreconstitution. The model of radiation-induced colitis (RIC) will be a useful tool to study the development of colitis and to investigate the mechanism(s) of MHC class II mediated protection from this disease and from immunopathology in general.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Mice

Wild-type C57BL/6 mice were obtained from Harlan Netherlands (Zeist, The Netherlands). Mice deficient for MHC class I expression (MHC I°) because of targeted disruption of the ß2-microglobulin gene (42) were obtained from Dr. B.-J. Fowlkes (National Institutes of Health, Bethesda, MD). These mutants had been crossed at least seven generations to C57BL/6 mice, after which intercrossing yielded mice homozygous for the disrupted allele. Mice of H-2b haplotype deficient in MHC class II expression (MHC II°) due to disruption of the I-A{alpha} gene in C57BL/6 stem cells (I-E{alpha}-) (43) were obtained from Dr. H. Bluethmann (Roche, Basel, Switzerland). MHC I° and MHC II° animals were interbred in our conventional animal facilities to obtain MHC I°II° mice.

Bone marrow chimeras

Hemopoietic chimeras were prepared essentially as described previously (44). In brief, age and sex-matched anti-NK1.1 treated (100 µg of PK136 i.p. (45)) hosts were lethally irradiated (1000 rad, 160 rad/min) using a Cs137 source and injected next day i.v. with 10–20 x 106 bone marrow cells depleted of T cells by complement killing using anti-Thy1 Ab AT83 (46). As a standard procedure in the generation of irradiation bone marrow chimeras, mice were kept on antibiotic (0.2% Bactrim; Roche, Basel, Switzerland) containing water for the duration of the experiment.

Histological analysis

Unfixed colon fragments were embedded in OCT medium (Tissue-Tek, Zoeterwonde, The Netherlands). Cryosections of 10 µm were fixed in 4% paraformaldehyde (PFA) and stained with a 1:1 mixture of May-Grünwald and Giemsa solution. At least 3 mice/group and 18 sections/mouse have been analyzed.

Antibodies

The following Abs were used for flow cytometry: anti-CD4-FITC (GK1.5, (47)), anti-CD8ß-FITC (H35-17.2, (48)), and anti-IFN-{gamma}-PE (XMG1.2, PharMingen, San Diego, CA). Immunohistochemistry was performed using anti-CD4 (H129-19.6, (49)), anti-CD8{alpha} (53.6.7, (50)), anti-I-Ab,d,q/I-Ed,k (clone M5/114.15.2, (51)), anti-CD11c (clone N-418, (52)), anti-rat Ig-HRP (Tago, Burlingame, CA), anti-hamster Ig-bio (Pierce, Rockford, IL), and streptavidin-HRP (Amersham, Little Chalfont, U.K.).

Immunohistochemistry

Colon cryosections (7 µm) were fixed in acetone. Endogenous peroxidase activity was extinguished by incubation in PBS/0.3% H2O2. Nonspecific Ab binding was blocked with 0.5% BSA complemented with 1% mouse and goat serum in PBS. Subsequently, sections were incubated with primary Ab followed by HRP-labeled secondary Ab or streptavidin in PBS/0.1% BSA. HRP activity was revealed by incubation in 0.2 mg/ml 3-amino-9-ethylcarbazole (AEC; Sigma, St. Louis, MO). Sections shown in Fig. 3Go were counterstained with Gill’s hematoxylin n.2 (Polysciences, Warrington, PA). At least three mice per group and five sections per mouse were analyzed.



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  		FIGURE 3. Lamina propria dendritic cells in MHC II° -> wt and MHC I°II° -> wt chimeras lack expression of MHC class II Ags. Serial transversal cryosections of colon from wt -> wt (a and b) and MHC I°II° -> wt (c and d) chimeras were fixed and stained with Abs to the DC marker CD11c (a and c) and MHC class II (b and d). Ab binding was revealed with HRP-labeled secondary reagents and AEC. Bar = 50 µm. LP, lamina propria; L, lumen; and E, epithelium.

 
Scoring of colitis

Sections were scored blindly by S.M. Hyperplasia was assessed by direct measurement of the size of the mucosa. Metaplasia was determined by counting the number of goblet cells in the epithelium. Infiltrating polymorphonuclear and mononuclear cells were counted. The ratio between values of individual mice and the average values of the wild-type (wt) -> wt mice were calculated. Values within mean ± SD of the controls were considered normal (-). Increased values are indicated as +, (mild); ++ (average); and +++ (maximal increase observed).

Intraepithelial lymphocytes (IEL) and lamina propria lymphocytes (LPL) isolation

Isolation of IEL and LPL was performed as described previously (53, 54). In brief, colons were flushed several times with PBS, opened longitudinally, and cut in pieces of ~5 mm. Fragments were incubated for 20 min at 37°C with stirring in HBSS without Ca2+ and Mg2+ (Life Technologies, Rockville, MD) supplemented with 4 mM NaHCO3 and 2 mM DTT. Supernatant was collected, and the fragments were incubated a second time using the same conditions. IEL in supernatants were enriched on a 40–80% Percoll gradient (Pharmacia Biotech, Uppsala, Sweden). For the isolation of LPL, gut fragments were subsequently washed for 30 min in HBSS without Ca2+ and Mg2+ supplemented with 1 mM EDTA, and incubated for 2 h in complete RPMI 1640 (Life Technologies) supplemented with 1 mM HEPES, 10% FCS, 1 mM DTT, and 90 U/ml collagenase (Sigma). Supernatant was collected and enriched for LPL on a 40–80% Percoll gradient.

