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The Journal of Immunology, 2001, 166: 2923-2928.
Copyright © 2001 by The American Association of Immunologists

Gastrointestinal Cells of IL-7 Receptor Null Mice Exhibit Increased Sensitivity to Irradiation1

Lisbeth A. Welniak*, Annette R. Khaled{dagger}, Miriam R. Anver§, Kristin L. Komschlies§, Robert H. Wiltrout{ddagger}, Scott Durum{dagger}, Francis R. Ruscetti*, Bruce R. Blazar and William J. Murphy2,§

* Laboratories of Leukocyte Biology, {dagger} Molecular Immunoregulation, and {ddagger} Experimental Immunology, Division of Basic Sciences, and § Intramural Research Support Program, Science Applications International Corporation Frederick, National Cancer Institute-Frederick Cancer Research and Development Center, Frederick, MD 21702; and Department of Bone Marrow Transplantation, University of Minnesota Cancer Center, Minneapolis, MN 55455


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
IL-7 is a critical cytokine in the development of T and B cells but little is known about its activity on nonhematopoietic cells. An unexpected finding was noted in allogeneic bone marrow transplant studies using IL-7 receptor null (IL-7R{alpha}-/-) mice as recipients. These mice exhibited a significantly greater weight loss after total body irradiation compared with wild type, IL-7R{alpha}+/+, mice. Pathological assessment indicated greater intestinal crypt damage in IL-7R{alpha}-/- recipients, suggesting these mice may be predisposed to gut destruction. Therefore, we determined the effect of the conditioning itself on the intestinal tract of these mice. IL-7R{alpha}-/- mice and IL-7R{alpha}+/+ mice were irradiated and examined for lesions and apoptosis within the small intestine. In moribund animals, IL-7R{alpha}-/- mice had extensive damage in the small intestine, including marked ablation of the crypts and extreme shortening of villi following 1500 cGy total body irradiation. In contrast, by 8 days after irradiation, the small intestines of IL-7R{alpha}+/+ mice had regenerated as distinguished by normal villus length and hyperplastic crypts. Following 750 cGy irradiation, IL-7R{alpha}-/- mice had a higher proportion of apoptotic cells in the crypts and an accompanying increase in the pro-apoptotic protein Bak was expressed in intestinal epithelial cells. These results demonstrate the increased radiosensitivity of intestinal stem cells within the crypts in IL-7R{alpha}-/- mice and a role for IL-7 in the protection of radiation-induced apoptosis in these same cells. This study describes a novel role of IL-7 in nonhematopoietic tissues.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
Interleukin-7 was originally identified as a bone marrow stroma-derived cytokine that induced proliferation and maintenance of B cell progenitors in culture (1). Later, IL-7 was shown to be produced by thymic stromal cells (2), dendritic cells (3), keratinocytes (4), and intestinal epithelial cells (IEC3; Ref. 5), and to promote the proliferation and survival of pro-T, pro-B, and Ag-stimulated mature T cells (6, 7, 8) as well as neuronal cells (9). Further, IL-7 has been shown to enhance the function of mature T cells (10) and to promote the development of hematopoietic cells (11, 12).

Generation of IL-7 null (13) and IL-7R{alpha} null (14) mice has demonstrated the critical, nonredundant role of IL-7 for the development of B and T cells. Both IL-7-/- and IL-7R{alpha}-/- mice are severely lymphopenic, but a few mature B and T cells are found in peripheral lymphoid tissues (13, 14). T cell development is blocked at the pro-T to pre-T cell transition stage 1 (13, 14). Reduced numbers of mature {alpha}{beta}+ T cells are produced in both knockout strains (13, 14). No mature {gamma}{delta}+ intestinal epithelial T-lymphocytes are detected in these mice (15, 16), demonstrating the critical requirement of IL-7 for successful thymic and extrathymic development of this subpopulation. B-lymphopoiesis is blocked at the pro-B to pre-B cell transition stage in IL-7 null mice (13). The block in development occurs in the pre-pro-B cell to pro-B cell transition in IL-7R null mice (14) suggesting that other cytokines may use the IL-7R. While the B and T cells in IL-7-/- mice demonstrate normal responses to LPS and Con A, respectively (13), peripheral T cells from IL-7R{alpha}-/- mice are functionally inactive and a large proportion undergo apoptosis after stimulation (17). However, T lymphopoieisis can be restored in IL-7R{alpha}-/- mice by the over expression of bcl-2 demonstrating that IL-7 is a critical survival factor in T-cells (18).

IL-7 receptors are also expressed constitutively or after activation on nonlymphoid cells including myeloid stem cells (19), and on nonhematopoietic tissues such as melanocytes (20), neuronal cells (9), and IEC (21). However, IL-7 and IL-7R{alpha}-/- mice do not exhibit lesions or deficiencies in the development of nonlymphoid cells or nonhematopoietic tissues (13). During the course of bone marrow transplant studies, we observed that IL-7R{alpha}-/- mice were more radiosensitive than controls and have investigated the mechanism. We hypothesized that radiation may impair survival or proliferation of various tissues in IL-7R{alpha}-/- mice that are masked by redundancy with other growth and survival factors under normal control conditions. A marked difference in IEC damage and recovery between the IL-7R{alpha}-/- mice and wild type C57BL/6 mice was observed following supralethal doses of {gamma}-irradiation. Consistent with IEC radiosensitivity in IL-7R{alpha}-/- mice, IEC in resting mice express more of the pro-apoptotic protein Bak and less Bcl-xL protein and increased numbers of apoptotic cells than cells from IL-7R{alpha}+/+ gut tissue. These findings demonstrate that IL-7 is important in the protection from radiation-induced apoptosis in nonlymphoid tissues.


