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Originally published In Press as doi:10.1074/jbc.M205300200 on July 22, 2002

J. Biol. Chem., Vol. 277, Issue 40, 37811-37819, October 4, 2002
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A Gnotobiotic Transgenic Mouse Model for Studying Interactions between Small Intestinal Enterocytes and Intraepithelial Lymphocytes*,

Indira U. MysorekarDagger , Robin G. Lorenz§, and Jeffrey I. GordonDagger ||

From the Departments of Dagger  Molecular Biology and Pharmacology and § Pathology and Immunology, Washington University School of Medicine, St. Louis, Missouri 63110

Received for publication, May 29, 2002, and in revised form, July 22, 2002

    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS AND DISCUSSION
REFERENCES

The mouse intestinal epithelium undergoes continuous renewal throughout life. Intraepithelial lymphocytes (IELs) represent a significant fraction of this epithelium and play an important role in intestinal mucosal barrier function. We have generated a germ-free transgenic mouse model to examine the effects of a genetically engineered proliferative abnormality in the principal epithelial cell lineage (enterocytes) on IEL census and on IEL-enterocytic cross-talk. SV40 large T antigen (TAgWt) or a mutant derivative (TAgK107/8) that does not bind pRB was expressed in small intestinal villus enterocytes under the control of elements from the intestinal fatty acid binding protein gene (Fabpi). Quantitative immunohistochemical and flow cytometric analyses of conventionally raised and germ-free FVB/N Fabpi-TAgWt, Fabpi-TAgK107/8, and nontransgenic mice disclosed that forced reentry of enterocytes into the cell cycle is accompanied by an influx of thymically educated alpha beta T cell receptor (TCR)+ CD4+ and alpha beta TCR+ CD8alpha beta + IELs and a decrease in intestinally derived gamma delta TCR+ CD8alpha alpha IELs. Real time quantitative reverse transcriptase-PCR studies of jejunal villus epithelium recovered from germ-free transgenic and normal mice by laser capture microdissection and gamma delta TCR+ jejunal IELs purified by flow cytometry disclosed that the proliferative abnormality is accompanied by decreased expression of enterocytic interleukin-7 as well as IEL interleukin-7Ralpha and transforming growth factor beta 3. The analysis also revealed that normal villus epithelium expresses Fms-like tyrosine kinase 3 (Flt3), a known regulator of hematopoietic stem cell proliferation and neuronal cell survival, and its ligand (Flt3L). Epithelial expression of this receptor and its ligand is reduced by the proliferative abnormality, whereas IEL expression of Flt3L remains constant. Together, these findings demonstrate that changes in the proliferative status of the intestinal epithelium affects maturation of gamma delta TCR+ IELs and produces an influx of alpha beta TCR+ IELs even in the absence of a microflora.

    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS AND DISCUSSION
REFERENCES

The adult mouse small intestine is a complex, spatially diversified ecosystem that maintains distinctive cephalocaudal differences in its various functions. This regional variation in function is accompanied by regional differences in the differentiation programs of its four continuously renewing epithelial cell lineages, in the composition of its mucosal immune system, and in the composition of its resident society of commensal/symbiotic microorganisms (reviewed in Refs. 1-3). A full understanding of how this ecosystem is organized and functions in health and how it is reorganized or disorganized in various disease states requires knowledge about the nature and regulation of interactions between its microflora, epithelium, and gut-associated lymphoid tissue (1, 4, 5). The molecular nature and significance of the signals exchanged between these components have been difficult to decipher because of the dynamic quality and complexity of the system. One way of approaching this problem is to simplify the ecosystem using inbred strains of mice with defined microbiological status (gnotobiotic animals). For example, comparative functional genomics studies of mice containing no resident microorganisms (germ-free), conventionally raised mice harboring an complete microflora, and germ-free animals that have been colonized with a single species from the normal microflora (ex-germ-free) have shown that indigenous commensal bacteria play an important role in regulating host nutrient processing, fortifying the epithelial barrier, and organizing/educating the mucosal immune system (5, 6).

The intestine contains a large population of intraepithelial lymphocytes (IELs),1 equivalent in size to the population of peripheral lymphocytes that resides in the spleen (7). IELs are distributed throughout the epithelium that overlies small intestinal villi (average of one IEL for every 6-10 epithelial cells (8)). Virtually all small intestinal IELs are T cells, but they are heterogeneous with respect to their surface phenotype. The majority are CD3+ and can be divided into alpha beta T cell receptor-positive (TCR+) and gamma delta TCR+ subsets (8). They can be further subdivided based on expression of CD8 (alpha alpha homodimer or alpha beta heterodimer) or CD4 coreceptors (i.e. (i) gamma delta TCR+ CD8alpha - CD8beta -; (ii) gamma delta TCR+ CD8alpha + CD8beta - (abbreviated gamma delta + TCR+ CD8alpha alpha ); (iii) alpha beta TCR+ CD4+; (iv) alpha beta TCR+ CD8alpha + CD8beta - (alpha beta TCR+ CD8alpha alpha ); and (v) alpha beta TCR+ CD8alpha + CD8beta + (alpha beta TCR+ CD8alpha beta )).