Surface and intracellular staining for flow cytometry

Intracellular staining for IFN-{gamma} was performed using a modified version of previously described protocols (55). In brief, freshly isolated IEL and LPL were incubated at 37°C in complete RPMI 1640 supplemented with 1 mM HEPES, 10% FCS, 50 ng/ml PMA, and 600 ng/ml ionomycin. After 2 h incubation, Brefeldin A (Sigma) was added to a final concentration of 10 µg/ml and cells were cultured for an additional 2 h. Subsequently, cells were incubated for 10 min on ice with anti-CD32 (2.4.G2) followed by anti-CD4-FITC or anti-CD8{alpha}-FITC for 20 min on ice in PBS/5% FCS/0.02% NaN3. After washing, cells were fixed in 2% PFA/1 µg ml-1 Brefeldin A, washed in PBS/5% FCS/0.5% Saponin (Sigma), incubated for 30 min at room temperature with whole rat IgG diluted in PBS/5% FCS/0.5% Saponin, and finally 1 h with anti-IFN-{gamma}-PE. Samples were analyzed on a FACScan flow cytometer, and data were analyzed using Lysis II and PC-Lysis software (Becton Dickinson, San Jose, CA).


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
MHC II° mice have been reported to develop colitis-like symptoms at 4–6 mo of age (1). To assess which cell type needs to be MHC class II deficient to allow colitis to develop, and to analyze the role of MHC class I molecules in this disease, we produced irradiation bone marrow chimeras. Lethally irradiated C57BL/6 mice were reconstituted with bone marrow derived from wt (wt -> wt chimeras), MHC I° (MHC I° -> wt), MHC II° (MCH II° -> wt), or MHC I°II° (MHC I°II° -> wt) C57BL/6 animals. After 6 wk, some MHC II° -> wt and MHC I°II° -> wt chimeras showed diarrhea and prostrated posture. While without exception all MHC I°II° -> wt chimeras showed prostrated posture by 8–12 wk postengraftment, all MHC II° -> wt mice systematically died between 8 and 9 wk after reconstitution. None of these signs have ever been observed by us in wt -> wt or MHC I° -> wt chimeras. For the analysis described below, chimeras were killed and analyzed between week 8 and 15 postreconstitution.

Chimeras lacking MHC class II expression by hemopoietic cells develop colitis

Macroscopic observation revealed swollen intestines in MHC II° -> wt and MHC I°II° -> wt chimeras as compared with wt -> wt and MHC I° -> wt mice (Fig. 1Go, a and b, and data not shown). Cryosections of the colon of chimeras were stained with May-Grünwald/Giemsa solution (Fig. 1Go, c–f). Control wt -> wt chimeras had a healthy colon morphology. Absence of MHC class I molecules on hemopoietic cells in MHC I° -> wt chimeras did not cause any morphological change. However, MHC II° -> wt and MHC I°II° -> wt chimera derived colons showed a striking hyperplasia of colon mucosa, accompanied by severe elongation of the crypts. Also, the density of goblet cells in the epithelium of diseased chimeras was significantly decreased. Finally, in MHC II° -> wt and MHC I°II° -> wt chimera derived colons an infiltration of the lamina propria by mononuclear and polymorphonuclear cells was evident (Fig. 1Go, c–f). This result indicates that MHC II° -> wt and MHC I°II° -> wt chimeras had developed a severe ulcerative colitis like disease.



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  		FIGURE 1. MHC II° -> wt and MHC I°II° -> wt, but not wt -> wt and MHC I° -> wt chimeras develop colitis. Lethally irradiated C57BL/6 (wt) hosts were reconstituted with wt (a and c), MHC I° (e), MHC II° (d), or MHC I°II° (b and f) bone marrow. Animals were sacrificed at 8 wk postengraftment. Macroscopic analysis revealed typical thickening of intestines from MHC I°II° -> wt (b) but not wt -> wt chimeras (a). Cryosections (10 µm) of the colon of chimeras were fixed in 4% PFA and stained in May-Grünwald solution. Representative transversal sections of the colon of wt -> wt (c), MHC II° -> wt (d), MHC I° -> wt (e), and MHC I°II° -> wt (f), chimeras are shown. Bar = 100 µm. S, serosa; LP, lamina propria; and L, lumen.

 
Colitis developed invariably within 2 mo, and all MHC II° -> wt and MHC I°II° -> wt chimeras were affected (Table IGo, and data not shown). Colitis has never been observed by us in wt -> wt and MHC I° -> wt chimeras.


