Anti-CD3 induces bi-phasic apoptosis in murine intestinal epithelial cells: possible involvement of the Fas/Fas ligand system in different T cell compartments
Naoko Miura1,
Masahiro Yamamoto1,
Masato Fukutake1,
Nobuhiro Ohtake1,
Seiichi Iizuka1,
Atsushi Ishige1,
Hiroshi Sasaki1,
Kazunori Fukuda2,
Tatsuo Yamamoto3 and
Satoshi Hayakawa3
1 Tsumura Research Institute, Tsumura & Co., 3586 Yoshiwara, Ami-machi, Inashiki-gun, Ibaraki 300-1192, Japan
2 Department of Oriental Medicine, Gifu University School of Medicine, Gifu, Japan
3 Department of Obstetrics and Gynaecology, Nihon University School of Medicine, Tokyo, Japan
Correspondence to: N. Miura; E-mail: miura_naoko{at}mail.tsumura.co.jp
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Abstract
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Recent studies have suggested that Fas-mediated apoptosis is involved in the pathogenesis of intestinal injury. In this study, we determined the role of Fas/Fas ligand (FasL) interactions in different T cell compartments using a murine model of small intestinal injury. An intraperitoneal injection of 145-2C11 (anti-CD3) antibody into C3H/HeN, BALB/c and MRL mice induced mucosal flattening and rapid, bi-phasic intestinal epithelial cell (IEC) apoptosis, which was detected by conventional light and electron microscopy and by terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling. In the first, early phase, villous apoptosis was observed up to 4 h after injection, and in the second, later phase, apoptotic crypt cells gradually accumulated for up to 24 h. The early and later phases of apoptosis were reduced in lpr/lpr and nude mice compared with those in control strains. In addition, the kinetics of Fas-mediated killer activity induced by the antibody injection were different between intestinal intraepithelial lymphocytes (IEL) and splenocytes (SPL) and seemed to correlate with the bi-phasic occurrence of the apoptosis. Finally, the transfer of intestinal IEL from euthymic to nude mice induced both phases of apoptosis, whereas SPL induced the second phase's crypt apoptosis only by the antibody injection. Together, these results suggest the involvement of Fas-mediated killer activity of thymus-derived T cells in different compartments. Namely, T cell populations in different compartments are differentially involved in the induction of IEC apoptosis and contribute to the complex pathogenesis of immune-mediated intestinal injury in which Fas/FasL interactions may play a critical role.
Keywords: crypt, intestinal injury, intestinal intraepithelial lymphocytes, splenocytes, villus
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Introduction
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A consensus is emerging that T cells play a central role in the development of intestinal inflammation. Activated T cells are involved in both regulatory and effector mechanisms in inflammatory responses. The effector mechanisms are important for defense against enterobacteria, but may also cause significant bystander tissue damage such as villous atrophy, which may be due to increased intestinal epithelial cell (IEC) apoptosis. Indeed, abnormal T cell activation has been reported in some enteropathies, such as Crohn's disease, ulcerative colitis and celiac disease (13).
Activated T cells mediate cytotoxicity through several mechanisms, of which, the Fas/Fas ligand (FasL) system plays an important role. Recent studies have demonstrated that the FasL molecule is expressed on either systemic and/or mucosal lymphocytes in enteropathies such as ulcerative colitis (4, 5) and celiac disease (610). In animal models of intestinal injury, several studies have shown that Fas/FasL-mediated cytotoxicity by mucosal lymphocytes may be partly responsible for the enteropathy (1115). The type and function of the immune components involved in intestinal damage are, however, still obscure, partly because many players including the immune components are involved in the intestinal immune system. The gut-associated lymphoid tissue comprises four compartments: intestinal epithelium, lamina propria, Peyer's patches and mesenteric lymph nodes (16). Gut tissue is an intersection of the mucosal and systemic immune systems, and interactions between different types of T cells may be critical for the integrity of the intestinal immune system. In the inflammatory state, the infiltration of peripheral leukocytes into the tissue makes the situation far more complicated.
The purpose of this study was to investigate the possible involvement of different T cell compartments in killing IEC in vivo using a model of small intestinal injury. T cell activation was evoked systemically by the intraperitoneal administration of anti-CD3 antibody. A direct administration was performed in order to avoid any possible interactions between the stimulator and intestinal contents (food, digestive enzymes and bacterial flora). Then we assessed the involvement of Fas using in vivo antibody injection to Fas-deficient mice and ex vivo measurements of Fas-mediated killer activity after antibody injection. Further, we determined whether thymus-derived T cells were involved in the observed intestinal injury using nude mice. Finally, we investigated the role of intraepithelial lymphocytes (IEL) and splenocytes (SPL) using cell transfer experiments.