Studies of Rag1-/- mice injected with bone marrow from nude mice or peripheral lymph node T cells from euthymic mice demonstrated that generation of alpha beta TCR+ CD4+ and CD8+ IELs is thymus-dependent, whereas gamma delta TCR+ CD8alpha alpha + IELs appeared in the absence of a thymus (9). One site of extrathymic maturation may be the crypts of Lieberkuhn. These distinct mucosal invaginations surround the base of each villus and contain long-lived multipotent stem cells (10) that give rise to the four epithelial lineages of the small intestine: enterocytes, goblet, and enteroendocrine cells, which differentiate as they migrate from the crypt up adjacent villi; and Paneth cells, which differentiate and remain at the crypt base (11-15). Crypts possess structures (cryptopatches) that contain clusters of c-Kit+ interleukin-7 receptor (IL-7R)+ Thy1+ lymphocytes (16). Mice with a truncated mutation of the common cytokine receptor chain (17) lack these cryptopatches and do not have gamma delta TCR+ CD8alpha alpha + IELs but contain thymus-dependent alpha beta TCR+ CD4+ and alpha beta TCR+ CD8alpha beta + IELs, suggesting a role for cryptopatches in maturation of extrathymically derived gamma delta TCR+ IELs (18-20).

The epithelium also appears to play a direct role in regulating IEL development. Epithelial cells produce stem cell factor (21), a ligand for the c-Kit receptor expressed on the surface of gamma delta TCR+ IELs (22). Mice deficient in either stem cell factor or c-Kit have reduced numbers of gamma delta TCR+ IELs (22). Furthermore, thyrotropin-releasing hormone stimulation of enterocytes results in local release of thyroid-stimulating hormone, which interacts with IEL-based thyroid-stimulating hormone receptor to promote IEL development (23) (e.g. hyt/hyt mice, which have a loss-of-function thyroid-stimulating hormone receptor mutation, have disrupted IEL maturation) (24, 25).

Epithelium-based IL-7 provides another regulatory signal for IEL proliferation (26). Studies of mice that lack IL-7 or the IL-7R have demonstrated that IL-7R-mediated signaling is essential for gamma delta TCR+ IEL development (26, 27). Moreover, Laky et al. (28) used transcriptional regulatory elements from the rat intestinal fatty acid-binding protein (Fabpi) to express IL-7 in the villus enterocytes of Il-7-/- mice. gamma delta TCR+ IELs were restored in the intestinal epithelium but remained absent from all other tissues, indicating that local production of IL-7 was sufficient for proper development/survival of this IEL subset.

Interactions between intestinal epithelial cells and IELs are reciprocal; IELs can influence epithelial cell biology. One illustration of this reciprocity is provided by TCRdelta subunit-deficient mice. These animals have reduced numbers of dividing cells in their crypts of Lieberkuhn and reduced crypt cellularity (29) and exhibit more severe intestinal epithelial damage following infection with the parasite Eimeria vermiformis (30). gamma delta TCR+ IELs produce keratinocyte growth factor, which affects epithelial cell growth and repair (31). These findings raise the question of whether gamma delta TCR+ IELs form part of a homeostatic surveillance mechanism that can detect and respond to perturbations in intestinal epithelial proliferation in order to maintain steady state cellular census in crypts and their associated villi.

Some workers have proposed that IELs are key elements in a "mucosal intranet," where they function to control epithelial integrity and immunologic homeostasis (32). Recent comparative DNA microarray-based studies of gene expression in gamma delta TCR+ IELs harvested from the small intestines of conventionally raised adult C57Bl6/J mice and alpha beta TCR+ cells harvested from their mesenteric lymph nodes have provided a list of candidate factors, preferentially expressed by gamma delta TCR+ IELs, that may support this mucosal intranet (33, 34).