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  		Table I. Scoring of RIC in bone marrow chimeras1

 
CD4+ and CD8+ cells infiltrate the lamina propria in MHC II° -> wt and MHC I°II° -> wt chimeras

The mononuclear cell infiltrates observed in diseased animals were characterized by immunohistochemistry (Fig. 2Go). Although some CD4+ T lymphocytes were clearly visible in the lamina propria of colon from healthy wt -> wt chimeras (Fig. 2Goa), their number was significantly increased in colitis-affected MHC II° -> wt (Fig. 2Goc) and MHC I°II° -> wt (Fig. 2Goe) chimeras. Lamina propria CD8{alpha}+ T cells were relatively rare in wt -> wt chimeras (Fig. 2Gob), but MHC II° -> wt (Fig. 2God) and MHC I°II° -> wt (Fig. 2Gof) colons showed important infiltration by these lymphocytes. These data show that the development of colitis in MHC II° -> wt and MHC I°II° -> wt chimeras was accompanied by lamina propria infiltration by CD4+ and CD8+ T lymphocytes.



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  		FIGURE 2. CD4+ and CD8+ lymphocytes infiltrate the lamina propria of chimeras affected with colitis. Transversal cryosections of colons from wt -> wt (a and b), MHC II° -> wt (c and d), and MHC I°II° -> wt (e and f) chimeras were fixed and stained with Abs specific for CD4 (a, c, and e) or CD8{alpha} (b, d, and f). Ab binding was revealed with HRP-labeled anti-rat Ig/AEC. Bar = 50 µm. S, serosa; LP, lamina propria; L, lumen; and E, epithelium.

 
Lamina propria dendritic cells do not express MHC class II in MHC II° -> wt and MHC I°II° -> wt chimeras

Infiltration of lamina propria by CD4+ and CD8+ T lymphocytes in chimeras lacking MHC class I and/or II expression on hemopoietic cells was rather unexpected. Therefore, we investigated whether lamina propria professional APC are in fact of donor origin. Immunohistological analysis using Abs specific for dendritic cells (DC)(CD11c, (52)) revealed the presence of CD11c+ cells in the lamina propria and epithelium of wt -> wt control and colitis-affected MHC I°II° -> wt chimeras (Fig. 3Go, a and c), as previously described in the rat (56). As expected, in MHC I°II° -> wt chimeras CD11c+ cells in the colon lacked expression of MHC class II molecules, and MHC class II expression was uniquely observed on intestinal epithelial cells (Fig. 3God). This result indicated that the APC were of donor origin and that the activation event (if any) that lead to infiltration by CD4+ and CD8+ lymphocytes in MHC I°II° -> wt chimeras was not mediated by MHC ligands expressed by colon DC.

LPL and IEL in chimeras affected with colitis produce IFN-{gamma}

To investigate whether the lamina propria infiltration by lymphocytes was accompanied by their activation, we analyzed production of the proinflammatory cytokine IFN-{gamma} by these cells. This cytokine is known to be instrumental in the development of colitis in SCID mice induced by injection of CD45RBhigh CD4+ T lymphocytes (57). LPL were isolated from affected and healthy animals and stimulated in vitro in the presence of the Golgi blocker Brefeldin A. Cells were subsequently analyzed for surface expression of CD4 and CD8ß and intracellular expression of IFN-{gamma} by flow cytometry (Fig. 4Go, A and B). Significantly more LPL produced IFN-{gamma} in affected (MHC II° -> wt and MHC I°II° -> wt) than in healthy (wt -> wt and MHC I° -> wt) chimeras (p < 0.01) (Fig. 4GoA). Most (88 ± 7%) of the IFN-{gamma} producers expressed CD4 or CD8ß (Fig. 4GoB and data not shown). Moreover, among CD4+ LPL, significantly more cells produced IFN-{gamma} in affected than in healthy chimeras (p < 0.05). The increased percentage of IFN-{gamma} producing LPL appears to be due to a higher fraction of CD4+ cells producing this cytokine and, in the case of MHC II° -> wt chimeras, to the significantly increased percentage of CD8+ T lymphocytes of which a high proportion produces IFN-{gamma} (Fig. 4GoB).



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  		FIGURE 4. A, IFN-{gamma} production by chimera derived LPL and IEL. LPL and IEL from chimeras were isolated, restimulated in vitro in the presence of Brefeldin A, and assessed for intracellular IFN-{gamma}. The percentage of IFN-{gamma} producing LPL and IEL from chimeras affected with colitis (MHC II° -> wt and MHC I°II° -> wt combined) was significantly higher than that of unaffected (wt -> wt and MHC I° -> wt combined) chimeras (p < 0.01 and p < 0.001, respectively (Student’s t test)). B, IFN-{gamma} production by chimera derived CD4+ and CD8ß+ LPL and IEL. LPL and IEL were isolated, restimulated in vitro in presence of Brefeldin A, and analyzed for surface CD4 and CD8 expression as well as intracellular IFN-{gamma} by flow cytometry. Data from representative experiments are shown. Numbers indicate percentage within indicated gate. The percentage of IFN-{gamma} producing CD4+ LPL and IEL from chimeras affected with colitis (MHC II° -> wt and MHC I°II° -> wt combined, n = 6) was significantly higher than that of unaffected (wt -> wt and MHC I° -> wt combined, n = 7 for LPL, n = 5 for IEL) chimeras (p < 0.05 and p < 0.01, respectively). The percentage of IFN-{gamma} producing CD8ß+ IEL was significantly higher in affected (n = 3) than in healthy (n = 4) chimeras (p < 0.01).