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Methods
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Animals and mAbs
Male C3H/HeN, MRL-+/+, MRL-lpr/lpr (Japan SLC Inc., Shizuoka, Japan), BALB/c and BALB/c nu/nu (Japan Charles River Laboratory, Yokohama, Japan) mice bred and maintained under specific pathogen-free conditions were used at 910 weeks of age. This study conformed to our institution's guidelines for the care and use of laboratory animals in research. Hamster anti-murine CD3 mAb (145-2C11) and control hamster IgG1
were obtained from BD Biosciences (San Jose, CA, USA). For flow cytometry, we used FITC-conjugated antibodies against CD3 (145-2C11), CD69 (H1.2F3), Fas (Jo2), control hamster IgG1
, IgG1
, IgG2
, PE-conjugated FasL (MFL3) and control hamster IgG1
(BD Biosciences).
Histology
Mice that received a single intraperitoneal injection of titrated concentrations of anti-CD3 antibody diluted in 100 µl of saline were sacrificed at different time points. The small intestines were removed, fixed and embedded in paraffin according to routine procedures. We quantified apoptotic cells by the method used in the previous studies of the intestinal injury model with minor modifications (1720). In brief, we have counted damaged cells that have condensing or fragmenting nuclei, classical and definitive features of apoptosis, by light microscopic observation of hematoxylin and eosin (H&E)-stained histological sections. Apoptotic cells in 21 pairs of an adjacent villus and crypt (hereafter the pair will be expressed as a villus per crypt unit) were counted in seven random high fields (x400 magnification) per mice. Data represent the number of apoptotic cells in a villus or crypt per unit separately. This classical method of detecting apoptosis is incomplete in terms of the determination of absolute number of apoptosis because of the presence of fragmented cell debris and the very early stage-apoptotic cells in which the changes in nuclei have not occurred yet, however, it can assess the degree of apoptosis at least enough to demonstrate the time- and site-dependent changes of apoptotic changes in small intestines. The terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling (TUNEL) assay (In situ apoptosis detection kit, Intergen Company, NY, USA) was used for only qualitative detection of DNA fragmentation because the TUNEL assay, when used to quantify IEC apoptosis, has been reported to give conflicting results (21). For electron microscopy, samples of small intestine were fixed in 2% glutaraldehyde and processed using routine methods.
Fas-mediated cytotoxicity assay
We analyzed the Fas-mediated cytotoxicity of lymphocytes obtained from mice after anti-CD3 injection. IEL, SPL, mesenteric lymph node cells (MLNC) and Peyer's patch lymphocytes (PPL) were prepared by the method of Taguchi et al. (22). The Fas-positive human cell line, Jurkat, was obtained from the American Type Culture Collection (Rockville, MD, USA). Fas-mediated cytotoxicity was assessed according to the method of Lin et al. (23), with modifications. This assay is based on the fact that both murine and human FasL can kill Jurkat cells equally well and that the observed cytotoxicity is exclusively dependent on Fas (24). Briefly, [3H]thymidine-labeled Jurkat cells (2 x 104 per well) were cultured with freshly isolated lymphocytes at different Effecter/Target (E/T) ratios in flat-bottomed 96-well plates, previously coated or not coated with 7.4 µg ml1 of anti-CD3 antibody. After cultivation, the cells were lysed and harvested onto two-layered filters: a diethylaminoethyl (DEAE) glass filter to trap fragmented DNA and an unmodified glass filter to trap intact chromatin (25). The percentage of DNA fragmentation was calculated as follows:
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The expression of cell surface markers was analyzed by flow cytometry (FACScan, BD Biosciences), and data were processed using the Macintosh CELLQuest program (BD Biosciences).
Adoptive transfer of immune cells
Experiment 1.
Freshly isolated SPL (5 x 107) or IEL (1 x 107) from 9- to 10-week-old BALB/c mice were transferred intravenously into BALB/c nu/nu mice. The following day, anti-CD3 antibody was injected intraperitoneally. Small intestines were removed at 4 and 24 h after the injection and prepared for histological analysis. In the dual-grafting experiment, donor SPL (5 x 107) and IEL (1 x 107) were successively engrafted into BALB/c nu/nu mice.
Experiment 2.
Freshly isolated IEL (1 x 107) from 6- to 11-week-old BALB/c mice were transferred intravenously into BALB/c nu/nu mice. After 5 weeks, anti-CD3 antibody was injected intraperitoneally. In the dual-grafting experiment, donor SPL (5 x 107) were engrafted at 24 h before the anti-CD3 antibody injection.
Statistical analysis
Statistical comparisons were made using Scheffe's method after analysis of variance. The results were considered significantly different when P < 0.05. All analyses were carried out using StatView version 5.0 (SAS Institute Inc., Cary, NC, USA).