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

Breeding pairs of C57BL/6J (B6) IL-7R{alpha}-/- mice were purchased from The Jackson Laboratory (Bar Harbor, ME). Mice were bred and maintained under specific pathogen-free conditions in our facility. BALB/c and C57BL/6N (B6) IL-7R{alpha}+/+ mice were purchased from the Animal Production Area (Frederick, MD). For some experiments, age-matched, female B6 IL-7R{alpha}-/- and IL-7R{alpha}+/+, B6 RAG1-/-, and B6 TCR{delta}-/- mice were purchased from The Jackson Laboratory.

Tissue preparation

Mice were euthanized by CO2 asphyxiation. Small intestines were removed and the lumen was flushed of its contents using a syringe fitted with a 20 gauge needle and filled with HBSS. IEC were isolated or the small intestines were prepared for histological evaluation using the swiss roll technique and fixed in 10% buffered neutral formalin.

Irradiation

For radiosensitivity studies, mice were irradiated with a single exposure of {gamma} irradiation from a 137Cs source (212 cGy/min) at total doses of 750, 1200, or 1500 cGy, and euthanized at predetermined time points or, in some studies, when they became moribund.

Induction of graft-vs-host disease (GVHD)

B6 IL-7R{alpha}-/- and IL-7R{alpha}+/+ recipients were irradiated with 1000 cGy total body irradiation. Recipients were then given 2 x 107 BALB/c splenocytes along with 107 BALB/c bone marrow cells by caudal vein injection in 0.5 ml volume. Mice were monitored and weighed weekly. All moribund animals were euthanized. Survival data were plotted by the Kaplan-Meier method and analyzed by the log-rank test.

Isolation and preparation of protein extracts from IEC

IEC were isolated from whole murine small intestines according to a method described by Rogler, et al. (22). Briefly, cut pieces of intestine were incubated in 10 mM DTT for 10 min with shaking at 37°C and centrifuged to pellet. Two additional 15-min incubations in 2 mM EDTA at 37°C released the IEC into the supernatant. The whole cell lysates were made by lysing IEC in a whole cell lysis buffer composed of 10 mM Tris, 150 mM NaCl, 1% Nonidet P-40, 0.4 mM EDTA, 10% glycerol, 10 mM NaF, and 1 mM Na3VO4, for 30 min on ice. Whole cell lysates underwent centrifugation at full speed in a microcentrifuge and the resulting pellet containing cellular debris was discarded. All extraction buffers contained the protease inhibitors, 1 mM PMSF and 1 µg/ml each of leupeptin, chymotrypsin, and pepstatin A (Sigma, St. Louis, MO). Protein concentrations were determined by the Bradford reagent microprotocol (Pierce, Rockford, IL) in which incompatible substances were diluted beyond the point of interference. Protein concentrations closely paralleled cell equivalents.

Histology

Tissues were embedded in paraffin, sectioned, and stained with hematoxylin and eosin for morphology or stained with an in situ apoptosis detection kit (Apotag; Oncor, Gaithersburg, MD) for evaluation of apoptotic cells. Tissue sections were evaluated and graded blind by a veterinary pathologist.

Western blots

To measure levels of Bak, Bcl-XL, and Bax protein expression in whole cell lysates, 30 µg each of total protein extract was loaded on a 12% SDS-PAGE minigel. Separated proteins were electrophoretically transferred to a nitrocellulose membrane, (Hybond ECL; Amersham Life Science, Arlington Heights, IL), and the membrane blocked with 5% nonfat milk (w/v). Bak protein was detected with a rabbit polyclonal primary Ab diluted 1:2000, followed by a peroxidase-conjugated goat anti-rabbit IgG Ab diluted 1:2000 (Santa Cruz Biotechnology, Santa Cruz, CA). Bcl-XL protein was detected with mouse primary Ab (PharMingen, San Diego, CA) diluted 1:2000, followed by a secondary Ab, peroxidase-labeled anti-mouse IgG, diluted 1:5000 (Amersham Life Science). Bax protein was detected with a mouse antiserum that was a kind gift of Dr. Richard J. Youle. (National Institute of Neurological Disorders and Stroke, National Institutes of Health, Bethesda, MD) and diluted 1:10 for use, followed by peroxidase-conjugated anti-mouse Ab (Amersham Life Science) diluted 1:4000. Ag-Ab complexes were visualized using the ECL Western blotting detection reagents (Amersham Life Sciences), following the manufacturer’s guidelines.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
IL-7R{alpha}-/- mice exhibit increased weight loss following an allogeneic bone marrow transplant and induction of GVHD