 
We also analyzed IFN-{gamma} production by IEL (Fig. 4Go). Significantly more IEL produced IFN-{gamma} in chimeras affected with colitis (MHC II° -> wt and MHC I°II° -> wt) than in healthy (wt -> wt and MHC I° -> wt) animals (p < 0.001, Fig. 4GoA), and most (76 ± 19%) of the IFN-{gamma} producers expressed either CD4 or CD8 (Fig. 4GoB, and data not shown). Moreover, the percentage of IFN-{gamma}-producing cells among CD4+ or CD8+ IEL was significantly higher in affected than in healthy chimeras (p < 0.01, Fig. 4GoB).


   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
In this report, we have described a novel murine colitis model, RIC, which is very reproducible and develops with complete penetrance. Irradiation hemopoietic chimeras that had been reconstituted with bone marrow derived from MHC class II expression deficient donors developed symptoms similar to those observed in human Crohn’s disease and ulcerative colitis: hyperplasia of the lamina propria and concomitant elongation of crypts, metaplasia, and infiltration of lamina propria by lymphocytes (of which a large proportion produces IFN-{gamma}), monocytes, and polymorphonuclear leukocytes. RIC develops in C57BL/6 mice, which allows the use of a large selection of induced mutants available on this genetic background.

The etiology of Crohn’s disease and ulcerative colitis remains unknown. Because several types of mutant mice develop colitis much later in life (see Introduction), in RIC the high dose gamma-irradiation is probably instrumental in disease development and in its complete penetrance. Because gamma-irradiation is known to cause damage to intestinal epithelium (58), Ags present in the gut lumen will presumably have more readily access to the lamina propria after irradiation. These Ags are foreign to the immune system, and inflammation followed by massive lymphocyte activation would be expected to occur. An uncontrolled inflammatory reaction may nonspecifically cause mucosal damage (59). The uncontrolled immune response may even develop into autoaggression (29, 30) because of molecular mimicry or bystander activation of normally tolerant (anergic) autospecific lymphocytes. While in animals with a normal immune system regulatory T lymphocytes are thought to control intestinal immunity (59, 60), the absence of MHC class II on APC in the MHC I°II° -> wt and MHC II° -> wt chimeras may preclude activation of such regulatory cells and thus cause colitis. The previously described development of colitis in allogenic, but not syngenic, bone marrow chimeras (61) may be due to the discrepancy between the haplotypes of MHC-encoded dimers involved in thymic (or peripheral) positive selection and peripheral activation of regulatory T lymphocytes. Similar mechanisms may apply for development of colitis in the absence of irradiation, although the way of entry of Ags is likely to be different and in that case possibly mediated by M cells (59, 62).

Many aspects of the above described model for colitis development in RIC remain to be experimentally addressed. First, because of the requirement for costimulation in activation of naive T lymphocytes, their activation in the absence of expression of MHC molecules on APC (in MHC I°II° -> wt chimeras) is rather surprising. APC could deliver costimulation in trans (63), and Ags may be presented by MHC expressing intestinal epithelial cells. Because T lymphocytes are efficiently killed by lethal irradiation and the T cells detected in chimeras must therefore have developed after reconstitution, the involvement of Ag-experienced T cells whose activation depends less on costimulation (64) seems rather unlikely. The use of costimulation-deficient bone marrow to reconstitute hosts would be useful to investigate these possibilities.

Lack of expression of MHC class II molecules on cells of hemopoietic origin is sufficient to render mice susceptible to RIC. Thymic positive selection is not expected to be affected in MHC II° -> wt and MHC I°II° -> wt chimeras because positively selecting MHC class II molecules seem to be exclusively expressed on radioresistant thymic epithelial cells (65). Lack of thymic clonal deletion because of the absence of MHC class II on thymic APC (66, 67) is an unlikely explanation because TCR{alpha}ß expressing lymphocytes are not even required for development of colitis (1). The most likely explanation appears to be that protective T lymphocytes require activation by APC of hemopoietic origin. Whatever the explanation, it appears that tolerization to enteric Ags is not mediated by MHC class II expressing epithelial cells but rather by APC of hemopoietic origin. It will be of interest to analyze in a similar chimeric mouse system if oral tolerance also depends on presentation by hemopoietic APC (68, 69).

The situation in the autoimmune models NOD, experimental autoimmune encephalomyelitis, and CIA is substantially different from that in RIC. In the former models, MHC expression is clearly required for disease development and therefore T lymphocytes are involved as effector cells. The protective effect of MHC class II molecules may therefore affect effector and/or protector T lymphocytes. For induction of murine colitis, T lymphocytes are definitively not required (1), and expression of protective MHC class II molecules therefore almost obligatorily affects protective T lymphocytes. RIC therefore seems to be an ideal system to study MHC class II mediated protection from immunopathology.

The mechanism(s) responsible for MHC class II mediated protection from colitis (and murine autoimmune disorders) remains a mystery. Based on our data, we favor the hypothesis that protective T lymphocytes require activation by APC. Whatever the precise mechanism(s), RIC is a very versatile and reproducible model that should prove useful for the elucidation of MHC class II mediated protection from colitis with possible implications for autoimmunity.