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Results
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Anti-CD3 antibody injection induces bi-phasic apoptosis in IEC
Anti-CD3 antibody induced apoptosis in many IEC in the small intestinal villus and crypts in a bi-phasic manner (i.e. early and late) (Figs 13
). In the first phase, apoptosis of ciliated villous IEC with condensed nuclei (Figs 2B, D, G and 3A) began to appear at 2 h after the injection, peaked at 4 h and declined thereafter (Fig. 1A). The intestine showed villous shortening and a decline in the number of IEC (Fig. 2C). In the second phase, apoptosis of crypt IEC (Figs 2C, E, H and 3B) increased gradually for 24 h. In addition to H&E staining, TUNEL staining (Fig. 2G and H) and electron microscopic findings (Fig. 3) at 4 and 24 h clearly showed that both phases of apoptosis were induced mainly in IEC, although TUNEL-positive cells were also observed in the lamina propria from 2 to 24 h. Apoptotic bodies in the villus and the crypt decreased to normal levels by days 3 and 4, respectively (data not shown). A low level of crypt hyperplasia and villous atrophy was observed at 4 h and markedly from 24 to 48 h. Inflammatory infiltrates of mononuclear cells were noted in the lamina propria at day 2, but the histological features returned almost to normal by day 4 (data not shown). Apoptotic bodies increased dose dependently, reaching a plateau at a dose of 12.5 µg per mouse in the villus at 4 h and 6.3 µg per mouse in the crypt at 24 h (Fig. 1B and C). Therefore, all further experiments were performed using a dose of 12.5 µg CD3 antibody per mouse. The administration of control hamster IgG1
had no effect on IEC apoptosis levels or histology (data not shown).

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Fig. 1. Time course (A) and dose dependency (B, C) of anti-CD3-induced apoptosis in the jejunum of C3H/HeN mice. Control mice received saline alone. Data represent the means ± SD of apoptosis per villus per crypt unit. (A) The antibody was administered intraperitoneally at a dose of 12.5 µg per mouse (number of mice = 67). Apoptotic cells with condensing/fragmenting nuclei appeared in the small intestine bi-phasically. ### P < 0.0005 versus the control group (villus) and *P < 0.05; ***P < 0.0005 versus the control group (crypt) at each time. (B) Four hours after and (C) 24 h after antibody injection (number of mice = 5). Apoptotic cells with condensing/fragmenting nuclei arose dose dependently in the villus at 4 h (B) and in the crypt at 24 h (C).
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Fig. 2. Histological sections of the jejunum of C3H/HeN mice. Anti-CD3 was administered at a dose of 12.5 µg per mouse. (AE) H&E staining. (FH) TUNEL assay. (A and F) Control mice receiving saline alone. (B, D and G) Four hours after the antibody injection. (C, D and G) Twenty-four hours after the antibody injection. Control mice have few apoptotic cells (A and F). Many apoptotic cells (arrow) are seen in the villus at 4 h (D and G) and in the crypt at 24 h (E and H) after anti-CD3 injection. Original magnification: AC, x25; D and E, x132; FH, x50.
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Fig. 3. Electron microscopic findings after anti-CD3 injection. (A) Condensed and fragmented nuclei (arrow) are seen in ciliated villous epithelial cells 4 h after anti-CD3 injection. The cytoplasm shows little change at this time. Neither apoptotic nor damaged lymphocyte infiltrate is observed. Little apoptosis occurs in IEL (unpublished observations in our laboratory). (B) Condensed and fragmented nuclei (arrow) and expanded mitochondria are seen in one crypt epithelial cell 24 h after anti-CD3 injection. The other crypt epithelial cells do not have typical condensed and/or fragmented nuclei, but nuclei are beginning to show damage. Neither apoptotic nor damaged lymphocyte infiltrate is present. The arrowhead indicates basal membrane. Little apoptosis occurs in lamina propria lymphocytes (unpublished observations in our laboratory). (C) Villus in naive mice and (D) crypt in naive mice. Naive mice have little apoptosis either in the villus or the crypt. The invasion of any microflora has not been observed in the small intestinal epithelia (AD and unpublished observations). Original magnification: x50 000.
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Apoptosis in IEC is induced by anti-CD3 via Fas/FasL interactions in vivo
IEC constitutively express Fas (11, 26), and IEL exert potent Fas-mediated killing activity after in vitro CD3 stimulation (27). To investigate whether Fas is involved in the apoptosis observed here, we used the Fas-deficient strain, MRL-lpr/lpr (Fig. 4). In MRL-+/+, normal Fas-expressing mice, bi-phasic apoptosis was induced by anti-CD3 injection to the same level as observed in C3H/HeN. On the other hand, in MRL-lpr/lpr, apoptosis in both phases was markedly reduced. These results suggest that the Fas/FasL system mediates a large portion of the observed apoptosis.

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Fig. 4. Differences in the number of apoptotic cells in MRL-+/+ and MRL-lpr/lpr mice after anti-CD3 (12.5 µg per mouse) administered intraperitoneally. Apoptotic bodies with clearly fragmented and/or condensed nuclei, the most classical and definitive feature of apoptosis, were counted as described in Histology in Methods. Data represent the means ± SD of apoptotic cells per villus per crypt unit 4 (A) and 24 h (B) after antibody administration (number of mice = 35). The number of apoptotic bodies in mice administered saline instead of anti-CD3 was <1.5 for both strains. *P < 0.05; ***P < 0.0005 versus the control group.