In the present study, we examine the cross-talk between IELs and epithelium using transgenic mice that express simian virus 40 large T antigen (TAgWt) in their villus-associated enterocytes. The rationale for our experimental approach was as follows. Fabpi-directed expression of TAgWt produces a proliferative abnormality restricted to villus enterocytes: Fabpi-reporter transgenes are not expressed in the IELs. Expression of the viral oncoprotein in postmitotic enterocytes induces their reentry into the cell cycle (35) and an associated p53-independent apoptosis (36) but is not accompanied by evidence of dysplasia during the 1-2-day interval that they take to complete their migration to the cellular extrusion zone located at the villus tip (36, 37). Fabpi-directed expression of a mutant TAg containing a Glu right-arrow Lys substitution at residues 107 and 108 (TAgK107/8) disrupts pRB binding that does not produce this proliferative abnormality. Thus, a three-way comparison of FVB/N Fabpi-TAgWt and Fabpi-TAgK107/8 transgenic mice and their age-matched nontransgenic littermates would allow direct assessment of whether a proliferative abnormality limited to the predominant intestinal epithelial lineage is accompanied by changes in the fractional representation of extrathymically educated or thymically derived IEL subsets. By performing this analysis in conventionally raised and germ-free mice, we could also determine whether the microflora contributed to any observed changes in IELs. Finally, by using laser capture microdissection (LCM) of small intestinal cryosections to harvest villus epithelium, flow cytometry to retrieve their IELs, and the DNA microarray-based data sets of IEL gene expression to direct quantitative reverse transcription-PCR measurements of the levels of specified mRNAs in each cell population, we could use this environmentally well defined system to identify enterocytic gene products affected by proliferative status that may impact on IEL development/survival.

Our results show that the engineered proliferative abnormality is accompanied by a microflora-independent reduction in extrathymically educated gamma delta TCR+ CD8alpha alpha + IELs. This change is accompanied by coordinate changes in the expression of enterocytic and gamma delta TCR +IEL gene products that probably help legislate the observed change in IEL representation.

    EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS AND DISCUSSION
REFERENCES

Generation and Maintenance of Conventionally Raised and Germ-free Transgenic Mice-- FVB/N mice hemizygous for a transgene containing nucleotides -1178 to +28 of rat Fabpi linked to TAgWt or TAgK107/8 are described in earlier reports (35, 36, 38). Conventionally raised animals were maintained in microisolators in a specified pathogen-free state.

Normal and transgenic mice were rederived as germ-free by Caesarian section of transgenic mothers and transfer of their embryonic day 19 fetuses to plastic gnotobiotic isolators (Standard Safety Equipment Co.) containing germ-free foster mothers. The protocol used for this rederivation is described in a recent publication (6). Both conventionally raised and germ-free mice were given sterilized BeeKay Autoclavable Diet (B & K Universal Inc.) ad libitum. All animals were maintained under a strict light cycle (lights on at 0600 h and off at 1800 h). Animals were genotyped using primers, tail DNA, and PCR conditions described in Ref. 36. Some mice received an intraperitoneal injection of an aqueous solution of 5-bromo-2'-deoxyuridine (BrdUrd; 120 mg/kg) and 5-fluoro-2'-deoxyuridine (12 mg/kg) (Sigma) 90 min prior to sacrifice. Only male mice were studied.

Quantitative Immunohistochemical Analysis of the IEL Subsets-- FVB/N transgenic mice and their wild type littermates were sacrificed at 6-8 weeks of age (n = 3 conventionally raised or germ-free animals/genotype/experiment; n = 3 independent experiments). The middle third of their small intestine (arbitrarily defined as jejunum) was immediately flushed with PBS and subdivided into five equal length segments. All were segments placed together in a tissue cassette, overlaid with OCT (Miles Scientific), and frozen in Cytocool II (Stephens Scientific). 100 serial 5-µm thick sections were cut parallel to the cephalocaudal axes of the segments. For each antibody surveyed, every 10th section was fixed for 20 min in methanol (at -20 °C), washed three times in PBS (3 min/cycle), and treated with PBS-blocking buffer (1% bovine serum albumin, 0.05% Triton X-100 in PBS) for 2 h at room temperature. Sections were subsequently treated three times with TNT wash buffer (0.1 M Tris, pH 7.5, 0.15 M NaCl, 0.05% Tween 20; three cycles; 5 min/cycle) and then incubated overnight at 4 °C with each of the following monoclonal antibodies (all from BD PharMingen, each diluted 1:1000 in TNB-blocking buffer (0.1 M Tris (pH 7.5), 0.15 M NaCl, and 0.5% blocking reagent from PerkinElmer Life Sciences)): (i) rat anti-mouse CD4 (clone H129.19); (ii) rat anti-mouse CD8alpha (clone 53-6.7); (iii) rat anti-mouse CD8beta (clone Ly-32); (iv) hamster anti-mouse alpha beta TCR (beta  chain; clone H57-597); (v) hamster anti-mouse gamma delta TCR (delta  chain; clone GL3); and (vi) hamster anti-mouse CD103 (integrin alpha IEL chain; clone 2E7).

