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Mucosal T Cells Bearing TCR Play a Protective Role in Intestinal Inflammation¹

Kyoko Inagaki-Ohara^2,*,, Takatoshi Chinen, Goro Matsuzaki, Atsuo Sasaki, Yukiko Sakamoto^*, Kenji Hiromatsu^*, Fukumi Nakamura-Uchiyama^*, Yukifumi Nawa^* and Akihiko Yoshimura^2,

^* Department of Infectious Diseases, Division of Parasitic Disease, University of Miyazaki, Miyazaki Medical College, Kiyotake, Miyazaki, Japan; Department of Immunology, Medical Institute of Bioregulation, Kyushu University, Higashi-ku, Maidashi, Fukuoka, Japan; and Division of Molecular Microbiology, Center of Molecular Biosciences, University of the Ryukyus, Senbaru, Nishihara, Okinawa, Japan

	Abstract

Top Abstract Introduction Materials and Methods Results Discussion References

 
Intestinal intraepithelial lymphocytes (IEL) bearing TCR represent a major T cell population in the murine intestine. However, the role of IEL in inflammatory bowel diseases (IBD) remains controversial. In this study, we show that IEL is an important protective T cell population against IBD. T cell-deficient (C^–/–) mice developed spontaneous colitis with age and showed high susceptibility to Th1-type 2,4,6-trinitrobenzene sulfonic acid (TNBS)-induced colitis at a young age. Transfer of IEL to C^–/– mice ameliorated TNBS-induced colitis, which correlated with decrease of IFN- and TNF- production and an increase of TGF- production by IEL. Furthermore, a high level of IL-15, which inhibits activation-induced cell death to terminate inflammation, was expressed more in intestinal epithelial cells (EC) from TNBS-treated C^–/– mice than in those from wild-type mice. EC from wild-type mice significantly suppressed the IFN- production of IEL from TNBS-treated C^–/– mice, whereas EC from TNBS-treated C^–/– mice did not. These data indicate that IEL play important roles in controlling IBD by regulating mucosal T cell activation cooperated with EC function. Our study suggests that enhancement of regulatory T cell activity is a possible new cell therapy for colitis.

	Introduction

Top Abstract Introduction Materials and Methods Results Discussion References

 
Intestinal mucosal lymphocytes, including both intestinal intraepithelial lymphocytes (IEL)³ and lamina propria lymphocytes (LPL), may serve a critical role in the mucosal immune system by providing immune surveillance of the epithelium (1, 2, 3). Murine IEL comprise an approximately equal frequency of population bearing TCR and TCR (4). IEL are considered to be a T cell population playing for the first line of host defense against a variety of Ags and pathogens (5, 6). Important roles of intestinal T cells in maintenance of homeostasis in intestine have been reported, such as production of keratinocyte growth factor, which stimulates epithelial cell (EC) proliferation (7) and the regulation of the generation and differentiation of EC (8). IEL also participate in immune regulation at epithelial site by contributing to the help for Ig class switching (9) and requirement for oral tolerance (10).

Inflammatory bowel diseases (IBD) are immune-mediated diseases that are induced by a complex chronic inflammatory process. The pathogenesis of IBD is deteriorated by proinflammatory cytokines such as IFN- and TNF-, and a dysregulation of T cell functions or cytokine balance has been suggested as a cause of colitis. CD4⁺CD45RB^high cells have been shown to develop spontaneous colitis (11). In contrast, transfer of CD4⁺CD45RB^low T cells with CD4⁺CD45RB^high cells prevents development of colitis mediated by involvement in TGF- and IL-10, regulatory cytokines (12, 13). However, little is known concerning the role of T cells in inflammatory disease, such as IBD. Some reports have indicated an increase in susceptibility to 2,4,6-trinitrobenzene sulfonic acid (TNBS), which is an inducer of Th1-type colitis, in T cell-depleted rats (14), and to dextran sodium sulfate, which is the inducer of T cell-independent colitis, in the C^–/– mice (15). Therefore, T cells seem to be involved in the regulation of colitis by T cell-dependent and/or -independent mechanisms. However, these protective roles against IBD remain to be investigated.

In this study, we evaluated whether intestinal T cells play a protective role and which mechanisms are involved in it. Our data suggest that IEL is a significant protective T cell population against colitis, because colitis induced in C^–/– mice is suppressed by IEL transfer mediated by TGF- production. Furthermore, aberrant cell communication between EC and IEL following TNBS administration is discussed.

	Materials and Methods

Top Abstract Introduction Materials and Methods Results Discussion References

 
Mice and induction of colitis

C57BL/6 mice were purchased at the age of 8 wk from the Japan SLC (Hamamatsu, Japan). TCR^–/– mice were purchased from the The Jackson Laboratory (Bar Harbor, ME). C^–/– mice were a kind gift from S. Itohara (Kyoto University, Kyoto, Japan). All mice were used between 8 and 12 wk of age and were approved by the institutional review board for animal experiments of Kyushu University and University of Miyazaki. Colitis was induced according to a modification of published methods (16). On days 0 and 7, the mice were administered a TNBS enema, and 3 days after the last administration, they were sacrificed to assess tissues and cells.

Detection of anaerobes

After the mice were scarified, the contents of their small intestines (SI) and large intestines (LI) were collected and suspended in saline. An aliquot of suspension was diluted, and 100 µl of the diluted suspension was plated on brain heart agar (BD Microbiology Systems, Sparks, MD). These plates were incubated in anaerobe chamber at 37°C overnight.