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The in vivo administration of anti-CD3 may result in the stimulation of different types of T lymphocytes. Therefore, to clarify which T cell compartments, IEL, PPL, MLNC and SPL, were activated and presented cytotoxicity by the anti-CD3 injection accompanied by intestinal injury, we analyzed ex vivo Fas-mediated cytotoxicity of these lymphocytes after anti-CD3 injection. At an E/T ratio of 1 : 20, freshly isolated IEL and SPL showed a weak spontaneous killing activity, with IEL stronger than SPL, but MLNC and PPL showed no detectable activity (Fig. 5A). When stimulated with anti-CD3 in vitro, cytotoxicity mediated by IEL and SPL was strongly induced, but was only marginally induced in MLNC and PPL, which is consistent with the data of Lin et al. (23) (Fig. 5B). Next, we analyzed the killer activity of lymphocytes isolated from mice injected with anti-CD3 antibody without in vitro stimulation (Fig. 5C). Similar to cells stimulated in vitro, the in vivo induction of cytotoxicity by anti-CD3 was mainly observed in IEL and SPL. The cytotoxicity of IEL increased rapidly and remained at the highest level from 0.5 to 4 h after the anti-CD3 injection and, thereafter, declined slowly. In contrast, SPL cytotoxicity rose more slowly, peaked from 4 to 8 h after the injection and then declined. The maximum cytotoxicities of IEL and SPL were similar to the maximum value obtained by in vitro stimulation, and the potency was IEL > SPL (approximately 60 versus 40% lysis at an E/T ratio of 1 : 20). The killer activity at 24 h was not analyzed because IEL were depleted by that time in this model. These results showed that the cytotoxicity of IEL and SPL was induced at different times and with different potency, which raised the possibility that IEL and SPL may be responsible for the induction of the different phases of IEC apoptosis.

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Fig. 5. Fas-mediated killer activity of lymphocytes. Lymphocytes isolated from naive C3H/HeN mice were cultured in the absence (A) or presence (B) of plate-bound anti-CD3 antibody. (C) Lymphocytes isolated from anti-CD3-injected C3H/HeN mice at different times after treatment were cultured in the absence of anti-CD3 antibody. Lymphocytes were pooled from three mice and a representative experiment of two performed is shown. The percentage of DNA fragmentation of Jurkat cells was measured and calculated as described in Fas-mediated cytotoxicity assay in Methods.
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We examined the expression of CD3, CD69, FasL and Fas on IEL and SPL from anti-CD3-treated and untreated mice (Fig. 6). FasL was constitutively expressed on IEL, but the number of FasL+ cells increased slightly at 4 h after injection of anti-CD3 and returned to the normal levels by 24 h. On the other hand, FasL was expressed at a very low level on SPL of naive mice and increased at 4 and 24 h after the antibody injection. The number of CD3+ cells and the density of CD3 expression on both IEL and SPL decreased at 4 h due to down-regulation of CD3, as previously reported (2830). However, these values recovered by 24 h after the injection. Conversely, the amount of the activation marker, CD69, on both IEL and SPL was increased at 4 h after the injection and decreased to normal levels at 24 h, suggesting a rapid, temporary T cell activation.

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Fig. 6. Expression of CD3, CD69, FasL and Fas on IEL and SPL. Lymphocytes were isolated from naive (black line) or anti-CD3-treated C3H/HeN mice 4 (red line) and 24 (blue line) h after the injection and pooled from three mice. A representative experiment of the three performed is shown. The dotted line represents the isotype control. Numbers indicate the percentages of positive cells.
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Thymus-derived lymphocytes are involved in both phases of IEC apoptosis induced by anti-CD3
To directly confirm the involvement of thymus-derived T cells in the apoptosis observed here, we injected anti-CD3 into nude mice (Fig. 7). In euthymic BALB/c, the antibody induced bi-phasic apoptosis similar to that observed in C3H/HeN. By contrast, in athymic nude mice, both phases of apoptosis were almost completely abrogated, being depressed to the same levels as observed in normal BALB/c that were not administered antibody.

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Fig. 7. Differences between the number of apoptotic cells in BALB/c and BALB/c nu/nu mice after intraperitoneal anti-CD3 (12.5 µg per mouse) administration. Apoptotic bodies with clearly fragmented and/or condensed nuclei, the most classical and definitive feature of apoptosis, were counted as described in Methods. Data represent the means ± SD of apoptotic cells per villus per crypt unit 4 (A) and 24 h (B) after antibody administration (number of mice = 4). The number of apoptotic bodies in mice administered with saline instead of anti-CD3 was <1.5 for both strains. The experiment was repeated three times with similar results. ***P < 0.0005.