   Acknowledgments
 
We thank G. Fischer and J. Bamat for expert help in histology and immunohistochemistry, I. Xenarios for help in intracellular cytokine detection, M. Allegrini for photography, P. Dubied for artwork, and Drs. B.-J. Fowlkes and H. Bluethmann for MHC-deficient mice.


   Footnotes
 
1 Current address: Department of Genetics and Microbiology, University of Geneva Medical School, Geneva, Switzerland. Back

2 Address correspondence and reprint requests at the current address to Dr. Joost P.M. van Meerwijk, Institut National de la Santé et de la Recherche Médicale U395, Purpan Hospital, BP 3028, 31024 Toulouse Cedex 3, France. E-mail address: Back

3 Abbreviations used in this paper: TNBS, 2,4,6-trinitrobenzene sulfonic acid; RIC, radiation-induced colitis; PFA, paraformaldehyde; MHC I°, MHC class I deficient; MHC II°, MHC class II deficient; MHC I°II°, MHC class I and II deficient; LPL, lamina propria lymphocytes; IEL, intraepithelial lymphocytes; NOD, nonobese diabetic; CIA, collagen-induced arthritis; AEC, 3-amino-9-ethylcarbazole; KO, knockout; wt, wild type; DC, dendritic cells. Back

Received for publication January 20, 1999. Accepted for publication July 22, 1999.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