Histological analysis

Intestines were fixed with 4% paraformaldehyde in PBS and embedded in paraffin. The paraffin sections were stained with H&E.

Preparation of IEL, EC, and LPL

Mucosal lymphocytes were isolated and prepared according to a modification of previously published methods (17, 18). Dissected small segments of the intestines were incubated at 37°C for 30 min in an RPMI 1640 medium (Sigma-Aldrich, St. Louis, MO) containing 10% FCS and 1 mM DTT with vigorous shaking. The tissue suspension was passed through a nylon mesh to remove debris and centrifuged through a 25/40/75% discontinuous Percoll (Sigma-Aldrich) gradient at 600 x g at 20°C for 20 min. The cells collected from the interface of 40/75% and 25/40% were IEL and EC, respectively. To isolate LPL, after removal of EC and IEL, tissues were incubated for 30 min at 37°C in RPMI 1640 containing collagenase type VIII (Sigma-Aldrich). The cell suspension was centrifuged through a 40/75% discontinuous Percoll gradient, and the cells at the interface were LPL. For the cell culture, CD4⁺, CD4⁺CD8⁺ double-positive (DP), and CD8⁺ IEL were separated by using anti-FITC MultiSort Kit (Miltenyi Biotec, Bergisch Gladbach, Germany), according to the manufacturer’s instruments. For the isolation of CD4⁺ and DP IEL, IEL were stained with anti-FITC-conjugated CD4 mAb (RM4-5), following MultiSort anti-FITC MicroBeads. All CD4⁺ IEL were positively selected using a MS⁺ column in MACS (Miltenyi Biotec). The MicroBeads were then released and stained with anti-PE CD8 mAb (53-6.7), following anti-PE microbeads. DP IEL were positively sorted, and CD4⁺CD8^– IEL were negatively sorted. For the isolation of CD4^–CD8⁺ IEL, IEL stained with anti-FITC-conjugated CD8 mAb were positively sorted, and then cells were restained with anti-PE-conjugated CD4 and were negatively sorted. For culture, EC were isolated, as described previously (19). Briefly, minced intestine was digested at 20°C for 30 min in a DMEM medium (Sigma-Aldrich) containing collagenase (type XI; Sigma-Aldrich) and dispase (neutral protease type I; Roche Diagnostic Systems, Indianapolis, IN). The tissue suspension was pipetted vigorously, transferred through a nylon mesh to remove debris, and washed at 200 x g for 2 min until the supernatant was completely clear.

Transfer of IEL

Donor and IEL from the SI and IEL from the LI were negatively sorted by MACS and labeled with or without 5 µM CFSE (Sigma-Aldrich). After incubating IEL for 15 min at 37°C, cells were washed and resuspended in 200 µl of sterile PBS. These IEL (3 x 10⁶) were injected i.p. into the recipient C^–/– mice, which were irradiated with 5 Gy from a cesium source irradiator.

FACS analysis

Cells were stained, as described previously (18). The mAbs used in this study were as follows: FITC-conjugated CD4 (RM4-5), TCR (GL3) mAb; PE-conjugated CD8 (53-6.7) and TCR (H57-597) mAb; biotin-conjugated TCR (H57-597) mAb, followed by streptavidin-allophycocyanin (AP); AP-conjugated CD3 (145-2C11) mAb. Intracellular FACS of TGF- in the small intestinal IEL was performed using Cytofix/Cytoperm Plus kit (BD Pharmingen, San Jose, CA), according to the manufacturer’s instructions. After permeabilization, IEL were stained with biotin-conjugated anti-TGF- mAb (A75-3.1), followed by streptavidin-PE. For the detection of apoptotic cells, IEL were stained with FITC-conjugated annexin V and propidium iodide (PI). All mAbs were purchased from BD Pharmingen. Flow cytometry analysis was performed on a FACSCalibur flow cytometer (BD Biosciences, Franklin Lakes, NJ).

Cell culture and cytokine analysis

Whole and sorted IEL (1 x 10⁶/ml) were added to a 96-well plate precoated with 2.5 µg of anti-CD3 mAb (145-2311; BD Pharmingen) and were cultured for 48 h in RPMI 1640 supplemented with 10% FCS, 100 U/ml penicillin, and 100 U/ml streptomycin. Isolated EC by collagenase/dispase digestion were plated on collagen (Cellmatrix type I-P; Nitta Gelatin, Osaka, Japan)-coated dish in DMEM containing 2.5% FCS. For the coculture of EC with IEL, EC were trypsinized and replated on 96-well plates with a 1:10 ratio of IEL for 48 h. The supernatants were collected to estimate the cytokine contents. The cytokine contents in the culture supernatant were assayed by an ELISA using mouse IFN-, IL-4, and TNF- (eBioscience, San Diego, CA) and TGF- (BioSource International, Camarillo, CA). ELISA systems were according to the manufacturer’s instructions. Intracellular staining of TGF- in the IEL was performed by using Cytofix/Cytoperm Plus kit (BD Pharmingen), according to the manufacturer’s instructions. After permeabilization, IEL were stained with biotin-conjugated anti-TGF- mAb (A75-3.1), followed by streptavidin-PE.