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Adoptive transfer reconstitutes IEC apoptosis in nude mice
These data suggested the following hypotheses: the first phase of apoptosis at 4 h after the injection was induced by thymus-derived IEL, and the second phase at 24 h was induced by peripheral lymphocytes. To test these hypotheses, we adoptively transferred SPL or IEL isolated from euthymic mice into nude mice at 1 day before the anti-CD3 injection (Fig. 8, experiment 1). Transfer of SPL followed by anti-CD3 resulted in a substantial reconstitution of the second phase of apoptosis with crypt hyperplasia without villous apoptosis/destruction (Fig. 8, experiment 1B). However, neither phase of apoptosis was induced by the antibody in mice into which IEL had been transferred (Fig. 8, experiment 1). In addition, we adoptively transferred SPL and IEL successively, then administered anti-CD3. Unexpectedly, dual grafting of SPL and IEL from euthymic mice induces neither the first phase nor the second phase of apoptosis in nude mice (Fig. 8, experiment 1B).

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Fig. 8. Adoptive transfer of BALB/c IEL and SPL into BALB/c nu/nu mice. Experiment 1: naive nude mice were respectively or serially injected with SPL (5 x 107) and IEL (1 x 107). The day after the transfer, anti-CD3 was administered at a dose of 12.5 µg per mouse. The number of apoptotic cells 4 (A) and 24 h (B) after antibody administration is shown (number of mice = 513). The experiment was repeated twice with similar results. When SPL and/or IEL were transferred into the mice without anti-CD3 administration, the number of apoptotic bodies was <1.5 for all graft groups. Data represent means ± SD per villus per crypt unit. ***P < 0.0005 versus non-transferred in crypt; ###P < 0.0005 versus SPL transferred alone in crypt. Experiment 2: IEL (1 x 107) from 6- to 11-week-old-BALB/c mice were transferred to BALB/c nu/nu mice of the same age. After 5 weeks, saline or SPL (5 x 107) was transferred to the IEL-grafted or naive BALB/c nu/nu mice, and on the next day anti-CD3 was administered at a dose of 12.5 µg per mouse. The number of apoptotic cells 4 (A) and 24 h (B) after antibody administration is shown (number of mice = 56). When SPL and/or IEL without anti-CD3 administration were transferred to the mice, the number of apoptotic bodies was <1.5 for all graft groups. Data represent means ± SD per villus per crypt unit. *P < 0.05 and ***P < 0.0005 versus non-transferred in the villus; #P < 0.05 and ###P < 0.0005 versus non-transferred in the crypt.
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In experiment 2, because the migration of transferred IEL into the intestinal epithelium has been reported to take more than several weeks (31, 32), we changed the timing of IEL transfer. We adoptively transferred IEL, and after 5 weeks, the anti-CD3 antibody was injected (Fig. 8, experiment 2). IEL transfer significantly induced not only the first phase of apoptosis at 4 h (Fig. 8, experiment 2A) but also the second phase of apoptosis at 24 h after the anti-CD3 injection (Fig. 8, experiment 2B). Further, substantial apoptosis was detected in the villus at 24 h, which had not been so prominent in anti-CD3 antibody-treated euthymic BALB/c mice. Finally, differing from the results of experiment 1, dual grafting of SPL and IEL showed additive effects on IEC apoptosis (Fig. 8, experiment 2).
In the transfer experiments without anti-CD3 injection, no aberrant morphological changes were observed and no aberrant IEC apoptosis occurred following adoptive transfer of IEL, SPL or both (data not shown).
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Discussion
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In the present study, the following results were obtained: (i) anti-CD3 injections induced the apoptosis of small intestinal IEC bi-phasically with different spatial and temporal patterns, (ii) each phase of apoptosis depended on Fas/FasL, (iii) anti-CD3 induced Fas-dependent cytotoxicity mediated by IEL rapidly, and by SPL gradually, (iv) each phase of apoptosis depended on thymus-derived T cells, (v) the adoptive transfer of SPL from euthymic mice to nude mice reconstituted only the second phase of IEC apoptosis, (vi) the adoptive transfer of IEL reconstituted both phases of IEC apoptosis, when anti-CD3 antibody was administered at 5 weeks after IEL transfer and (vii) dual grafting of SPL and IEL showed either abrogative or additive effects on IEC apoptosis, depending on the engrafting protocol.