  1. Mombaerts, P., E. Mizoguchi, M. J. Grusby, L. H. Glimcher, A. K. Bhan, S. Tonegawa. 1993. Spontaneous development of inflammatory bowel disease in T cell receptor mutant mice. Cell 75:274.[Medline]
  2. Sadlack, B., H. Merz, H. Schorle, A. Schimpl, A. C. Feller, I. Horak. 1993. Ulcerative colitis-like disease in mice with a disrupted interleukin-2 gene. Cell 75:253.[Medline]
  3. Kuhn, R., J. Lohler, D. Rennick, K. Rajewsky, W. Muller. 1993. Interleukin-10-deficient mice develop chronic enterocolitis. Cell 75:263.[Medline]
  4. Shull, M. M., I. Ormsby, A. B. Kier, S. Pawlowski, R. J. Diebold, M. Yin, R. Allen, C. Sidman, G. Proetzel, D. Calvin, et al 1992. Targeted disruption of the mouse transforming growth factor-ß1 gene results in multifocal inflammatory disease. Nature 359:693.[Medline]
  5. Morrissey, P. J., K. Charrier, S. Braddy, D. Liggitt, J. D. Watson. 1993. CD4+ T cells that express high levels of CD45RB induce wasting disease when transferred into congenic severe combined immunodeficient mice: disease development is prevented by cotransfer of purified CD4+ T cells. J. Exp. Med. 178:237.[Abstract/Free Full Text]
  6. Powrie, F., M. W. Leach, S. Mauze, L. B. Caddle, R. L. Coffman. 1993. Phenotypically distinct subsets of CD4+ T cells induce or protect from chronic intestinal inflammation in C.B-17 scid mice. Int. Immunol. 5:1461.[Abstract/Free Full Text]
  7. Powrie, F., R. Correa-Oliveira, S. Mauze, R. L. Coffman. 1994. Regulatory interactions between CD45RBhigh and CD45RBlow CD4+ T cells are important for the balance between protective and pathogenic cell-mediated immunity. J. Exp. Med. 179:589.[Abstract/Free Full Text]
  8. Podolsky, D. K.. 1997. Lessons from genetic models of inflammatory bowel disease. Acta Gastroenterol. Belg. 60:163.[Medline]
  9. Kim, H. S., A. Berstad. 1992. Experimental colitis in animal models. Scand. J. Gastroenterol. 27:529.[Medline]
  10. Neurath, M. F., I. Fuss, B. L. Kelsall, E. Stuber, W. Strober. 1995. Antibodies to interleukin 12 abrogate established experimental colitis in mice. J. Exp. Med. 182:1281.[Abstract/Free Full Text]
  11. Neurath, M. F., I. Fuss, B. L. Kelsall, D. H. Presky, W. Waegell, W. Strober. 1996. Experimental granulomatous colitis in mice is abrogated by induction of TGF-ß-mediated oral tolerance. J. Exp. Med. 183:2605.[Abstract/Free Full Text]
  12. Powrie, F., J. Carlino, M. W. Leach, S. Mauze, R. L. Coffman. 1996. A critical role for transforming growth factor-ß but not interleukin 4 in the suppression of T helper type 1-mediated colitis by CD45RBlow CD4+ T cells. J. Exp. Med. 183:2669.[Abstract/Free Full Text]
  13. Groux, H., A. O’Garra, M. Bigler, M. Rouleau, S. Antonenko, J. E. de Vries, M. G. Roncarolo. 1997. A CD4+ T-cell subset inhibits antigen-specific T-cell responses and prevents colitis. Nature 389:737.[Medline]
  14. Nishimoto, H., H. Kikutani, K. Yamamura, T. Kishimoto. 1987. Prevention of autoimmune insulitis by expression of I-E molecules in NOD mice. Nature 328:432.[Medline]
  15. Slattery, R. M., L. Kjer-Nielsen, J. Allison, B. Charlton, T. E. Mandel, J. F. Miller. 1990. Prevention of diabetes in non-obese diabetic I-Ak transgenic mice. Nature 345:724.[Medline]
  16. Lund, T., L. O’Reilly, P. Hutchings, O. Kanagawa, E. Simpson, R. Gravely, P. Chandler, J. Dyson, J. K. Picard, A. Edwards, et al 1990. Prevention of insulin-dependent diabetes mellitus in non-obese diabetic mice by transgenes encoding modified I-A ß-chain or normal I-E {alpha}-chain. Nature 345:727.[Medline]
  17. Gonzalez-Gay, M. A., G. H. Nabozny, M. J. Bull, E. Zanelli, III J. Douhan, M. M. Griffiths, L. H. Glimcher, H. S. Luthra, C. S. David. 1994. Protective role of major histocompatibility complex class II Eßd transgene on collagen-induced arthritis. J. Exp. Med. 180:1559.[Abstract/Free Full Text]
  18. Mustafa, M., C. Vingsbo, T. Olsson, S. Issazadeh, A. Ljungdahl, R. Holmdahl. 1994. Protective influences on experimental autoimmune encephalomyelitis by MHC class I and class II alleles. J. Immunol. 153:3337.[Abstract]
  19. Christadoss, P., C. S. David, M. Shenoy, S. Keve. 1990. Ek {alpha} transgene in B10 mice suppresses the development of myasthenia gravis. Immunogenetics 31:241.[Medline]
  20. Merino, R., M. Iwamoto, L. Fossati, P. Muniesa, K. Araki, S. Takahashi, J. Huarte, K. Yamamura, J. D. Vassalli, S. Izui. 1993. Prevention of systemic lupus erythematosus in autoimmune BXSB mice by a transgene encoding I-E {alpha} chain. J. Exp. Med. 178:1189.[Abstract/Free Full Text]
  21. Reich, E. P., R. S. Sherwin, O. Kanagawa, Jr C. A. Janeway. 1989. An explanation for the protective effect of the MHC class II I-E molecule in murine diabetes. Nature 341:326.[Medline]
  22. Schmidt, D., J. Verdaguer, N. Averill, P. Santamaria. 1997. A mechanism for the major histocompatibility complex-linked resistance to autoimmunity. J. Exp. Med. 186:1059.[Abstract/Free Full Text]
  23. Slattery, R. M., J. F. Miller, W. R. Heath, B. Charlton. 1993. Failure of a protective major histocompatibility complex class II molecule to delete autoreactive T cells in autoimmune diabetes. Proc. Natl. Acad. Sci. USA 90:10808.[Abstract/Free Full Text]