Semiquantitative RT-PCR

Total RNA was extracted from isolated cells using the RNeasy Mini Kit (Qiagen, Valencia, CA) and primed with 20 pmol of a random primer in mixtures for reverse transcription. The synthesized cDNA was amplified by PCR using primers specific for the murine IL-15 and -actin cDNA sequence. The primer sets were as follows: IL-15 sense, 5'-GGA AGG CTG AGT TCC ACA TC-3'; IL-15 antisense, AGG GAG ACC TAC ACT GAC AC-3'; -actin sense, 5'-TGG AAT CCT GTG GCA TCC ATG AAA C-3'; -actin antisense, 5'-TAA AAC GCA GCT CAG TAA CAG TCC G-3'.

Western blotting analysis

Lysates of EC from both small and large intestinal tissues of wild-type (WT) and C^–/– mice were obtained, according to a modification of previously published methods (20). Goat anti-mouse IL-15 Ab was purchased from R&D Systems (Minneapolis, MN), and mouse anti--actin Ab from Sigma-Aldrich.

Statistics analysis

Student’s t test was used to determine the significant difference. A p value of <0.05 was taken as significant. Kaplan-Meier analysis (log-rank test) was used to determine the significant difference of survival following TNBS administration.

	Results

Top Abstract Introduction Materials and Methods Results Discussion References

 
Susceptibility of C^–/– mice to colitis induction

We found that 5% of C^–/– mice over 8 mo of age developed colitis with soft stool, diarrhea, and rectal prolapse when housed in a conventional environment (data not shown). When healthy young C^–/– mice were housed with the aged mice with rectal prolapse, the young mice showed no signs or symptoms. Thus, these observations suggested that the colitis of C^–/– mice was not induced by infection in the colony, and C^–/– mice spontaneously develop colitis with age.

Development of spontaneous colitis in aged C^–/– mice led us to examine whether young C^–/– mice are susceptible to colitis induction. The ability of T cells to protect the intestinal mucosa from TNBS has been reported in Lewis rats treated with anti-TCR Ab (14). Thus, we used C^–/– mice to investigate significance of T cells for suppression of TNBS-induced colitis. In advance to this experiment, we optimized the dose of TNBS required to induce colitis in C57BL/6 mice because this strain is considered to be resistant to TNBS-induced colitis, and C^–/– mice are C57BL/6 background. Intrarectal inoculation of TNBS at 20 µg/g body weight induced no colitis, and 80 µg/g body weight was a lethal dose. The TNBS inoculation at 40 µg/g body weight induced mild loss of body weight without lethality (Table I). Thus, we used the dose of 40 µg/g body weight of TNBS in the following experiments. As shown in Fig. 1A, 50% of young C^–/– mice died within 14 days after TNBS administration, whereas WT mice survived. The TNBS-treated C^–/– mice showed a more severe thickening and shortening of the colon than the TNBS-treated WT mice. In addition, the SI was also impaired by TNBS administration (Fig. 1B), because the tissue wall became thin and fragile. As it was found that the TNBS-treated C^–/– mice showed diarrhea and it has been reported that anaerobe number was increased in IBD patient (21), we examined changes of colony number of anaerobe. Anaerobe number was greater in C^–/– mice than WT mice following TNBS administration (Fig. 1C). These results suggest a strong correlation between the high sensitivity to TNBS-induced colitis and the lack of T cells in the intestine; they also indicate that the T cells have a beneficial role for protection against TNBS-induced colitis.

View larger version (50K): [in this window] [in a new window]   FIGURE 1. Susceptibility of C–/– mice to TNBS. A, C–/– mice and WT mice (n = 10) were administered 40 µg of TNBS per gram of body weight. Survival rates were obtained daily after the first TNBS administration. B, Gross photographs of dissected SI and LI of WT and C–/– mice on day 10 after TNBS administration. C, Anaerobe number on day 10 after TNBS administration. Values represent the mean ± SD of three individual experiments.

Preventive role of IEL against TNBS-induced colitis

To investigate the regulatory role of T cells against colitis, we examined whether transfer of intestinal IEL protects C^–/– mice from TNBS-induced colitis. Transferred small intestinal IEL prepared from the WT mice were repopulated in the SI of C^–/– mice at the level comparable to WT mice (Fig. 2A). The small intestinal IEL also repopulated in the LI of the C^–/– mice, although the number is lower than that of WT mice. However, the transferred T cells were not detected in other tissues such as the liver and spleen. The large intestinal IEL transferred to C^–/– mice repopulated preferentially in LI, but not in SI (Fig. 2A). When small intestinal IEL-transferred mice were administered with TNBS, they exhibited significant increase of survival rate (Fig. 2B). In contrast, small intestinal IEL-transferred C^–/– mice showed exacerbated colitis. Interestingly, IEL prepared from LI did not improve the odds of survival against TNBS-induced colitis. On gross examination, nontransferred C^–/– mice showed severe shortening and thickening of colonic wall after TNBS treatment. In contrast, small intestinal IEL-transferred C^–/– mice showed milder thickening of the colon after TNBS treatment compared with the nontransferred C^–/– mice (Fig. 2C, left). Small intestinal IEL- and large intestinal IEL-transferred C^–/– mice exaggerated thickening. In histological analysis, we observed the thickening of intestinal wall and the ulcers reaching the muscle layer, diffuse cell infiltration, and distortion of crypts in the TNBS-treated C^–/– mice that had not received any IEL and TNBS-treated small intestinal IEL- or large intestinal IEL-transferred C^–/– mice (Fig. 2C, right). However, small intestinal IEL-transferred C^–/– mice showed only mild thickening and cell infiltration, although distortion of crypts was observed. These results suggest that small intestinal IEL suppress colitis when transferred to colitis-prone mice with no IEL.