Following incubation with these primary antibodies, sections were washed in TNT buffer (three cycles, each 5 min). Biotin-conjugated mouse anti-rat IgG1/IgG2a (BD PharMingen) or biotin-conjugated mouse anti-hamster IgG mixture (BD PharMingen) was added (final dilution of each = 1:100 in TNB blocking buffer). After a 30-min incubation with the secondary antibodies at room temperature, sections were treated three times with TNT wash buffer (5 min/wash cycle). The sections were then incubated for 30 min at room temperature with streptavidin-horseradish peroxidase (PerkinElmer Life Sciences; 1:1000 in TNB) followed by three washes of 5 min each in TNT buffer. The final steps consisted of (i) adding biotinyl-tyramide (PerkinElmer Life Sciences; diluted 1:100 in 1× amplification diluent from the same manufacturer) for 10 min; (ii) washing three times with TNT buffer (5 min/cycle); (iii) incubating the section with indocarbocyanine (Cy3)-conjugated streptavidin (PerkinElmer Life Sciences; diluted 1:500 in TNB) for 30 min, and (iv) performing three final rinses in TNT buffer. Two controls were performed to verify the specificity of the signals produced: (i) direct amplification of endogenous peroxidase activity alone without the addition of primary or secondary antibodies but with the addition of biotinyl-tyramide; (ii) direct amplification of endogenous peroxidase activity followed by omission of each primary antibody but with inclusion of all other steps and reagents.

Only well oriented jejunal crypt-villus units were scored. "Well oriented" was defined as sectioned parallel to the crypt-villus axis with an unbroken epithelial column extending from the crypt base to the villus tip. The data were compiled as the number of IELs of a particular type per 1000 villus epithelial cells or per 100 crypt epithelial cells. A minimum of 100 jejunal crypt-villus units were scored per mouse. Data obtained with each antibody from all mice of a given genotype (germ-free or conventional) were averaged.

Multilabel immunohistochemical studies were performed on sections of normal and transgenic mouse jejunums using rabbit anti-TAg (1:1000 in PBS-blocking buffer; kindly provided by Doug Hanahan, University of California, San Francisco, CA) and goat anti-BrdUrd (1:1000) (38, 39). Antigen-antibody complexes were detected with Cy3-labeled donkey anti-rabbit Ig and fluorescein isothiocyanate (FITC)-labeled donkey anti-goat Ig (1:500; Jackson ImmunoResearch).

FACS Analysis of IELs-- 6-8-week-old transgenic mice and their normal littermates were sacrificed, and their jejunums were recovered (n = 3 germ-free and 3 conventionally raised mice/genotype/experiment; three independent experiments). Peyer's patches were identified by inspecting the serosal surfaces of the jejunal segment and were then excised. Each jejunal segment was subsequently opened with a longitudinal incision, washed in PBS, and cut into 1-cm pieces that were placed in 40 ml of ice-cold sterile PBS. The pooled segments from all three animals/genotype/experiment were washed five times in PBS (vigorous shaking), allowed to settle by gravity, and resuspended in 25 ml of R2 medium (RPMI 1640 buffer containing 5% fetal calf serum (Sigma), 1 mM sodium pyruvate, 1 mM sodium bicarbonate, 1% nonessential amino acids (Sigma), and 0.1% 2-mercaptoethanol). The mixture was shaken gently for 30 min at 37 °C and then rigorously for 2 min at room temperature. The intestinal segments were allowed to settle by gravity, and the supernatant was collected and passed through a Nytex filter (Becton Dickinson). The flow-through, containing IELs and epithelial cells, was passed over a column of dimethyldichlorosilane-treated glass wool fiber (0.5 g/10-ml syringe) preequilibrated in R2 medium. The flow-through was spun at 1500 × g for 5 min, and the resulting cell pellet, highly enriched for IELs, was resuspended in 10 ml of R2 medium. The suspension was centrifuged at 1500 × g for 5 min, and the pellet resuspended to a final concentration of 107 cells/ml of FACS staining buffer (RPMI, 1% bovine serum albumin (Sigma), 1 mg/ml human IgG (Sigma)).

IELs were stained with the following antibodies in various combinations (all from BD PharMingen; all diluted 1:100 in FACS staining buffer): (i) phycoerythrin (PE)-conjugated hamster anti-mouse alpha beta TCR (beta  chain; clone H57-597); (ii) PE-conjugated hamster anti-mouse gamma delta TCR (delta  chain; clone GL3); (iii) PE-conjugated rat anti-mouse CD8beta .2 (clone 53-5.8); (iv) FITC-conjugated rat anti-mouse CD8alpha (clone 53-6.7); (v) FITC- or PE-conjugated rat anti-mouse CD4 (clone RM4-5); (vi) FITC-conjugated rat anti-mouse CD45 (clone 30-F11); and (vii) biotinylated hamster anti-mouse CD103 (integrin alpha IEL chain; clone 2E7). Biotinylated primary antibodies were visualized with FITC-streptavidin or PE-streptavidin (BD PharMingen). Idiotype as well as secondary antibody alone controls were also performed. Following incubation with these reagents (60-90 min on ice), cells were spun for 5 min at 1500 × g, washed with sterile ice-cold PBS, and examined by flow cytometry (FACScalibur; Becton Dickinson).