  24. Bohme, J., B. Schuhbaur, O. Kanagawa, C. Benoist, D. Mathis. 1990. MHC-linked protection from diabetes dissociated from clonal deletion of T cells. Science 249:293.[Abstract/Free Full Text]
  25. Luhder, F., J. Katz, C. Benoist, D. Mathis. 1998. Major histocompatibility complex class II molecules can protect from diabetes by positively selecting T cells with additional specificities. J. Exp. Med. 187:379.[Abstract/Free Full Text]
  26. Singer, S. M., R. Tisch, X. D. Yang, H. O. McDevitt. 1993. An Aßd transgene prevents diabetes in nonobese diabetic mice by inducing regulatory T cells. Proc. Natl. Acad. Sci. USA 90:9566.[Abstract/Free Full Text]
  27. Asano, M., M. Toda, N. Sakaguchi, S. Sakaguchi. 1996. Autoimmune disease as a consequence of developmental abnormality of a T cell subpopulation. J. Exp. Med. 184:387.[Abstract/Free Full Text]
  28. Deng, H., R. Apple, M. Clare-Salzler, S. Trembleau, D. Mathis, L. Adorini, E. Sercarz. 1993. Determinant capture as a possible mechanism of protection afforded by major histocompatibility complex class II molecules in autoimmune disease. J. Exp. Med. 178:1675.[Abstract/Free Full Text]
  29. Mizoguchi, A., E. Mizoguchi, C. Chiba, G. M. Spiekermann, S. Tonegawa, C. Nagler-Anderson, A. K. Bhan. 1996. Cytokine imbalance and autoantibody production in T cell receptor-{alpha} mutant mice with inflammatory bowel disease. J. Exp. Med. 183:847.[Abstract/Free Full Text]
  30. Wen, L., S. J. Roberts, J. L. Viney, F. S. Wong, C. Mallick, R. C. Findly, Q. Peng, J. E. Craft, M. J. Owen, A. C. Hayday. 1994. Immunoglobulin synthesis and generalized autoimmunity in mice congenitally deficient in {alpha}ß+ T cells. Nature 369:654.[Medline]
  31. Duchmann, R., I. Kaiser, E. Hermann, W. Mayet, K. Ewe, K. H. Meyer zum Buschenfelde. 1995. Tolerance exists towards resident intestinal flora but is broken in active inflammatory bowel disease (IBD). Clin. Exp. Immunol. 102:448.[Medline]
  32. Duchmann, R., E. Schmitt, P. Knolle, K. H. Meyer zum Buschenfelde, M. Neurath. 1996. Tolerance towards resident intestinal flora in mice is abrogated in experimental colitis and restored by treatment with interleukin-10 or antibodies to interleukin-12. Eur. J. Immunol. 26:934.[Medline]
  33. Contractor, N. V., H. Bassiri, T. Reya, A. Y. Park, D. C. Baumgart, M. A. Wasik, S. G. Emerson, S. R. Carding. 1998. Lymphoid hyperplasia, autoimmunity, and compromised intestinal intraepithelial lymphocyte development in colitis-free gnotobiotic IL-2-deficient mice. J. Immunol. 160:385.[Abstract/Free Full Text]
  34. Dianda, L., A. M. Hanby, N. A. Wright, A. Sebesteny, A. C. Hayday, M. J. Owen. 1997. T cell receptor-{alpha}ß-deficient mice fail to develop colitis in the absence of a microbial environment. Am. J. Pathol. 150:91.[Abstract]
  35. Mahler, M., I. J. Bristol, E. H. Leiter, A. E. Workman, E. H. Birkenmeier, C. O. Elson, J. P. Sundberg. 1998. Differential susceptibility of inbred mouse strains to dextran sulfate sodium-induced colitis. Am. J. Physiol. 274:G544.[Abstract/Free Full Text]
  36. Axelsson, L. G., E. Landstrom, T. J. Goldschmidt, A. Gronberg, A. C. Bylund-Fellenius. 1996. Dextran sulfate sodium (DSS) induced experimental colitis in immunodeficient mice: effects in CD4+-cell depleted, athymic and NK-cell depleted SCID mice. Inflamm. Res. 45:181.[Medline]
  37. Dieleman, L. A., B. U. Ridwan, G. S. Tennyson, K. W. Beagley, R. P. Bucy, C. O. Elson. 1994. Dextran sulfate sodium-induced colitis occurs in severe combined immunodeficient mice. Gastroenterology 107:1643.[Medline]
  38. Grusby, M. J., L. H. Glimcher. 1995. Immune responses in MHC class II-deficient mice. Annu. Rev. Immunol. 13:417.[Medline]
  39. Takeda, S., H. R. Rodewald, H. Arakawa, H. Bluethmann, T. Shimizu. 1996. MHC class II molecules are not required for survival of newly generated CD4+ T cells, but affect their long-term life span. Immunity 5:217.[Medline]
  40. Beutner, U., H. R. MacDonald. 1998. TCR-MHC class II interaction is required for peripheral expansion of CD4 cells in a T cell-deficient host. Int. Immunol. 10:305.[Abstract/Free Full Text]
  41. Swain, S. L.. 1983. T cell subsets and the recognition of MHC class. Immunol. Rev. 74:129.[Medline]
  42. Koller, B. H., P. Marrack, J. W. Kappler, O. Smithies. 1990. Normal development of mice deficient in ß2M, MHC class I proteins, and CD8+ T cells. Science 248:1227.[Abstract/Free Full Text]
  43. Koentgen, F., G. Suess, C. Stewart, M. Steinmetz, H. Bluethmann. 1993. Targeted disruption of the MHC class II A{alpha} gene in C57BL/6 mice. Int. Immunol. 5:957.[Abstract/Free Full Text]
  44. van Meerwijk, J. P. M., E. M. O’Connell, R. N. Germain. 1995. Evidence for lineage commitment and initiation of positive selection by thymocytes with intermediate surface phenotypes. J. Immunol. 154:6314.[Abstract]
  45. Koo, G. C., J. R. Peppard. 1984. Establishment of monoclonal anti-Nk-1.1 antibody. Hybridoma 3:301.[Medline]
  46. Dialynas, D. P., M. R. Loken, A. L. Glasebrook, F. W. Fitch. 1981. Lyt-2-/Lyt-3- variants of a cloned cytolytic T cell line lack an antigen receptor functional in cytolysis. J. Exp. Med. 153:595.[Abstract/Free Full Text]