View larger version (51K): [in this window] [in a new window]   FIGURE 2. Suppressive role of IEL in the development of colitis. A, Reconstitution of transferred IEL in the recipient C–/– mice. or IEL in the SI and IEL in the LI of WT mice were transferred into irradiated C–/– mice, as described in Materials and Methods. Three weeks later, IEL in both the SI and LI were prepared in these IEL-transferred recipient mice for FACS analysis. Representative data show IEL staining in mice, which are IEL transferred. The numbers indicate the percentages of fluorescence-positive cells in the corresponding squares. B, Survival rate of C–/– mice. None of IEL or SI IEL were transferred to C–/– mice (n = 12). SI (n = 10) or LI IEL (n = 9) were transferred to C–/– mice. After the transfer of cells, the mice were administered 40 µg of TNBS per gram of body weight. The survival rates were observed daily after the first TNBS administration. Data shown represent the survival in terms of percentage obtained by Kaplan-Meier analysis. *, p < 0.05 was determined by the log-rank test (Mantel-Cox). C, The gross appearance (left) and H&E staining of transverse sections of the colon in each group on day 14 (right, magnification, x100).

View larger version (40K): [in this window] [in a new window]   FIGURE 3. Changes of cytokine production following TNBS administration. A, Cytokine profiles in IEL in both SI and LI before TNBS administration. IEL were prepared before TNBS administration and on day 10 after the first TNBS enema. Freshly isolated IEL were cultured with plate-bound anti-CD3 mAb for 48 h, and the supernatants were collected to determine the concentration of each cytokine by ELISA for IFN-, IL-4, TNF-, and TGF-. B, Cytokine production of the SI and LI IEL in C–/– mice following TNBS administration. Intracellular FACS of TGF- was performed in the SI IEL of the recipient C–/– mice, which received CFSE-labeled SI IEL (right). Values represent the mean ± SD of three individual experiments. *, Represents a statistically significant difference (p < 0.05).

Increase in CD4⁺CD8⁺ DP IEL in C^–/– mice treated with TNBS

We also noticed that CD4⁺CD8⁺ (DP) IEL in the SI increased more in C^–/– mice than it did in WT mice at 20 and 40 wk of age (Fig. 4A). Similar changes were observed in LPL in the SI (Fig. 4A) and IEL in the LI (data not shown). As the increase of DP T cells was most marked in the IEL of the SI, we focused on the IEL in the SI in the following experiments. We examined whether the increased number of DP T cells in C^–/– mice contributes to increased susceptibility to TNBS-induced colitis in the mice. When C^–/– mice were treated with TNBS, an increase in DP IEL was observed even in young mice, but TNBS administration did not induce DP IEL in WT mice (Fig. 4B). The transfer of IEL into C^–/– mice reduced increase of DP IEL after TNBS administration. No such increase of DP IEL was observed in TCR^–/– mice, which develop spontaneous Th2-type colitis with age. Even though TCR^–/– mice showed symptoms of colitis, such as prolapse at 30 wk, they did not show the alterations of the DP population (Fig. 4C). These results suggest that the T cell is involved in the regulation of DP cells, and increase of DP cells may be associated with Th1-type, but not Th2-type, colitis.

View larger version (50K): [in this window] [in a new window]   FIGURE 4. Age- or TNBS-dependent changes of intestinal T cells in C–/– mice. A, CD4 and CD8 expression of SI IEL and LPL cells in WT and C–/– mice at 8, 20, and 40 wk of age. These CD4 and CD8 expressions are gated on CD3+ cells. B, Expression of CD4 and CD8 on SI IEL. SI IEL obtained from WT, C–/–, and C–/– mice transferred with SI IEL in WT mice, which were treated with or without TNBS. C, The expression of CD4 and CD8 of SI IEL in TCR–/– mice.

Next, we tried to identify the cytokine produced by the DP IEL, and the functional differences between the DP IEL in aged WT mice and those induced in TNBS in C^–/– mice. As shown in Fig. 5, the DP IEL from aged WT mice and TNBS-treated C^–/– mice produced a high level of TGF- as well as CD8⁺ IEL from the mice. IFN- and TNF- were predominantly produced by CD8⁺ IEL rather than DP IEL, and TNF- production in C^–/– mice was much more than that in aged WT mice. In contrast, CD4⁺ IEL were not involved in the production of these cytokines. These results suggest that the increase of DP IEL in C^–/– mice after TNBS administration may play a protective role against colitis because these cells produced TGF-. We speculate that the TGF--producing DP IEL increased in C^–/– mice to compensate for the lack of a regulatory function of IEL.

View larger version (43K): [in this window] [in a new window]   FIGURE 5. Comparison of cytokine production between aged WT mice and C–/– mice treated with TNBS. Concentrations of each cytokine in whole, CD4+, CD4+CD8+, and CD8+ subsets are shown. Values represent the mean ± SD of three individual experiments.