IL-7R{alpha}-/- mice were employed in studies to examine the role the role of IL-7 in the induction of GVHD following myeloablative radiation and allogeneic bone marrow transplantation. IL-7 has been suggested to be a survival factor protecting cells from apoptosis and it was of interest to ascertain whether the tissues of the mice have an altered susceptibility to destruction during GVHD. IL-7R{alpha}-/- and IL-7R{alpha}+/+ B6 (H-2b) mice were irradiated with 1000 cGy followed by infusion of allogeneic bone marrow and splenocytes from BALB/c (H-2d) mice. As shown in Fig. 1Go, IL-7R{alpha}-/- mice exhibited a significant reduction (p < 0.001) in body weight compared with IL-7R{alpha}+/+ mice. A trend toward significant differences (p = 0.06) in weight loss after irradiation were also observed in irradiated mice that received an allogeneic bone marrow transplant without splenocytes (data not shown). These results suggested that while induction of GVHD enhanced weight loss, sensitivity of the gastrointestinal tract to irradiation may be the primary factor.



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  		FIGURE 1. Weight loss in IL-7R{alpha}-/- and IL-7R{alpha}+/+ B6 (H-2b) recipient mice 4 days after allogeneic bone marrow transplant and induction of GVHD. Each treatment group consists of 11 animals. Mice received 1000 cGy followed by i.v. administration of 10 million bone marrow cells and 20 million splenocytes from BALB/c (H-2d) donors. IL-7R{alpha} -/- B6 recipients had significantly (p < 0.001, Student’s t test) lower weights vs IL-7R{alpha}+/+ B6 recipient mice post transplant. Weight loss presented as the change in body weight on day 4 post transplant from the mean body weights at day 0. Results expressed as the mean ± SEM.

 
Intestinal epithelial damage is more extensive following supralethal {gamma}-irradiation in IL-7R{alpha}-/- mice

IL-7R{alpha} expression can be induced in human IEC (21) and cell lines (21) by epidermal growth factor (21) or bacterial invasion (23). This may suggest that IL-7 may have biological effects on epithelial cells perhaps acting following tissue injury. If this were the case, loss of IL-7R would result in increased epithelial cell damage and/or delayed recovery that may account for the increased susceptibility to GVHD in these mice. To test this hypothesis, IL-7R{alpha}-/- and IL-7R{alpha}+/+ mice received a single dose of 1500 cGy {gamma}-irradiation. The small intestines of these mice were examined microscopically when mice became moribund. The results are summarized in Table IGo. At 8 days postirradiation, the IL-7R{alpha}+/+ mice had villi of normal length with some piling up of epithelium. Crypt height was increased, epithelium was basophilic, and mitotic figures were present high in the crypts demonstrating regenerative hyperplasia following {gamma}-irradiation. However, the small intestines of IL-7R{alpha}-/- mice reveal extensive damage in both the crypts and villi (Fig. 2Go) at day 7 postirradiation. Villi were extremely shortened with karyomegalic epithelial cells. Many intestinal crypts were ablated. Remaining crypts were dilated with attenuated epithelium or were hyperplastic with pseudostratified epithelium. In the remaining crypts, proliferative changes were evident. These late effects imply extensive damage to the intestinal epithelial stem cells within the crypts of IL-7R{alpha}-/- mice following {gamma}-irradiation that result in an impaired ability of these mice to regenerate the intestinal epithelium.


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  		Table I. Histological lesions in small intestine from moribund mice following 1500 cGy irradiationa

 


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  		FIGURE 2. Photomicrograph of small intestines after 1500 cGy whole body {gamma}-irradiation. Sections were stained with hematoxylin and eosin. A, IL-7R{alpha}+/+. Villi (v) are of normal length; tall crypts (c) are present. B, IL-7R{alpha}-/-. Villi (v) are markedly shortened and distorted; many crypts have been ablated. Magnification of A and B is x25. C, IL-7R{alpha}+/+. Hyperplastic crypts (c) with mitotic figures and piled up epithelial cells. D, IL-7R{alpha}-/-. Ablated crypts. Remaining crypts (c) are distorted with focal epithelial cell hyperplasia. Magnification of C and D is x70.

 
To determine whether IL-7R{alpha}-/- impaired intestinal epithelial stem cell regeneration following {gamma}-irradiation is a consequence of the immunodeficiency in the knockout animal, gastrointestinal radiosensitivity in IL-7R{alpha}-/- mice were compared with RAG1-/- mice, which are deficient in both B and T cells, or TCR{delta}-/- mice, which lack {gamma}{delta} TCR+ T cells. The immunodeficient mice received a single dose of 1500 cGy {gamma}-irradiation. The small intestines of these mice were then examined microscopically when mice became moribund. The results are summarized in Table IIGo. IL-7R{alpha}-/- mice showed increased ablation of intestinal crypts following whole body {gamma}-irradiation compared with either IL7R{alpha}+/+ immunodeficient mouse strain (RAG1-/- or TCR{delta}-/-). These results demonstrate that the absence of {gamma}{delta} TCR+ T cells or the paucity of other lymphocytes in the gastrointestinal tract is not responsible for the increased radiosensitivity observed in intestinal epithelial crypt cells in IL-7R{alpha}-/- mice.