A few studies have characterized the pathology and pathogenesis of anti-CD3 antibody-induced small intestinal lesions (33, 34). Merger et al. (17) demonstrated that multiple pathways of cytotoxicity, including tumor necrosis factor-
(TNF-
)-, Fas/FasL-, and perforin-mediated apoptosis, were associated with architectural damage. We have employed a model of enteropathy induced by a protocol similar to theirs, but some different results were obtained. Our data demonstrated that the antibody-induced bi-phasic apoptosis differed in spatiotemporal distribution, which was not previously noted. In addition, in our study, Fas/FasL-mediated killing played a major role in T cell-induced mucosal damage, whereas Merger et al. (17) indicated that the absence of Fas/FasL did not reduce apoptosis. This discrepancy may be due to differences in the dose of antibody used. In various studies, antibody was injected intravenously at relatively high doses (from 50 to 400 µg per mouse), whereas we used a dose of 12.5 µg per mouse intraperitoneally (17, 33, 34). At this dose, the number of apoptotic cells had already reached the maximum value (Fig. 1B and C). However, we also observed that the intestinal tissue damage that developed in our experimental setting was milder and recovered more rapidly than that at higher antibody doses (data not shown). Further, in contrast to previous studies, serum TNF-
was not detected at any time point up to 24 h after the injection (data not shown; detection limit: 50 pg ml1). These data suggest that the Fas/FasL pathway is important for the T cell-mediated IEC apoptosis following a weaker stimulation by anti-CD3 and that higher doses of the antibody stimulate various additional immunological responses and result in more complicated, long-term and serious enteropathy. Nonetheless, other cytotoxic pathways, for example perforin, suggested to be a very important cytotoxic pathway by Merger et al. (17), may contribute to IEC apoptosis in the present study because it was incompletely inhibited in the absence of Fas (in lpr/lpr mice). Thus, our protocol may be useful to discriminate and analyze the involvement of different cytotoxic mechanisms causing intestinal damage.
In the present study, in addition to the analysis using lpr/lpr mice, we quantified the Fas-mediated killing activity of lymphocytes from different compartments isolated from mice administered anti-CD3 antibody and found that the enhanced cytotoxicity of IEL and SPL was seen at different times. The enhanced killer activity of IEL and SPL at different times may, therefore, result in the induction of apoptosis at different times and sites. Complete abrogation of both phases of apoptosis in nude mice suggests an essential contribution of thymus-derived T cells. Indeed,
ßIEL, which contain a large portion of thymus-derived T cells, showed stronger killer activity than 
IEL, which consist mainly of gut-derived T cells (data not shown). These data suggest the hypothesis that the first phase of apoptosis is mediated mainly by thymus-derived IEL and the second phase is mediated by peripheral lymphocytes. Lymphocyte transfer experiments provided data that were, at least partially, consistent with this hypothesis. Namely, the adoptive transfer of SPL from euthymic mice to nude mice before anti-CD3 injection resulted in only the second phase of apoptosis, whereas IEL caused both (Fig. 8). Compared with anti-CD3 antibody-induced apoptosis in euthymic mice, the apoptosis induced in IEL-engrafted nude mice was more prominent in the villus at 24 h (Figs 7 and 8, experiment 2). This discrepancy remains to be clarified in the future, but the following should be considered. The rapid massive villous apoptosis induced in euthymic mice resulted in an extensive loss of intestinal villus by 24 h and, therefore, the number of observable villous apoptotic bodies may have decreased. A relatively longer villus (i.e. greater integrity) was observed in the intestine of dual-grafted nude mice (data not shown).
The concomitant transfer of IEL and SPL abrogated the SPL-induced second phase of apoptosis, but SPL transfer at 5 weeks after IEL transfer had an additive effect on the second phase of apoptosis at 24 h (Fig. 8). Clarification of this discrepancy also requires further investigation but may be explained by the following. In experiment 1, a substantial amount of transferred IEL may have accumulated in the spleen because the cells did not have sufficient time to repopulate the intestinal epithelium. In such a case, upon CD3 activation, FasL+IEL may kill Fas+SPL, including dendritic cells, which would result in the suppression of any T cell activation. By contrast, in experiment 2, many of the transferred IEL will have migrated into the intestine by 5 weeks after transfer, and therefore, the killing of SPL by IEL in the spleen may not occur when anti-CD3 antibody is injected. The killer activity of both IEL and SPL can thus be expressed additively, resulting in the observed enhanced killing of IEC.
It has been reported that 78.9% of IEC express the Fas molecule and the expression is not uniform (11). In addition, the sensitivity of cell death may be different between the villus and crypt because P53-independent cell death occurred by ischemia-reperfusion is induced in the villus to a greater extent than in the crypt (20). One explanation for bi-phasic apoptosis may be that villous IEC is more sensitive than crypt IEC for FasL signaling. The differences in the sensitivity of IEC to apoptosis may contribute to the bi-phasic apoptosis when mediated by IEL, especially. However, this hypothesis appears unsatisfactory because it does not provide a full explanation of the data presented in Figure 8, experiments 1 and 2, in that the anti-CD3-injection to SPL-grafting nude mice resulted in the second phase apoptosis only.
In summary, we have described a model of small intestinal injury induced by systemic T cell activation and characterized by IEC apoptosis with at least two distinct phases mediated mainly by the Fas/FasL system of thymus-derived T cells. Differential involvement of both peripheral and mucosal lymphocytes in the induction of the apoptosis has been demonstrated. Despite the many unresolved issues raised by these experiments, the demonstration of the possible involvement of different T cell compartments and their interactions in IEC apoptosis provides an important advance and useful tools in the effort to determine the components and mechanisms by which immune responses are regulated in normal and pathological intestinal mucosa.