EC play an essential role in the onset and development of colitis, and IEL and EC regulate each other (8, 22). Therefore, we next analyzed EC-mediated regulation of IEL in C^–/– mice. As an EC-derived immunoregulator, we first analyzed the expression of IL-15 by EC because: 1) IL-15 is a cytokine produced by various cells including EC (18, 23); 2) IL-15 affects the survival of T cells by inhibiting activation-induced cell death (AICD) (24, 25); and 3) IL-15 mRNA increased in the inflamed intestinal tissue of IBD (26, 27). Although the C^–/– and WT mice showed similar expression levels before TNBS administration in the SI, the C^–/– mice showed an enhanced IL-15 mRNA level after TNBS administration, whereas WT mice showed only a slight increase (Fig. 6A). The C^–/– mice also showed a slight increase of the IL-15 message in the LI following TNBS administration. We next examined expression of IL-15 at the protein level by Western blotting analysis. In accordance with the result of RT-PCR, larger amount of IL-15 was produced by the EC of the C^–/– mice than those of WT mice following TNBS administration (Fig. 6B). We further analyzed AICD of intestinal T cells in the C^–/– mice. IEL of the C^–/– mice contained 30% of apoptotic annexin V-positive PI-negative cells, but those of WT mice contained 60% of apoptotic cells (Fig. 6C), indicating that less IEL of C^–/– mice undergo AICD than those of WT mice. These results suggest that EC of C^–/– mice protect proinflammatory intestinal T cells from AICD through expression of IL-15 at a high level. These results also suggest that the sustained activation of IEL could be caused by a failure to terminate the inflammation.

View larger version (29K): [in this window] [in a new window]   FIGURE 6. Increased IL-15 level in EC and following TNBS administration. A, Expression of IL-15 mRNA in both EC of SI and LI in WT and C–/– mice with or without TNBS administration. Semiquantitative RT-PCR was performed by using cDNA diluted with 4-fold serially as templates. B, Lysates from EC in WT and C–/– mice were prepared and were subsequently analyzed by Western blot for IL-15. C, IEL were incubated with FITC-conjugated annexin V, PI, and AP conjugated with TCR mAb.

View larger version (20K): [in this window] [in a new window]   FIGURE 7. Inhibition of the activation of IEL stimulated with anti-CD3 mAb by EC. IEL were cultured with EC in the presence of immobilized anti-CD3 mAb at EC to IEL ratios of 1:10. These cells were then incubated for 48 h at 37°C to collect supernatants for the determination of cytokine production by ELISA for IFN- and TGF-. Values represent the mean ± SD of three individual experiments. *, Represents a statistically significant difference (p < 0.05).

	Discussion

Top Abstract Introduction Materials and Methods Results Discussion References

Germfree mice rarely develop IBD, which implies that the presence of microbes influences the development of IBD. Our results showed that the intestine of C^–/– mice with TNBS-induced colitis contained an increased number of anaerobes (Fig. 1C). These results could be supported by the fact that the pathology of colitis is associated with a high level of anaerobe (21) and a reduction of T cell (31). It has been also reported that a decrease in the humoral immune response is associated with an increase of several species of enteric bacteria (32, 33). It was also demonstrated that mice with a deficiency in Ig class switch and IgA production showed an increase of anaerobes in the intestine (34). Because colitis-prone C^–/– mice showed a decrease of TGF- production and TGF- is an important cytokine in the IgA class switch, it is possible that the reduction of IgA secretion in C^–/– mice causes an increase of anaerobes and the resultant colitis. The hypothesis is now under investigation.

As IL-15 is produced by the EC and is a growth factor of IEL, the possible roles of IL-15 in the mucosal intranet between EC and IEL have been investigated (18, 35). In contrast, negative regulatory systems controlling IL-15 production may be required because IL-15 is also an inflammatory cytokine. The expression of IL-15 mRNA was found to be increased in the inflamed intestinal tissue of IBD (26, 27). IL-15 has been reported to be overexpressed in rheumatoid arthritis, and IL-15-recruited and -activated autoreactive T cells in the synoviral membrane have led to TNF- secretion in synoviral fluid (36, 37). Localized overexpression of intestinal IL-15 led to the development of a unique subset of AICD-resistant Th1-type CD8⁺NK1.1⁺ T cells and the induction of inflammation in the SI (38). The TNBS-treated mice showed a high level of inflammatory cytokines from IEL (Figs. 3 and 5) and a high level of IL-15 mRNA expression and protein (Fig. 6, A and B). IL-15 sustains the Bcl-2 level of IEL (18), causing IEL to survive and become resistant against apoptosis in IBD. Collectively, the activation observed in C^–/– mice might be due to effete or aberrant cells failing to undergo apoptosis.

The only barrier between the Ag-rich luminal environment and the mucosal immune system is the single layer of EC. EC would not only form a purely physical barrier, but would also be capable of producing a series of cytokines, chemokines, and defensins (39, 40). Cell-to-cell interactions between IEL and EC would be mediated by cell surface molecules, suggesting that these interactions prevent potential inflammatory responses at the intestinal mucosal surface (22). We demonstrated the significance of EC in the regulation of IEL function. The activation of IEL in TNBS-treated mice was suppressed by normal EC (Fig. 7). However, EC from TNBS-treated C^–/– mice failed to suppress IFN- production of IEL. Therefore, T cell-mediated acquisition of EC-suppressive function can be an important mechanism of T cell-mediated suppression of IEL. In this study, we showed the suppressive effect of T cells on colitis. However, T cells are not only anti-inflammatory cells in the intestine. Our data also demonstrated that DP IEL produce TGF-. We speculate that the DP IEL may also participate in suppression of colitis, which is in accordance with previous reports showing regulatory function of DP IEL against a colitis model (41). In addition, intestinal EC express CD1d. CD1d-restricted NKT cells have been reported to suppress dextran sodium sulfate-induced colitis (42). It is assumed that multiple regulatory T cell subsets are necessary for protecting against colitis. To elucidate the different reactivity of these regulatory T cells is valuable in therapeutics for colitis.