Isolation of RNA from gamma delta TCR+ IELs-- gamma delta TCR+ IELs were isolated from jejunal segments that had been removed from 6-week-old germ-free male transgenic mice and their normal littermates. The gamma delta TCR+, CD103+ lymphocyte population was sorted (FACS Vantage; Becton Dickinson), collected in sterile cold PBS, and recovered by centrifugation (1000 × g for 5 min at room temperature). RNA was isolated using the RNAeasy kit (Qiagen) (5 mice/IEL preparation; n = 10 preparations/genotype). RNA was also isolated from intact jejunal segments (n = 10 germ-free mice/genotype).

Laser Capture Microdissection (LCM) of Jejunal Villus Epithelium-- LCM was conducted using jejunal cryosections that had been stained briefly with eosin Y and methyl green. Dissection of villus epithelium was restricted to well oriented crypt-villus units and was accomplished using the PixCell II system (Arcturus; 7.5-µm diameter laser spot), CapSure HS LCM Caps (Arcturus), and protocols described in Ref. 40. ~10,000 jejunal villus epithelial cells were harvested from each germ-free normal and TAg mouse (n = 3 animals/group). RNA was prepared from captured cells from each mouse in each group using the PicoPure RNA Isolation Kit (Arcturus). The concentration of each preparation was defined (RiboGreen RNA quantitation kit; Molecular Probes, Inc., Eugene, OR), and equally sized aliquots from members of a group of animals were pooled.

Analysis of Previously Published DNA Microarray Data Sets-- Data sets of gene expression profiles from gamma delta TCR+ IELs and the alpha beta TCR+ cells were a generous gift from Aude Fahrer and Y-H. Chien (Dept. of Microbiology and Immunology, Stanford University) (33). These data sets were obtained using an early manufactured version of a high density, oligonucleotide-based DNA microarray containing probe sets representing 6352 mouse genes or expressed sequence tag clusters (Mu6K GeneChip; Affymetrix).

We used GeneChip software (version 4.0; Affymetrix) to compute an average fluorescence intensity across all probe sets on the GeneChips prior to conducting pairwise chip-to-chip comparisons (41) of gamma delta TCR+ IEL versus alpha beta T cell transcript levels (the alpha beta TCR+ cell GeneChip was designated as "base line"). We then extracted all mRNAs fulfilling the following selection criteria: (i) called "present" in either the base-line or partner chip; (ii) >= 2-fold difference in transcript level in the two RNA populations (increased or decreased); and (iii) the increase or decrease was reproduced in duplicate GeneChip comparisons

SYBR Green-based Real Time Quantitative PCR (qRT-PCR)-- qRT-PCR was used to examine changes in levels of selected mRNAs in RNAs prepared from the intact jejunums, LCM villus epithelium, and/or sorted gamma delta TCR+ IELs harvested from 6-8-week-old male germ-free normal and TAgWt mice. cDNAs were generated from each pooled RNA preparation (see above) using reagents and protocols described in Ref. 40. cDNA was added to 25 µl of qRT-PCRs containing 12.5 µl of 2× SYBR Green master mix (Applied Biosystems), 900 nM gene-specific primers (see Table I in the supplemental material) and 0.25 units UDP-N-glycosidase (Invitrogen). A melting curve was used to identify a temperature where only the amplicon, and not primer dimers, accounted for the SYBR Green-bound fluorescence (6). Assays were performed in triplicate with an ABI Prism 7700 sequence detector (Applied Biosystems). All data were normalized to an internal standard (glyceraldehyde-3-phosphate dehydrogenase mRNA; Delta Delta CT method, User Bulletin 2, Applied Biosystems). For the gamma delta TCR+ IEL mRNA analysis, 18 S rRNA was used as the internal standard.

    RESULTS AND DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS AND DISCUSSION
REFERENCES

Forced Expression of TAgWt in Villus Enterocytes Causes a Change in the Representation of IEL Subsets within the Small Intestinal Epithelium-- As noted in the Introduction, transcriptional regulatory elements from the Fabpi gene were used to direct expression of TAgWt in small intestinal villus enterocytes of adult FVB/N transgenic mice (Fig. 1A). There was no detectable TAgWt in the crypt epithelium, the mesenchyme underlying crypt-villus units (Fig. 1A), the organized gut-associated lymphoid tissue (Peyer's patch lymphocytes plus smaller submucosal lymphoid aggregates), or in the spleen and thymus (data not shown). Other than villus enterocytes, the only other site of transgene expression was the follicle-associated epithelium overlying Peyer's patches (Fig. 1B). An identical pattern of transgene expression was observed in FVB/N mice from the reference control pedigree containing Fabpi-TAgK107/108 (data not shown).