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Acknowledgements
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The authors thank Dr Yasuhiro Komatsu, an ex-chief manager of Kampo & Pharmacognosy Laboratory of Tsumura & Co., for his valuable discussions.
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Abbreviations
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DEAE | diethylaminoethyl |
E/T | Effecter/Target |
FasL | Fas ligand |
H&E | hematoxylin and eosin |
IEC | intestinal epithelial cells |
IEL | intestinal intraepithelial lymphocytes |
MLNC | mesenteric lymph node cells |
PPL | Peyer's patch lymphocytes |
SPL | splenocytes |
TNF | tumor necrosis factor |
TUNEL | terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling |
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Notes
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Transmitting editor: H. Kikutani
Received 8 April 2003,
accepted 3 February 2005.
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References
|
---|
- Fiocchi, C. 1998. Inflammatory bowel disease: etiology and pathogenesis. Gastroenterology 115:182.[ISI][Medline]
- Radford-Smith, G. 1997. Ulcerative colitis: an immunological disease? Bailliere's Clin. Gastroenterol. 11:35.[CrossRef][ISI][Medline]
- Schuppan, D. 2000. Current concepts of celiac disease pathogenesis. Gastroenterology 119:234.[ISI][Medline]
- Ueyama, H., Kiyohara, T., Sawada, N. et al. 1998. High Fas ligand expression on lymphocytes in lesions of ulcerative colitis. Gut 43:48.[Abstract/Free Full Text]
- Suzuki, A., Sugimura, K., Ohtsuka, K. et al. 2000. Fas/Fas ligand expression and characteristics of primed CD45RO+ T cells in the inflamed mucosa of ulcerative colitis. Scand. J. Gastroenterol. 35:1278.[CrossRef][ISI][Medline]
- Ciccocioppo, R., Di Sabatino, A., Parroni, R. et al. 2000. Cytolytic mechanisms of intraepithelial lymphocytes in coeliac disease (CoD). Clin. Exp. Immunol. 120:235.[CrossRef][ISI][Medline]
- Di Sabatino, A., D'Alo, S., Millimaggi, D. et al. 2001. Apoptosis and peripheral blood lymphocyte depletion in coeliac disease. Immunology 103:435.[CrossRef][ISI][Medline]
- Di Sabatino, A., Ciccocioppo, R., D'Alo, S. et al. 2001. Intraepithelial and lamina propria lymphocytes show distinct patterns of apoptosis whereas both populations are active in Fas based cytotoxicity in coeliac disease. Gut 49:380.[Abstract/Free Full Text]
- Ciccocioppo, R., Di Sabatino, A., Parroni, R. et al. 2001. Increased enterocyte apoptosis and Fas-Fas ligand system in celiac disease. Am. J. Clin. Pathol. 115:494.[CrossRef][ISI][Medline]
- Ciccocioppo, R., D'Alo, S., Di Sabatino, A. et al. 2002. Mechanisms of villous atrophy in autoimmune enteropathy and coeliac disease. Clin. Exp. Immunol. 128:88.[CrossRef][ISI][Medline]
- Sakai, T., Kimura, Y., Inagaki-Ohara, K., Kusugami, K., Lynch, D. H. and Yoshikai, Y. 1997. Fas-mediated cytotoxicity by intestinal intraepithelial lymphocytes during acute graft-versus-host disease in mice. Gastroenterology 113:168.[ISI][Medline]
- Bonhagen, K., Thoma, S., Leithauser, F., Moller, P. and Reimann, J. 1998. A pancolitis resembling human ulcerative colitis (UC) is induced by CD4+ TCR alphabeta T cells of athymic origin in histocompatible severe combined immunodeficient (SCID) mice. Clin. Exp. Immunol. 112:443.[CrossRef][ISI][Medline]
- Miwa, K., Hashimoto, H., Yatomi, T., Nakamura, N., Nagata, S. and Suda, T. 1999. Therapeutic effect of an anti-Fas ligand mAb on lethal graft-versus-host disease. Int. Immunol. 11:925.[Abstract/Free Full Text]
- Kataoka, Y., Iwasaki, T., Kuroiwa, T. et al. 2001. The role of donor T cells for target organ injuries in acute and chronic graft-versus-host disease. Immunology 103:310.[CrossRef][ISI][Medline]
- Arnold, D., Wasem, C., Juillard, P. et al. 2002. IL-18-independent cytotoxic T lymphocyte activation and IFN-gamma production during experimental acute graft-versus-host disease. Int. Immunol. 14:503.[Abstract/Free Full Text]