We demonstrated in the present study that DP IEL increase in C^–/– mice from early stage of ontogeny, although such increase of DP IEL was observed at later stage of ontogeny in WT mice. Although the mechanism of the early increase of DP IEL in C^–/– mice is not clear at this moment, there are two possible mechanisms for the increase. The increase of anaerobic bacteria in the intestinal lumen of C^–/– mice may induce the increase of DP IEL because it has been reported that the increase of DP IEL depends on the recognition of bacterial Ags (43, 44). Alternatively, inflammation in the intestine recruits CD4⁺ T cells to the intestine and converts its phenotype to DP. The second possibility is supported by the observation that splenic CD4⁺CD8^– T cells change their phenotype to DP when transferred to RAG-1^–/– mice (41). We need further information to prove the hypotheses.

In summary, our study demonstrates that T cells have protective ability against developing colitis, and suggests the possibility of a novel therapeutic strategy using regulatory T cells for the suppression of colitis, including transfer of the T cells or activation of the cells in vivo. In this study, we proposed that the protective role of T cells against colitis is mediated by TGF- production. Furthermore, T cell-mediated regulation of the immunoregulatory function of EC is also suggested to be involved in the regulation of colitis. However, it remains to be determined when and how T cells develop and are induced as regulatory cells during the development of colitis, and whether there is communication or a relationship between T cells and other regulatory cells such as DP cell. Therefore, it will be important to characterize T cells as regulatory T cells.

	Acknowledgments

 
We thank Drs. Keisuke Nakayama and Kaori Ohtani (University of Miyazaki) for providing advice about anaerobe cultures. We also thank Chiharu Yamauchi and Joko Shigenobu for expert technical assistance, and Mika Sasaki and Ayumi Tanaka for their steadfast support.

	Footnotes

 
¹ This work was supported by grants-in-aid (to A.Y.) and the Japan Health Science Foundation, the Human Frontier Science Program, and 21st Century Center of Excellence program (University of Miyazaki) from the Japanese Ministry of Education, Science, Technology, Sports, and Culture, and Public Trust Haraguchi Memorial Cancer Research Fund (to K.I.-O.).

² Address correspondence and reprint requests to Dr. Kyoko Inagaki-Ohara, Department of Infectious Diseases, Division of Parasitic Disease, University of Miyazaki, Miyazaki Medical College, Kiyotake, Miyazaki 889-1692, Japan (E-mail address: INAGAKI{at}med.miyazaki-u.ac.jp); or Dr. Akihiko Yoshimura, Division of Molecular and Cellular Immunology, Medical Institute of Bioregulation, Kyushu University, Fukuoka 812-8582, Japan (E-mail address: yakihiko{at}bioreg.kyushu-u.ac.jp).

³ Abbreviations used in this paper: IEL, intestinal intraepithelial lymphocytes; AICD, activation-induced cell death; AP, allophycocyanin; DP, double positive; EC, epithelial cell; IBD, inflammatory bowel disease; LI, large intestine; LPL, lamina propria lymphocyte; PI, propidium iodide; SI, small intestine; TNBS, 2,4,6-trinitrobenzene sulfonic acid; WT, wild type.

Received for publication January 27, 2004. Accepted for publication May 14, 2004.

	References

Top Abstract Introduction Materials and Methods Results Discussion References

 