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  		Fig. 1.   TAgWt expression in the jejunal villus epithelium of conventionally raised adult FVB/N Fabpi-TAgWt transgenic mice. Multilabel immunohistochemical study of a 6-week-old mouse that had received an intraperitoneal injection of BrdUrd (BrdU) 90 min prior to sacrifice. A, section of jejunum stained with rabbit antibodies to TAg, Cy3-labeled donkey anti-rabbit Ig, goat anti-BrdUrd, and FITC-conjugated donkey anti-goat Ig. TAgWt-positive nuclei appear red/orange. BrdUrd-positive nuclei appear green. Co-expression of TAg and BrdUrd produces yellow staining of nuclei (e. g. arrowhead). TAg is not expressed in the crypt epithelium (nuclei are green; arrows). B, TAgWt expression in the follicle-associated epithelium (FAE) overlying Peyer's patches. The section was incubated with antibodies to TAgWt and Cy3-donkey anti-rabbit Ig, resulting in magenta-colored TAgWt-positive nuclei in the follicle-associated epithelium. The lymphoid population underlying the follicle-associated epithelium does not express detectable levels of TAgWt; their nuclei appear blue after counterstaining with bis-benzidine. Bars, 25 µm.

Age-matched 6-8-week-old Fabpi-TAgWt and Fabpi-TAgK107/108 male mice as well as their nontransgenic littermates were given an intraperitoneal injection of BrdUrd, 1.5 h prior to sacrifice (n = 2-3 mice/genotype). Expression of the wild type viral oncoprotein induced villus enterocytes to reenter the cell cycle (Fig. 1A). In contrast, the jejunal villus epithelium and follicle-associated epithelium were not labeled with BrdUrd in either wild type or Fabpi-TAgK107/108 mice (data not shown). To determine whether the proliferative abnormality induced by TAgWt caused a change in the composition or spatial organization of IELs, these cells were isolated from the jejunal epithelium of each group of conventionally raised mice and subjected to flow cytometry. There were no statistically significant differences in the purity of the lymphocyte preparations from each group of mice; >80% of the gated lymphocytes expressed the IEL-specific marker, CD103 (Fig. 2A). The total yield of lymphocytes was similar in each group (5-7 × 107).


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  		Fig. 2.   Expression of TAgWt results in an increase in alpha beta TCR+ and a decrease in gamma delta TCR+ IELs. IELs from the jejunums of conventionally raised 6-8-week-old FVB/N Fabpi-TAgWt, Fabpi-TAgK107/8, and normal mice were analyzed by flow cytometry (n = 3 mice/group/experiment; three experiments). Mean values ± S.E. are plotted. A, gated lymphocytes positive for the IEL-specific marker, CD103. The results reveal no statistically significant differences in the purity of the lymphocyte preparations between groups. B, sorted IELs double positive for CD103 and CD45, a panlymphocyte marker. The total yield of lymphocytes is similar in each group. C, results showing a statistically significant increase in the percentage of alpha beta TCR+ IELs in Fabpi-TAgWt mice (asterisk; p < 0.05 relative to normal mice). D, evidence for a statistically significant decrease in gamma delta TCR+ IELs in Fabpi-TAgWt transgenics.

The majority of the IELs were also positive for CD45, a pan-lymphocyte marker (Fig. 2B). However, there was a statistically significant increase in the fractional representation of alpha beta TCR+ IELs in Fabpi-TAgWt mice compared with their normal littermate controls (p < 0.05; Student's t test) and a statistically significant decrease in gamma delta TCR+ IELs (p < 0.05) (Fig. 2, C and D). In contrast, there were no differences in the percentages of these IEL subsets in Fabpi-TAgK107/8 versus normal animals (Fig. 2, C and D).

We performed a quantitative immunohistochemical study of jejunal crypt-villus units to determine whether the change in alpha beta TCR+ and gamma delta TCR+ IEL representation in Fabpi-TAgWt mice was restricted to the villus epithelium, where the proliferative abnormality was evident, or whether the change extended to the crypt epithelium, where there was no change in proliferative status. An analysis of sections of jejunum indicated that there were no significant differences in the total number of CD103+ IELs per 1000 villus epithelial cells between age-matched Fabpi-TAgWt, Fabpi-TAgK107/108, and normal FVB/N mice (Fig. 3A). However, there was a significant increase in the density of alpha beta TCR+ IELs, and a significant reduction in the density gamma delta TCR+ IELs in TAgWt mice compared with the other two groups (p < 0.05) (Fig. 3, B and C).