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  		Table II. Histological lesions in small intestine from moribund mice following 1500 cGy irradiationa

 
Increased apoptosis in small intestinal crypt cells following sublethal {gamma}-irradiation

To confirm the hypersensitivity of IL-7R{alpha}-/- crypt cells to {gamma}-radiation, labeling of 3OH-DNA ends was used to examine the presence of apoptotic cells in the small intestinal crypts of IL-7R{alpha}-/- and IL-7R{alpha}+/+ mice 8 h post {gamma}-irradiation (Fig. 3Go). Based upon previous reports, radiation-induced apoptosis plateaus at a dose of ~1000 cGy in the small intestine of normal mice (24). In our studies, 8 h after a single dose of 750 cGy total body {gamma}-irradiation, histological evaluation of the small intestine revealed mild irradiation damage in IL-7R{alpha}-/- and IL-7R{alpha}+/+ mice in which a higher proportion of apoptotic cells was observed in the crypts of the small intestine of IL-7R{alpha}-/- compared with IL-7R{alpha}+/+ mice (Fig. 3Go). These findings demonstrate that the epithelial stem/progenitor cells within the crypts of small intestines of IL-7R{alpha}-/- mice are more sensitive to radiation-induced apoptosis than their wild type counterparts.



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  		FIGURE 3. Apoptosis in small intestinal crypts. Mice received 750 cGy whole body {gamma}-irradiation. At 8 h postirradiation, the animals were euthanized and apoptotic cells were detected in tissue sections of small intestines by the TUNEL method. A, Representative photomicrograph of apoptotic cells in crypts (c) of IL-7R{alpha}+/+. B, Representative photomicrograph with increased numbers of apoptotic cells in crypts (c) of IL-7R{alpha}-/-. Tissues counterstained with methyl green (40 times). C, Apoptotic cells were enumerated in 20 crypts of the proximal and 20 crypts of the distal portions of the small intestine for each specimen. Only crypts where both the paneth cells and the crypt-villus junction were evident were scored. Results represent the mean number of cells per crypt ± SEM of three mice per group. *, p < 0.0001 comparing IL-7R{alpha}-/- and IL-7R{alpha}+/+ within the same time point (Student’s t test).

 
Increased expression of proapoptotic gene products following sublethal {gamma}-irradiation in IL-7R{alpha}-/- mouse IEC

IL-7 protects lymphoid progenitors from apoptosis at least partially through the induction of Bcl-2 (25). To determine whether IEC from IL-7R{alpha}-/- mice are more sensitive to irradiation injury because of a shift in the balance between anti-apoptotic and/or pro-apoptotic proteins of the bcl-2 family, we examined isolated IEC from resting and irradiated mice. Mice were irradiated with 750 cGy and lysates were prepared from isolated IEC collected from mice euthanized at several time points. Evaluation of baseline apoptotic protein expression (Fig. 4Go) revealed a low constitutive expression of Bak protein in IEC of untreated IL-7R{alpha}-/- but not IL-7R{alpha}+/+ mice. While the pro-apoptotic protein Bak was elevated in the IL-7R{alpha}-/- mice, the converse was true for the anti-apoptotic protein, Bcl-xL. Bcl-xL is expressed at higher concentrations in the epithelial cells of untreated IL-7R{alpha}+/+ compared with IL-7R{alpha}-/- mice. After irradiation, Bak protein levels increased in the IEC of IL-7R{alpha}-/- mice peaking at 12 h (Fig. 5Go). Bak protein levels remained elevated through 48 h. Bak protein was also induced in IEC of IL-7R{alpha}+/+ mice following irradiation though the levels were markedly lower than IL-7R{alpha}-/- IEC. A detectable level of Bax protein was induced following irradiation in IL-7R{alpha}-/- IEC though IL-7R{alpha}+/+ mice (Fig. 5Go). These findings demonstrate that exposure of IL-7R{alpha}-/- mice to {gamma}-irradiation results in increased Bak production in the IEC with subsequent increases in apoptosis and lesions. Thus, signaling through the IL-7 receptor appears to play a role in protecting the intestinal tract from {gamma}-irradiation, in part through the regulation of apoptotic proteins.



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  		FIGURE 4. Differences in IEC expression of Bcl-2 family members in IL-7R{alpha}-/- and IL-7R{alpha}+/+ mice. Epithelial cells from the small intestine were isolated from resting IL-7R{alpha}-/- and IL-7R{alpha}+/+ mice and protein expression detected by immunoblot using Abs specific for Bak and Bcl-xL. Results from one of two independent experiments are shown.

 


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  		FIGURE 5. Immunoblot analysis of Bak and Bax protein in IEC from irradiated mice. IL-7R{alpha}-/- and IL-7R{alpha}+/+ were exposed to 750 cGy {gamma}-irradiation. Mice were euthanized at various time points and epithelial cells from the small intestine of two mice per group were isolated and pooled. Protein samples (30 µg per lane) from whole cell lysates were subjected to SDS-PAGE, transferred to nitrocellulose and blotted with Abs specific for Bak and Bax.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
Using IL-7R{alpha} null mice, stimulation of IL-7R{alpha} was found to deliver an endogenous radioprotective signal to IEC. This is the first demonstration that ligation of IL-7R has a biological effect on the gut. Whereas the lymphoid system depends on IL-7 for essential developmental functions, the normal development/function of the gut does not appear to require IL-7. However, IL-7 appears to have an important role in gut protection/regeneration specifically following irradiation damage. The radioprotective effect of signaling through the IL-7R{alpha} in gut parallels that previously reported for mature lymphoid cells (26). Because the studies were performed in IL-7R{alpha}-/- mice, signaling through the IL-7R{alpha} could be through binding of IL-7 or thymic stromal cell-derived lymphopoietin (27) and it is unclear which ligand is responsible for the radioprotective activity.