1. Janeway, C. A., Jr, B. Jones, A. Hayday. 1988. Specificity and function of T cells bearing receptors. Immunol. Today 9:73.[Medline]
2. Panja, A., R. S. Blumberg, S. P. Balk, L. Mayer. 1993. CD1d is involved in T cell-intestinal epithelial cell interactions. J. Exp. Med. 178:1115.[Abstract/Free Full Text]
3. Targan, S. R., R. L. Deem, M. Liu, S. Wang, A. Nel. 1995. Definition of a lamina propria T cell responsive state: enhanced cytokine responsiveness of T cells stimulated through the CD2 pathway. J. Immunol. 154:664.[Abstract]
4. Guy-Grand, D., N. Cerf-Bensussan, B. Malissen, M. Malassis-Seris, C. Briottet, P. Vassalli. 1991. Two gut intraepithelial CD8⁺ lymphocyte populations with different T cell receptors: a role for the gut epithelium in T cell differentiation. J. Exp. Med. 173:471.[Abstract/Free Full Text]
5. Allison, J. P., W. L. Havran. 1991. The immunobiology of T cells with invariant antigen receptors. Annu. Rev. Immunol. 9:679.[Medline]
6. Kaufmann, S. H.. 1996. / and other unconventional T lymphocytes: what do they see and what do they do?. Proc. Natl. Acad. Sci. USA 93:2272.[Abstract/Free Full Text]
7. Boismenu, R., W. L. Havran. 1994. Modulation of epithelial cell growth by intraepithelial T cells. Science 266:1253.[Abstract/Free Full Text]
8. Komano, H., Y. Fujiura, M. Kawaguchi, S. Matsumoto, Y. Hashimoto, S. Obana, P. Mombaerts, S. Tonegawa, H. Yamamoto, S. Itohara, et al 1995. Homeostatic regulation of intestinal epithelia by intraepithelial T cells. Proc. Natl. Acad. Sci. USA 92:6147.[Abstract/Free Full Text]
9. Fujihashi, K., T. Taguchi, W. K. Aicher, J. R. McGhee, J. A. Bluestone, J. H. Eldridge, H. Kiyono. 1992. Immunoregulatory functions for murine intraepithelial lymphocytes: / T cell receptor-positive (TCR⁺) T cells abrogate oral tolerance, while / TCR⁺ T cells provide B cell help. J. Exp. Med. 175:695.[Abstract/Free Full Text]
10. Ke, Y., K. Pearce, J. P. Lake, H. K. Ziegler, J. A. Kapp. 1997. T lymphocytes regulate the induction and maintenance of oral tolerance. J. Immunol. 158:3610.[Abstract]
11. Powrie, F., R. Correa-Oliveira, S. Mauze, R. L. Coffman. 1994. Regulatory interactions between CD45RB^high and CD45RB^low CD4⁺ T cells are important for the balance between protective and pathogenic cell-mediated immunity. J. Exp. Med. 179:589.[Abstract/Free Full Text]
12. Read, S., V. Malmstrom, F. Powrie. 2000. Cytotoxic T lymphocyte-associated antigen 4 plays an essential role in the function of CD25⁺CD4⁺ regulatory cells that control intestinal inflammation. J. Exp. Med. 192:295.[Abstract/Free Full Text]
13. Annacker, O., R. Pimenta-Araujo, O. Burlen-Defranoux, T. C. Barbosa, A. Cumano, A. Bandeira. 2001. CD25⁺ CD4⁺ T cells regulate the expansion of peripheral CD4 T cells through the production of IL-10. J. Immunol. 166:3008.[Abstract/Free Full Text]
14. Hoffmann, J. C., K. Peters, S. Henschke, B. Herrmann, K. Pfister, J. Westermann, M. Zeitz. 2001. Role of T lymphocytes in rat 2,4,6-trinitrobenzene sulphonic acid (TNBS) induced colitis: increased mortality after T cell depletion and no effect of T cell depletion. Gut 48:489.[Abstract/Free Full Text]
15. Chen, Y., K. Chou, E. Fuchs, W. L. Havran, R. Boismenu. 2002. Protection of the intestinal mucosa by intraepithelial T cells. Proc. Natl. Acad. Sci. USA 99:14338.[Abstract/Free Full Text]
16. Dohi, T., K. Fujihashi, H. Kiyono, C. O. Elson, J. R. McGhee. 2000. Mice deficient in Th1- and Th2-type cytokines develop distinct forms of hapten-induced colitis. Gastroenterology 119:724.[Medline]
17. Harriman, G. R., E. Hornqvist, N. Y. Lycke. 1992. Antigen-specific and polyclonal CD4⁺ lamina propria T-cell lines: phenotypic and functional characterization. Immunology 75:66.[Medline]
18. Inagaki-Ohara, K., H. Nishimura, A. Mitani, Y. Yoshikai. 1997. Interleukin-15 preferentially promotes the growth of intestinal intraepithelial lymphocytes bearing T cell receptor in mice. Eur. J. Immunol. 27:2885.[Medline]
19. Evans, G. S., N. Flint, A. S. Somers, B. Eyden, C. S. Potten. 1992. The development of a method for the preparation of rat intestinal epithelial cell primary cultures. J. Cell Sci. 101:219.[Abstract/Free Full Text]
20. Sasaki, A., H. Yasukawa, A. Suzuki, S. Kamizono, T. Syoda, I. Kinjyo, M. Sasaki, J. A. Johnston, A. Yoshimura. 1999. Cytokine-inducible SH2 protein-3 (CIS3/SOCS3) inhibits Janus tyrosine kinase by binding through the N-terminal kinase inhibitory region as well as SH2 domain. Genes Cells 4:339.[Abstract]
21. Cummings, J. H., G. T. Macfarlane, S. Macfarlane. 2003. Intestinal bacteria and ulcerative colitis. Curr. Issues Intest. Microbiol. 4:9.[Medline]
22. Yamamoto, M., K. Fujihashi, K. Kawabata, J. R. McGhee, H. Kiyono. 1998. A mucosal intranet: intestinal epithelial cells down-regulate intraepithelial, but not peripheral, T lymphocytes. J. Immunol. 160:2188.[Abstract/Free Full Text]