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  		Fig. 3.   Quantitative immunohistochemical studies of alpha beta TCR+ and gamma delta TCR+ IELs in conventionally raised transgenic and normal mice. Sections, prepared from the jejunums of 6-8-week-old male Fabpi-TAgWt, Fabpi-TAgK107/108, and normal animals, were stained with antibodies to CD103 and the beta  chain or the delta  chain of TCR. A, evidence that there are no significant differences in the total number of CD103+ IELs per 1000 villus epithelial cells among the three groups. Mean values ± S.E. are shown. B, data indicating that there is a statistically significant increase in the density of alpha beta TCR+ IELs in Fabpi-TAgWt mice (asterisk; p < 0.05 when compared with normal FVB/N mice). C, results showing a statistically significant reduction in gamma delta TCR+ IELs in Fabpi-TAgWt animals.

In the crypt epithelium of conventionally raised normal male 6-8-week-old FVB/N mice, the densities of CD103+, alpha beta TCR+, and gamma delta TCR+ lymphocytes are 10 ± 1, 5 ± 1, and 4 ± 1 per 100 epithelial cells, respectively. There were no statistically significant differences in the numbers of these cells among the three groups of mice, indicating that the proliferative abnormality produced by TAgWt had a "local" effect on villus IELs that did not extend to the crypt.

The Increase in alpha beta TCR+ and Decrease in gamma delta TCR+ IELs Observed in Conventionally Raised TAgWt Transgenics Is Recapitulated in Germ-free Mice-- One question raised by these findings is whether the intestinal microflora was exerting an influence on the composition of the villus IEL population, e.g. from a potential epithelial barrier disruption associated with the engineered proliferative abnormality, or as a direct consequence of a cross-talk between components of the microflora and the epithelium. To address this question, we rederived our pedigrees of Fabpi-TAgWt and Fabpi-TAgK107/108 transgenic mice and their normal littermates as germ-free. The cellular patterns of expression of TAgWt and TAgK107/108 were not affected when the microflora was removed. An epithelial proliferative abnormality extending from the base to the tips of the villi was evident in 6-8-week-old germ-free FVB/N Fabpi-TAgWt but not in Fabpi-TAgK107/108 or normal animals (Fig. 4A plus data not shown).


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  		Fig. 4.   Quantitative immunohistochemical studies of IEL populations in germ-free transgenic and normal mice. A, a section of jejunum, harvested from a 6-week-old male germ-free Fabpi-TAgWt mouse, was stained exactly as described in the legend to Fig. 1. Enterocytes distributed from the base to the tips of jejunal villi have reentered the cell cycle (red, BrdUrd; green, TAg; yellow, colocalization). B and C, immunohistochemical study using antibodies to the delta  chain of TCR, showing that Fabpi-TAgWt mice have a marked reduction in the density of jejunal villus gamma delta TCR+ IELs (magenta) compared with normal littermates. Nuclei are stained blue with bis-benzimide. D-F, sections of jejunum were stained with antibodies specific for CD103 and the beta  chain or the delta  chain of TCR. The number of CD103+, gamma delta TCR+, and alpha beta TCR+ IELs was scored per 1000 villus epithelial cells. Mean values ± S.E. for each subset are plotted (asterisk, p < 0.05 when compared with normal mice; n = 3 animals/group/experiment; three experiments).

Quantitative immunohistochemical studies also disclosed that Fabpi-TAgWt mice, like their conventionally raised counterparts, had a reduction in the density of their villus gamma delta TCR+ IELs when compared with age- and gender-matched FVB/N Fabpi-TAgK107/108 or normal mice (p < 0.05; Fig. 4, B-D). There was also a modest increase in alpha beta TCR+ IELs associated with the TAgWt-induced proliferative abnormality, although the differences were not statistically significant compared with the other two groups of mice (Fig. 4E). The density of all IELs (i.e. CD103+ cells) in the jejunal villus epithelium was similar in all three groups of germ-free mice (Fig. 3F) but severalfold less than in conventionally raised animals (compare Figs. 3A and 4F). As in conventionally raised mice, production of TAgWt in the villus epithelium did not result in any changes in the number of crypt-associated CD103+, alpha beta TCR+, or gamma delta TCR+ IELs (data not shown).

FACS analysis of germ-free jejunal IELs confirmed the results of our quantitative immunohistologic survey and established that TAgWt expression produced a statistically significant increase in alpha beta TCR+ and a significant decrease in gamma delta TCR+ IELs (p < 0.05 in comparison with age-matched normal or TAgK107/108 mice (Fig. 5, A-C).


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  		Fig. 5.   FACS analysis of IELs harvested from the jejunums of germ-free Fabpi-TAgWt mice demonstrates an increase in thymically derived CD4+ and CD8alpha beta + TCR IELs and a decrease in intestinally derived gamma delta TCR+ CD8alpha alpha IELs compared with normal littermates. Mean values ± S.E. are plotted for each group of mice (n = 3 mice/group/experiment; three experiments).