- Mowat, A. M. and Vinley, J. L. 1997. The anatomical basis of intestinal immunity. Immunol. Rev. 156:145.[ISI][Medline]
- Merger, M., Viney, J. L., Borojevic, R. et al. 2002. Defining the roles of perforin, Fas/FasL, and tumour necrosis factor alpha in T cell induced mucosal damage in the mouse intestine. Gut 51:155.[Abstract/Free Full Text]
- Tsuzuki, T., Yoshikai, Y., Ito, M., Mori, N., Ohbayashi, M. and Asai, J. 1994. Kinetics of intestinal intraepithelial lymphocytes during acute graft-versus-host disease in mice. Eur. J. Immunol. 24:709.[ISI][Medline]
- Okudela, K., Ito, T., Mitsui, H. et al. 1999. The role of p53 in bleomycin-induced DNA damage in the lung. A comparative study with the small intestine. Am. J. Pathol. 155:1341.[Abstract/Free Full Text]
- Coopersmith, C. M., O'Donnell, D. and Gordon, J. I. 1999. Bcl-2 inhibits ischemia-reperfusion-induced apoptosis in the intestinal epithelium of transgenic mice. Am. J. Physiol. 276:G677.[ISI][Medline]
- Pritchard, D. M. and Watson, A. J. 1996. Apoptosis and gastrointestinal pharmacology. Pharmacol. Ther. 72:149.[CrossRef][ISI][Medline]
- Taguchi, T., McGhee, J. R., Coffman, R. L. et al. 1990. Analysis of Th1 and Th2 cells in murine gut-associated tissues. Frequencies of CD4+ and CD8+ T cells that secrete IFN-gamma and IL-5. J. Immunol. 145:68.[Abstract/Free Full Text]
- Lin, T. S., Brunner, T., Tietz, B. et al. 1998. Fas ligand-mediated killing by intestinal intraepithelial lymphocytesparticipation in intestinal graft-versus-host disease. J. Clin. Investig. 101:570.[Abstract/Free Full Text]
- Ramsdell, F., Seaman, M. S., Miller, R. E., Tough, T. W., Alderson, M. R. and Lynch, D. H. 1994. gld/gld mice are unable to express a functional ligand for Fas. Eur. J. Immunol. 24:928.[ISI][Medline]
- Yamamoto, M., Ogawa, K., Morita, M., Fukuda, K. and Komatsu, Y. 1996. The herbal medicine Inchin-ko-to inhibits liver cell apoptosis induced by transforming growth factor beta 1. Hepatology 23:552.[ISI][Medline]
- Yukawa, M., Iizuka, M., Horie, Y. et al. 2002. Systemic and local evidence of increased Fas-mediated apoptosis in ulcerative colitis. Int. J. Colorectal Dis. 17:70.[CrossRef][ISI][Medline]
- Inagaki-Ohara, K., Nishimura, H., Sakai, T., Lynch, D. H. and Yoshikai, Y. 1997. Potential for involvement of Fas antigen Fas ligand interaction in apoptosis of epithelial cells by intraepithelial lymphocytes in murine small intestine. Lab. Investig. 77:421.[ISI][Medline]
- Ellenhorn, J. D., Hirsch, R., Schreiber, H. and Bluestone, J. A. 1988. In vivo administration of anti-CD3 prevents malignant progressor tumor growth. Science 242:569.[ISI][Medline]
- Hirsch, R., Eckhaus, M., Auchincloss, H., Jr., Sachs, D. H. and Bluestone, J. A. 1988. Effects of in vivo administration of anti-T3 monoclonal antibody on T cell function in mice. I. Immunosuppression of transplantation responses. J. Immunol. 140:3766.[Abstract/Free Full Text]
- Hirsch, R., Gress, R. E., Pluznik, D. H., Eckhaus, M. and Bluestone, J. A. 1989. Effects of in vivo administration of anti-CD3 monoclonal antibody on T cell function in mice. II. In vivo activation of T cells. J. Immunol. 142:737.[Abstract/Free Full Text]
- Poussier, P., Edouard, P., Lee, C., Binnie, M. and Julius, M. 1992. Thymus-independent development and negative selection of T cells expressing T cell receptor alpha/beta 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]
- Suzuki, S., Sugahara, S., Shimizu, T. et al. 1998. Low level of mixing of partner cells seen in extrathymic T cells in the liver and intestine of parabiotic mice: its biological implication. Eur. J. Immunol. 28:3719.[CrossRef][ISI][Medline]
- Ferran, C., Dy, M., Sheehan, K. et al. 1991. Inter-mouse strain differences in the in vivo anti-CD3 induced cytokine release. Clin. Exp. Immunol. 86:537.[ISI][Medline]
- Ferran, C., Dy, M., Sheehan, K. et al. 1991. Cascade modulation by anti-tumor necrosis factor monoclonal antibody of interferon-gamma, interleukin-3 and interleukin-6 release after triggering of the CD3/T cell receptor activation pathway. Eur. J. Immunol. 21:2349.[ISI][Medline]