23. Giri, J. G., M. Ahdieh, J. Eisenman, K. Shanebeck, K. Grabstein, S. Kumaki, A. Namen, L. S. Park, D. Cosman, D. Anderson. 1994. Utilization of the and chains of the IL-2 receptor by the novel cytokine IL-15. EMBO J. 13:2822.[Medline]
24. Marks-Konczalik, J., S. Dubois, J. M. Losi, H. Sabzevari, N. Yamada, L. Feigenbaum, T. A. Waldmann, Y. Tagaya. 2000. IL-2-induced activation-induced cell death is inhibited in IL-15 transgenic mice. Proc. Natl. Acad. Sci. USA 97:11445.[Abstract/Free Full Text]
25. Ku, C. C., M. Murakami, A. Sakamoto, J. Kappler, P. Marrack. 2000. Control of homeostasis of CD8⁺ memory T cells by opposing cytokines. Science 288:675.[Abstract/Free Full Text]
26. Kirman, I., O. H. Nielsen. 1996. Increased numbers of interleukin-15-expressing cells in active ulcerative colitis. Am. J. Gastroenterol. 91:1789.[Medline]
27. Vainer, B., O. H. Nielsen, J. Hendel, T. Horn, I. Kirman. 2000. Colonic expression and synthesis of interleukin 13 and interleukin 15 in inflammatory bowel disease. Cytokine 12:1531.[Medline]
28. Ehrhardt, R. O., W. Strober, G. R. Harriman. 1992. Effect of transforming growth factor (TGF)-1 on IgA isotype expression: TGF-1 induces a small increase in sIgA⁺ B cells regardless of the method of B cell activation. J. Immunol. 148:3830.[Abstract]
29. Poussier, P., T. Ning, D. Banerjee, M. Julius. 2002. A unique subset of self-specific intraintestinal T cells maintains gut integrity. J. Exp. Med. 195:1491.[Abstract/Free Full Text]
30. Peng, S. L., J. M. McNiff, M. P. Madaio, J. Ma, M. J. Owen, R. A. Flavell, A. C. Hayday, J. Craft. 1997. T cell regulation and CD40 ligand dependence in murine systemic autoimmunity. J. Immunol. 158:2464.[Abstract]
31. Fukushima, K., T. Masuda, H. Ohtani, I. Sasaki, Y. Funayama, S. Matsuno, H. Nagura. 1991. Immunohistochemical characterization, distribution, and ultrastructure of lymphocytes bearing T-cell receptor / in inflammatory bowel disease. Gastroenterology 101:670.[Medline]
32. Elsaghier, A., C. Prantera, C. Moreno, J. Ivanyi. 1992. Antibodies to Mycobacterium paratuberculosis-specific protein antigens in Crohn’s disease. Clin. Exp. Immunol. 90:503.[Medline]
33. Darfeuille-Michaud, A., C. Neut, N. Barnich, E. Lederman, P. Di Martino, P. Desreumaux, L. Gambiez, B. Joly, A. Cortot, J. F. Colombel. 1998. Presence of adherent Escherichia coli strains in ileal mucosa of patients with Crohn’s disease. Gastroenterology 115:1405.[Medline]
34. Fagarasan, S., M. Muramatsu, K. Suzuki, H. Nagaoka, H. Hiai, T. Honjo. 2002. Critical roles of activation-induced cytidine deaminase in the homeostasis of gut flora. Science 298:1424.[Abstract/Free Full Text]
35. Reinecker, H. C., R. P. MacDermott, S. Mirau, A. Dignass, D. K. Podolsky. 1996. Intestinal epithelial cells both express and respond to interleukin 15. Gastroenterology 111:1706.[Medline]
36. McInnes, I. B., J. al-Mughales, M. Field, B. P. Leung, F. P. Huang, R. Dixon, R. D. Sturrock, P. C. Wilkinson, F. Y. Liew. 1996. The role of interleukin-15 in T-cell migration and activation in rheumatoid arthritis. Nat. Med. 2:175.[Medline]
37. McInnes, I. B., B. P. Leung, R. D. Sturrock, M. Field, F. Y. Liew. 1997. Interleukin-15 mediates T cell-dependent regulation of tumor necrosis factor- production in rheumatoid arthritis. Nat. Med. 3:189.[Medline]
38. Ohta, N., T. Hiroi, M. N. Kweon, N. Kinoshita, M. H. Jang, T. Mashimo, J. Miyazaki, H. Kiyono. 2002. IL-15-dependent activation-induced cell death-resistant Th1 type CD8 ⁺NK1.1⁺ T cells for the development of small intestinal inflammation. J. Immunol. 169:460.[Abstract/Free Full Text]
39. Campbell, N., X. Y. Yio, L. P. So, Y. Li, L. Mayer. 1999. The intestinal epithelial cell: processing and presentation of antigen to the mucosal immune system. Immunol. Rev. 172:315.[Medline]
40. O’Neil, D. A., E. M. Porter, D. Elewaut, G. M. Anderson, L. Eckmann, T. Ganz, M. F. Kagnoff. 1999. Expression and regulation of the human -defensins hBD-1 and hBD-2 in intestinal epithelium. J. Immunol. 163:6718.[Abstract/Free Full Text]
41. Das, G., M. M. Augustine, J. Das, K. Bottomly, P. Ray, A. Ray. 2003. An important regulatory role for CD4⁺CD8 T cells in the intestinal epithelial layer in the prevention of inflammatory bowel disease. Proc. Natl. Acad. Sci. USA 100:5324.[Abstract/Free Full Text]
42. Saubermann, L. J., P. Beck, Y. P. De Jong, R. S. Pitman, M. S. Ryan, H. S. Kim, M. Exley, S. Snapper, S. P. Balk, S. J. Hagen, et al 2000. Activation of natural killer T cells by -galactosylceramide in the presence of CD1d provides protection against colitis in mice. Gastroenterology 119:119.[Medline]
43. Takeuchi, M., H. Miyazaki, K. Mirokawa, T. Yokokura, Y. Yoshikai. 1993. Age-related changes of T cell subsets in intestinal intraepithelial lymphocytes of mice. Eur. J. Immunol. 23:1409.[Medline]
44. Nakamura, T., G. Matsuzaki, H. Takimoto, K. Nomoto. 1995. Age-associated changes in the proliferative response of rat intestinal intraepithelial leukocytes to bacterial antigens. Gastroenterology 109:748.[Medline]

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