Initial reports on the IL-7R{alpha}-/- mouse phenotype described the severe combined immunodeficiency of these animals (13, 14). There are few mature {alpha}{beta} T-cells and no mature {gamma}{delta} T-cells within the intestinal tract (13, 14), and Peyer’s patches do not form (28). The lack of lymphoid cells within the GI tract has the potential to sensitize the mice to radiation damage due to an increased bacterial burden and/or loss of cells that could indirectly influence the radiosensitivity of the epithelium. Helicobacter species have been implicated in spontaneous colitis in immunodeficient mice (29). Rare spirochetes were observed in the colons of both wild type and null experimental animals. No differences in helicobacter burden was observed between IL-7R{alpha}-/- and IL-7R{alpha}+/+ nor was colitis observed in any of the animals (data not shown). Observations in the studies reported here were confined to the small intestine and did not include colonic epithelium.

In contrast to previous reports that {gamma}{delta} T cells are important in the regulation of IEC and that mutant mice lacking {gamma}{delta} T cells have hypocellular intestinal crypts (30), we observed no histological differences in the small intestinal crypts of IL-7R{alpha}-/- and IL-7R{alpha}+/+ mice. In addition, the absence of either B and T lymphocytes or {gamma}{delta} T cells in RAG-1-/- or TCR {delta}-/- IL-7R{alpha}+/+ mice, respectively, did not confer increased sensitivity of intestinal crypt stem cells to {gamma}-radiation as was observed in the IL-7R{alpha}-/- mice.

The lining of the intestine is generated by rapidly proliferating epithelial cells. Proliferation is confined to the crypts that are composed of gut stem and progenitor cells. The epithelial cells differentiate as they migrate from the base to the top of the villus and are eventually turned over in ~5 days (31, 32, 33, 34). In the unirradiated animals, no difference was detectable in the histology or incidence of apoptosis within the crypts of IL-7R{alpha}-/- mice, relative to IL-7R{alpha}+/+ mice. Unlike apoptosis that occurs as part of normal homeostasis, radiation-induced death of crypt cells of the small intestine has been shown to be mediated by p53 (31, 32, 33, 34, 35, 36) which, among other activities, acts as a transcription factor that induces expression of genes whose products are involved in cell cycle arrest (e.g., p21) and in cell death (e.g., Bax). p53 protein levels rise quickly after irradiation in crypt cells and the cells that survive can continue to show elevated p53 protein for up to 6 days post irradiation (37). Therefore, the radioprotective action induced through the IL-7R{alpha} could be due to interference with the activation of p53 or with its action or the action of its downstream effectors.

The trophic action of IL-7 on pro-T cells is associated with Bcl-2 family members, probably the Bcl-2/Bax ratio (38, 39). In the gut, the anti-apoptotic action of IL-7R{alpha} also correlated with Bcl-2 family members (but different ones than in thymocytes), possibly the Bcl-xL/Bak ratio. We observed a sustained elevation of Bak (up to 9 days) in IEC preparations following irradiation of IL-7R{alpha}-/- mice. This sustained Bak elevation could be induced by p53, which shows a long-lived elevation following irradiation of crypt cells, comparable to the induction of the p21 gene by p53 which, like Bak, is sustained for some days after irradiation in the small intestine (37). The various Bcl-2 family members have been thought to play similar roles in different cell types, i.e., whereas Bcl-2 plays a key survival role in lymphoid cells, Bcl-xL has a parallel survival function in neurons. Our results suggest the potential for Bak in triggering death of IEC, which is supported by previous reports (40, 41), serving a parallel function to that which Bax performs.

Many studies have demonstrated the role cytokines can play in radioprotection of the gut. Not only epithelial cell growth factors (e.g., epidermal growth factor (40, 41, 42, 43), TGF-{beta} (44), and keratinocyte growth factor (45)) but also cytokines associated with hematopoiesis and immune function such as stem cell factor (46), IL-1 (47, 48), and IL-11 (49). Demonstration of the radioprotective effects, by administration of cytokines to animals, usually requires treatment before irradiation. In this scenario, cytokines protect the gut through increases in the intestinal mucosa (50, 51) and/or by enhancing crypt survival (44, 45). The absence of IL-7R signaling has no apparent effect on the development of the intestinal epithelium. If IL-7 has any role in development and homeostasis of the gut, it is masked by other factors except for a increase in basal levels of bak in intestinal epithelium. However, while other cytokines can enhance crypt survival after cytotoxic treatment, IL-7R ligand(s) has a nonredundant role in the protection of these epithelial stem cells.

This study describes a novel function for the IL-7 receptor in the gut. These results demonstrate the increased radiosensitivity of stem cells within the crypts in IL-7R{alpha} -/- mice and conversely a role for IL-7R ligand(s) in the protection of radiation-induced apoptosis in these same cells.


   Acknowledgments
 
We gratefully acknowledge the expert technical assistance provided by Steve Stull and Roberta Smith. We thank Laura Knott for outstanding secretarial services.