  47. Dialynas, D. P., Z. S. Quan, K. A. Wall, A. Pierres, J. Quintans, M. R. Loken, M. Pierres, F. W. Fitch. 1983. Characterization of the murine T cell surface molecule, designated L3T4, identified by monoclonal antibody GK1.5: similarity of L3T4 to the human Leu-3/T4 molecule. J. Immunol. 131:2445.[Abstract]
  48. Golstein, P., C. Goridis, A.-M. Schmitt-Verhulst, B. Hayot, A. Pierres, A. van Agthoven, Y. Kaufmann, Z. Eshhar, M. Pierres. 1982. Lymphoid cell surface interaction structures detected using cytolysis-inhibiting monoclonal antibodies. Immunol. Rev. 68:5.[Medline]
  49. Pierres, A., P. Naquet, A. Van Agthoven, F. Bekkhoucha, F. Denizot, Z. Mishal, A. M. Schmitt-Verhulst, M. Pierres. 1984. A rat anti-mouse T4 monoclonal antibody (H129.19) inhibits the proliferation of Ia-reactive T cell clones and delineates two phenotypically distinct (T4+, Lyt-2,3-, and T4-, Lyt-2,3+) subsets among anti-Ia cytolytic T cell clones. J. Immunol. 132:2775.[Abstract]
  50. Ledbetter, J. A., L. A. Herzenberg. 1979. Xenogeneic monoclonal antibodies to mouse lymphoid differentiation antigens. Immunol. Rev. 47:63.[Medline]
  51. Bhattacharya, A., M. E. Dorf, T. A. Springer. 1981. A shared alloantigenic determinant on Ia antigens encoded by the I-A and I-E subregions: evidence for I region gene duplication. J. Immunol. 127:2488.[Abstract]
  52. Metlay, J. P., M. D. Witmer-Pack, R. Agger, M. T. Crowley, D. Lawless, R. M. Steinman. 1990. The distinct leukocyte integrins of mouse spleen dendritic cells as identified with new hamster monoclonal antibodies. J. Exp. Med. 171:1753.[Abstract/Free Full Text]
  53. Poussier, P., P. Edouard, C. Lee, M. Binnie, M. Julius. 1992. Thymus-independent development and negative selection of T cells expressing T cell receptor {alpha}ß in the intestinal epithelium: evidence for distinct circulation patterns of gut- and thymus-derived T lymphocytes. J. Exp. Med. 176:187.[Abstract/Free Full Text]
  54. Simpson, S. J., G. A. Hollander, E. Mizoguchi, D. Allen, A. K. Bhan, B. Wang, C. Terhorst. 1997. Expression of pro-inflammatory cytokines by TCR {alpha}ß+ and TCR {gamma}{delta}+ T cells in an experimental model of colitis. Eur. J. Immunol. 27:17.[Medline]
  55. Prussin, C., D. D. Metcalfe. 1995. Detection of intracytoplasmic cytokine using flow cytometry and directly conjugated anti-cytokine antibodies. J. Immunol. Methods 188:117.[Medline]
  56. Maric, I., P. G. Holt, M. H. Perdue, J. Bienenstock. 1996. Class II MHC antigen (Ia)-bearing dendritic cells in the epithelium of the rat intestine. J Immunol 156:1408.[Abstract]
  57. Powrie, F., M. W. Leach, S. Mauze, S. Menon, L. B. Caddle, R. L. Coffman. 1994. Inhibition of Th1 responses prevents inflammatory bowel disease in scid mice reconstituted with CD45RBhi CD4+ T cells. Immunity 1:553.[Medline]
  58. Novak, J. M., J. T. Collins, M. Donowitz, J. Farman, D. G. Sheahan, H. M. Spiro. 1979. Effects of radiation on the human gastrointestinal tract. J. Clin. Gastroenterol. 1:9.[Medline]
  59. Mowat, A. M., J. L. Viney. 1997. The anatomical basis of intestinal immunity. Immunol. Rev. 156:145.[Medline]
  60. Powrie, F.. 1995. T cells in inflammatory bowel disease: protective and pathogenic roles. Immunity 3:171.[Medline]
  61. van Bekkum, D. W., O. Vos, W. W. H. Weyzen. 1959. The pathogenesis of the secondary disease after foreign bone-marrow transplantation in X-irradiated mice. J. Natl. Cancer Inst. 23:75.
  62. Kerneis, S., A. Bogdanova, J. P. Kraehenbuhl, E. Pringault. 1997. Conversion by Peyer’s patch lymphocytes of human enterocytes into M cells that transport bacteria. Science 277:949.[Abstract/Free Full Text]
  63. Harding, F. A., J. G. McArthur, J. A. Gross, D. H. Raulet, J. P. Allison. 1992. CD28-mediated signalling co-stimulates murine T cells and prevents induction of anergy in T-cell clones. Nature 356:607.[Medline]
  64. Croft, M., L. M. Bradley, S. L. Swain. 1994. Naive versus memory CD4 T cell response to antigen. Memory cells are less dependent on accessory cell costimulation and can respond to many antigen-presenting cell types including resting B cells. J. Immunol. 152:2675.[Abstract]
  65. Markowitz, J. S., H. J. Auchincloss, M. J. Grusby, L. H. Glimcher. 1993. Class II-positive hematopoietic cells cannot mediate positive selection of CD4+ T lymphocytes in class II-deficient mice. Proc. Natl. Acad. Sci. USA 90:2779.[Abstract/Free Full Text]
  66. van Meerwijk, J. P. M., S. Marguerat, R. K. Lees, R. N. Germain, B. J. Fowlkes, H. R. MacDonald. 1997. Quantitative impact of thymic clonal deletion on the T cell repertoire. J. Exp. Med. 185:377.[Abstract/Free Full Text]
  67. van Meerwijk, J. P. M., H. R. MacDonald. 1999. In vivo T lymphocyte tolerance in the absence of thymic clonal deletion mediated by hematopoietic cells. Blood 93:3856.[Abstract/Free Full Text]
  68. Garside, P., A. M. Mowat. 1997. Mechanisms of oral tolerance. Crit. Rev. Immunol. 17:119.[Medline]
  69. Weiner, H. L., A. Friedman, A. Miller, S. J. Khoury, A. al-Sabbagh, L. Santos, M. Sayegh, R. B. Nussenblatt, D. E. Trentham, D. A. Hafler. 1994. Oral tolerance: immunologic mechanisms and treatment of animal and human organ-specific autoimmune diseases by oral administration of autoantigens. Annu. Rev. Immunol. 12:809.[Medline]



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