   Footnotes
 
1 This project has been funded in whole or in part with Federal funds from the National Cancer Institute, National Institutes of Health under Contract No. N01-CO-56000. The content of this publication does not necessarily reflect the review or policies of the Department of Health and Human Services, nor does mention of trade names, commercial products, or organizations imply endorsement by the U.S. Government. Animals were cared for humanely according to the U.S. Public Health Policy on the Care and Use of Animals, and the Guide for the Care and Use of Laboratory Animals. National Cancer Institute-Frederick Cancer Research and Development Center facilities are accredited by the Association for Assessment and Accreditation of Laboratory Animal Care International. Back

2 Address correspondence and reprint requests to Dr. William J. Murphy, Intramural Research Support Program, Science Applications International Corporation Frederick, National Cancer Institute-Frederick Cancer Research and Development Center, Building 567, Room 210, Frederick, MD 21702-1201. Back

3 Abbreviations used in this paper: IEC, intestinal epithelial cells; B6, C57BL/6J; GVHD, graft-vs-host disease. Back

Received for publication May 5, 2000. Accepted for publication December 8, 2000.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

  1. Namen, A. E., S. Lupton, K. Hjerrild, J. Wignall, D. Y. Mochizuki, A. Schmierer, B. Mosley, C. J. March, D. Urdal, S. Gillis. 1988. Stimulation of B-cell progenitors by cloned murine interleukin-7. Nature 333:571.[Medline]
  2. Wiles, M. V., P. Ruiz, B. A. Imhof. 1992. Interleukin-7 expression during mouse thymus development. Eur. J. Immunol. 22:1037.[Medline]
  3. Kropf, P., R. Etges, L. Schopf, C. Chung, J. Sypek, I. Muller. 1997. Characterization of T cell-mediated responses in nonhealing and healing Leishmania major infections in the absence of endogenous IL-4. J. Immunol. 159:3434.[Abstract]
  4. Heufler, C., G. Topar, A. Grasseger, U. Stanzl, F. Koch, N. Romani, A. E. Namen, G. Schuler. 1993. Interleukin 7 is produced by murine and human keratinocytes. J. Exp. Med. 178:1109.[Abstract/Free Full Text]
  5. Watanabe, M., Y. Ueno, T. Yajima, Y. Iwao, M. Tsuchiya, H. Ishikawa, S. Aiso, T. Hibi, H. Ishii. 1995. Interleukin 7 is produced by human intestinal epithelial cells and regulates the proliferation of intestinal mucosal lymphocytes. J. Clin. Invest. 95:2945.
  6. Morrissey, P. J., R. G. Goodwin, R. P. Nordan, D. Anderson, K. H. Grabstein, D. Cosman, J. Sims, S. Lupton, B. Acres, S. G. Reed. 1989. Recombinant interleukin 7, pre-B cell growth factor, has costimulatory activity on purified mature T cells. J. Exp. Med. 169:707.[Abstract/Free Full Text]
  7. Murray, R., T. Suda, N. Wrighton, F. Lee, A. Zlotnik. 1989. IL-7 is a growth and maintenance factor for mature and immature thymocyte subsets. Int. Immunol. 1:526.[Abstract/Free Full Text]
  8. Samaridis, J., G. Casorati, A. Traunecker, A. Iglesias, J. C. Gutierrez, U. Muller, R. Palacios. 1991. Development of lymphocytes in interleukin 7-transgenic mice. Eur. J. Immunol. 21:453.[Medline]
  9. Michaelson, M. D., M. F. Mehler, H. Xu, R. E. Gross, J. A. Kessler. 1996. Interleukin-7 is trophic for embryonic neurons and is expressed in developing brain. Dev. Biol. 179:251.[Medline]
  10. Komschlies, K. L., T. A. Gregorio, M. E. Gruys, T. C. Back, C. R. Faltynek, R. H. Wiltrout. 1994. Administration of recombinant human IL-7 to mice alters the composition of B-lineage cells and T cell subsets, enhances T cell function, and induces regression of established metastases. J. Immunol. 152:5776.[Abstract]
  11. Damia, G., K. L. Komschlies, C. R. Faltynek, F. W. Ruscetti, R. H. Wiltrout. 1992. Administration of recombinant human interleukin-7 alters the frequency and number of myeloid progenitor cells in the bone marrow and spleen of mice. Blood 79:1121.[Abstract/Free Full Text]
  12. Boerman, O. C., T. A. Gregorio, K. J. Grzegorzewski, C. R. Faltynek, J. J. Kenny, R. H. Wiltrout, K. L. Komschlies. 1995. Recombinant human IL-7 administration in mice affects colony-forming units-spleen and lymphoid precursor cell localization and accelerates engraftment of bone marrow transplants. J. Leukocyte Biol. 58:151.[Abstract]
  13. von Freeden-Jeffry, U., P. Vieira, L. A. Lucian, T. McNeil, S. E. Burdach, R. Murray. 1995. Lymphopenia in interleukin (IL)-7 gene-deleted mice identifies IL-7 as a nonredundant cytokine. J. Exp. Med. 181:1519.[Abstract/Free Full Text]
  14. Peschon, J., P. Morrissey, K. Grabstein, F. Ramsdell, E. Maraskovsky, B. Gliniak, L. Park, S. Ziegler, D. Williams, C. Ware, et al 1994. Early lymphocyte expansion is severely impaired in interleukin 7 receptor-deficient mice. J. Exp. Med. 180:1955.[Abstract/Free Full Text]
  15. Moore, T. A., U. von Freeden-Jeffry, R. Murray, A. Zlotnik. 1996. Inhibition of {gamma}{delta} T cell development and early thymocyte maturation in IL-7-/- mice. J. Immunol. 157:2366.[Abstract]
  16. Maki, K., S. Sunaga, Y. Komagata, Y. Kodaira, A. Mabuchi, H. Karasuyama, K. Yokomuro, J. I. Miyazaki, K. Ikuta. 1996. Interleukin 7 receptor-deficient mice lack {gamma}{delta} T cells. Proc. Natl. Acad. Sci. USA 93:7172.[Abstract/Free Full Text]
  17. Maraskovsky, E., M. Teepe, P. Morrissey, S. Braddy, R. Miller, D. Lynch, J. Peschon. 1996. Impaired survival and proliferation in IL-7 receptor-deficient peripheral T cells. J. Immunol. 157:5315.[Abstract]
  18. Maraskovsky, E., L. O’Reilly, M. Teepe, L. Corcoran, J. Peschon, A. Strasser. 1997. Bcl-2 can rescue T lymphocyte development in interleukin-7 receptor-deficient mice but not in mutant rag-1-/- mice. Cell 89:1011.[Medline]
  19. Jacobsen, F. W., O. P. Veiby, C. Skjonsberg, S. E. Jacobsen. 1993. Novel role of interleukin 7 in myelopoiesis: stimulation of primitive murine hematopoietic progenitor cells. J. Exp. Med. 178:1777.[Abstract/Free Full Text]
  20. Kirnbauer, R., B. Charvat, E. Schauer, A. Kock, A. Urbanski, E. Forster, P. Neuner, I. Assmann, T. A. Luger, T. Schwarz. 1992. Modulation of intercellular adhesion molecule-1 expression on human melanocytes and melanoma cells: evidence for a regulatory role of IL-6, IL-7, TNF {beta}, and UVB light. J. Invest. Dermatol. 98:320.[Medline]
  21. Reinecker, H., D. Podolsky. 1995. Human intestinal epithelial cells express functional cytokine receptors sharing the common {gamma}c chain of the interleukin 2 receptor. Proc. Natl. Acad. Sci. USA 92:8353.[Abstract/Free Full Text]
  22. Rogler, G., R. Daig, E. Aschenbrenner, D. Vogl, K. Schlottmann, W. Falk, V. Gross, J. Scholmerich, T. Andus. 1998. Establishment of long-term primary cultures of human small and large intestinal epithelial cells. Lab Invest. 78:889.[Medline]
  23. Yamada, K., M. Shimaoka, K. Nagayama, T. Hiroi, H. Kiyono, T. Honda. 1997. Bacterial invasion induces interleukin-7 receptor expression in colonic epithelial cell line, T84. Eur. J. Immunol. 27:3456.[Medline]
  24. Potten, C. S.. 1977. Extreme sensitivity of some intestinal crypt cells to X and {gamma} irradiation. Nature 269:518.[Medline]
  25. Griffiths, S., D. Goodhead, S. Marsden, E. Wright, S. Krajewski, J. Reed, S. Korsmeyer, M. Greaves. 1994. Interleukin 7-dependent B lymphocyte precursor cells are ultrasensitive to apoptosis. J. Exp. Med. 179:1789.[Abstract/Free Full Text]
  26. Boise, L. H., A. J. Minn, C. H. June, T. Lindsten, C. B. Thompson. 1995. Growth factors can enhance lymphocyte survival without committing the cell to undergo cell division. Proc. Natl. Acad. Sci. USA 92:5491.[Abstract/Free Full Text]
  27. Friend, S. L., S. Hosier, A. Nelson, D. Foxworthe, D. E. Williams, A. Farr. 1994. A thymic stromal cell line supports in vitro development of surface IgM+ B cells and produces a novel growth factor affecting B and T lineage cells. Exp Hematol 22:321.[Medline]
  28. Laky, K., L. Lefrancois, U. von Freeden-Jeffry, R. Murray, L. Puddington. 1998. The role of IL-7 in thymic and extrathymic development of TCR {gamma}{delta} cells. J. Immunol. 161:707.[Abstract/Free Full Text]
  29. Ward, J. M., M. R. Anver, D. C. Haines, J. M. Melhorn, P. Gorelick, L. Yan, J. G. Fox. 1996. Inflammatory large bowel disease in immunodeficient mice naturally infected with Helicobacter hepaticus. Lab. Anim. Sci. 46:15.[Medline]
  30. 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 {gamma}{delta} T cells. Proc. Natl. Acad. Sci. USA 92:6147.[Abstract/Free Full Text]
  31. Adachi, S., H. Yoshida, K. Honda, K. Maki, K. Saijo, K. Ikuta, T. Saito, S. Nishikawa. 1998. Essential role of IL-7 receptor {alpha} in the formation of Peyer’s patch anlage. Int. Immunol. 10:1.[Abstract/Free Full Text]
  32. Cheng, H., C. P. Leblond. 1974. Origin, differentiation and renewal of the four main epithelial cell types in the mouse small intestine. V. Unitarian Theory of the origin of the four epithelial cell types. Am. J. Anat. 141:537.[Medline]
  33. Cheng, H., C. P. Leblond. 1974. Origin, differentiation and renewal of the four main epithelial cell types in the mouse small intestine. I. Columnar cell. Am. J. Anat. 141:461.[Medline]
  34. Schmidt, G. H., M. M. Wilkinson, B. A. Ponder. 1985. Cell migration pathway in the intestinal epithelium: an in situ marker system using mouse aggregation chimeras. Cell 40:425.[Medline]
  35. Merritt, A. J., C. S. Potten, C. J. Kemp, J. A. Hickman, A. Balmain, D. P. Lane, P. A. Hall. 1994. The role of p53 in spontaneous and radiation-induced apoptosis in the gastrointestinal tract of normal and p53-deficient mice. Cancer Res. 54:614.[Abstract/Free Full Text]
  36. Clarke, A. R., S. Gledhill, M. L. Hooper, C. C. Bird, A. H. Wyllie. 1994. p53 dependence of early apoptotic and proliferative responses within the mouse intestinal epithelium following {gamma}-irradiation. Oncogene 9:1767.[Medline]
  37. Wilson, J. W., D. M. Pritchard, J. A. Hickman, C. S. Potten. 1998. Radiation-induced p53 and p21WAF-1/CIP1 expression in the murine intestinal epithelium: apoptosis and cell cycle arrest. Am. J. Pathol. 153:899.[Abstract/Free Full Text]
  38. Candeias, S., K. Muegge, S. K. Durum. 1997. IL-7 receptor and VDJ recombination: trophic versus mechanistic actions. Immunity 6:501.[Medline]
  39. Kim, K., C. K. Lee, T. J. Sayers, K. Muegge, S. K. Durum. 1998. The trophic action of IL-7 on pro-T cells: inhibition of apoptosis of pro-T1, -T2, and -T3 cells correlates with Bcl-2 and Bax levels and is independent of Fas and p53 pathways. J. Immunol. 160:5735.[Abstract/Free Full Text]
  40. Moss, S., B. Agarwal, N. Arber, R. Guan, M. Krajewska, S. Krajewski, J. Reed, P. Holt. 1996. Increased intestinal Bak expression results in apoptosis. Biochem. Biophys. Acta 223:199.
  41. Hague, A., G. Diaz, D. Hicks, S. Krajewski, J. Reed, C. Paraskeva. 1997. bcl-2 and bak may play a pivotal role in sodium butyrate-induced apoptosis in colonic epithelial cells; however overexpression of bcl-2 does not protect against bak-mediated apoptosis. Int. J. Cancer 72:898.[Medline]
  42. McKenna, K. J., S. Ligato, Jr G. L. Kauffman, A. B. Abt, J. A. Stryker, R. L. Conter. 1994. Epidermal growth factor enhances intestinal mitotic activity and DNA content after acute abdominal radiation. Surgery 115:626.[Medline]
  43. Lindegaard, J. C., L. Vinter-Jensen, J. Overgaard. 1997. Epidermal growth factor and acute radiation damage in CDF1 mice in vivo. Acta Oncol. 36:393.[Medline]
  44. Potten, C. S., D. Booth, J. D. Haley. 1997. Pretreatment with transforming growth factor {beta}-3 protects small intestinal stem cells against radiation damage in vivo. Br. J. Cancer 75:1454.[Medline]
  45. Khan, W. B., C. Shui, S. Ning, S. J. Knox. 1997. Enhancement of murine intestinal stem cell survival after irradiation by keratinocyte growth factor. Radiat. Res. 148:248.[Medline]
  46. Leigh, B. R., W. Khan, S. L. Hancock, S. J. Knox. 1995. Stem cell factor enhances the survival of murine intestinal stem cells after photon irradiation. Radiat. Res. 142:12.[Medline]
  47. Wu, S. G., T. Miyamoto. 1990. Radioprotection of the intestinal crypts of mice by recombinant human interleukin-1{alpha}. Radiat. Res. 123:112.[Medline]
  48. Hancock, S. L., R. T. Chung, R. S. Cox, R. F. Kallman. 1991. Interleukin 1{beta} initially sensitizes and subsequently protects murine intestinal stem cells exposed to photon radiation. Cancer Res. 51:2280.[Abstract/Free Full Text]
  49. Potten, C. S.. 1995. Interleukin-11 protects the clonogenic stem cells in murine small-intestinal crypts from impairment of their reproductive capacity by radiation. Int. J. Cancer 62:356.[Medline]
  50. Farrell, C. L., J. V. Bready, K. L. Rex, J. N. Chen, C. R. DiPalma, K. L. Whitcomb, S. Yin, D. C. Hill, B. Wiemann, C. O. Starnes, et al 1998. Keratinocyte growth factor protects mice from chemotherapy and radiation-induced gastrointestinal injury and mortality. Cancer Res. 58:933.[Abstract/Free Full Text]
  51. Potten, C. S., J. W. Wilson, C. Booth. 1997. Regulation and significance of apoptosis in the stem cells of the gastrointestinal epithelium. Stem Cells 15:82.[Abstract/Free Full Text]




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