Based on these findings, we concluded that the alterations in these two IEL populations occurred independently of the microflora and were ascribable to the proliferative effects of TAgWt rather than to other functions mediated by regions of the viral oncoprotein located outside of its pRB pocket protein binding domain.

Expression of TAgWt Leads to a Decrease in Accumulation of Intestinally Derived gamma delta TCR+ CD8alpha alpha IELs and an Increase in Thymically Derived alpha beta TCR+ CD8alpha beta IELs-- As noted in the Introduction, intestinal IELs are derived from two sources. The vast majority of alpha beta TCR+ CD4+ and alpha beta TCR+ CD8alpha beta + IELs are thymically derived, whereas all CD8alpha alpha + cells, whether they express alpha beta TCR or gamma delta TCR, are derived from extrathymic sites (42). The phenotype produced by TAgWt-induced proliferation of villus enterocytes in germ-free mice could reflect subtle disruptions of epithelial barrier function with resulting presentation of nonmicrobial luminal antigens (e.g. from the diet) to components of the gut-associated lymphoid tissue. If this were the case, one would expect an increased influx of thymically derived, antigen-induced alpha beta TCR+ IELs.

We addressed this hypothesis in two ways. First, germ-free Fabpi-TAgWt mice and their normal littermates were given an intraperitoneal injection of BrdUrd 1.5 h prior to sacrifice to label intestinal epithelial cells in S phase. Sections of jejunal crypt-villus units were then stained with antibodies to BrdUrd and E-cadherin, the principal epithelial cadherin and an important regulator of cell adhesion in this system (43, 44). Expression of TAgWt and/or entry of jejunal enterocytes into the cell cycle produced no detectable changes in the steady state cellular levels or intracellular compartmentalization of E-cadherin (data not shown; n = 2 germ-free mice/genotype). Second, FACS analysis of jejunal IELs demonstrated that the TAgWt-associated increase in alpha beta TCR+ IELs involved both the CD4 and CD8alpha beta subsets (Fig. 5, D and E). There were no changes in the CD8alpha alpha subtype of alpha beta TCR IELs (data not shown). These findings confirm that the proliferative abnormality engineered in enterocytes is associated with an influx of thymically derived IELs.

FACS analysis also established that expression of TAgWt, but not TAgK107/108, in germ-free villus epithelium leads to a significant (p < 0.05) reduction in intestinally derived gamma delta TCR+ CD8alpha alpha IELs compared with normal littermate controls (Fig. 5F). Immunostaining of intestinally derived gamma delta TCR+ CD8alpha alpha IELs and thymically derived alpha beta TCR+ CD4+ and CD8alpha beta subsets obtained by flow cytometry revealed that they did not contain detectable levels of TAg (data not shown plus see below).

Taken together, these findings demonstrate that TAgWt-dependent reentry of villus enterocytes into the cell cycle produces a specific decrease in the gamma delta TCR+ CD8alpha alpha IEL populations that normally develop in the intestine.

qRT-PCR Analysis of TAgWt-dependent Regulation of IL-7 Expression-- Previous studies have established that the majority of intestinal IELs are maintained in G0 of the cell cycle (45). In addition, some reports have suggested that epithelial cells may act as antigen-presenting cells for induction and activation of these resting IELs (46, 47). Thus, the proliferative abnormality produced by TAgWt could result in suppression of critical trophic factors necessary for the appropriate development and activation of gamma delta TCR+ CD8alpha alpha IELs, leading to their diminution in the epithelium.

IL-7 is one such trophic factor: it is produced by the epithelium and required for generation of mature gamma delta TCR+ IELs (see Introduction). gamma delta TCR+ IELs express the receptor for this cytokine, IL-7R (48). We tested the hypothesis that TAgWt-induced reentry of villus enterocytes into the cell cycle is accompanied by reduced IL-7 expression by performing a qRT-PCR analysis of RNAs isolated from intact jejunum as well as LCM jejunal villus epithelium (Fig. 6A). The results revealed a 12-fold lower steady state concentration of IL-7 mRNA in the intact jejunum of germ-free Fabpi-TAgWt mice compared with germ-free normal littermates and a 4-fold reduction in levels in their LCM villus epithelium (Fig. 6B). Control qRT-PCR assays of LCM epithelial RNA documented a 2-fold reduction in TCRdelta mRNA (Fig. 6C), consistent with the reduced representation of gamma delta TCR+ IELs in transgenic mouse jejunum documented by quantitative immunohistochemical and flow cytometry analyses (Figs. 4D and 5F).


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  		Fig. 6.   qRT-PCR studies of gene expression in laser capture microdissected jejunal villus epithelium obtained from germ-free Fabpi-TAgWt mice and their normal littermates. A, LCM of jejunal villus epithelium. 5-µm-thick cryosections were prepared from jejunal segments of a normal mouse, fixed in 70% ethanol, and stained with eosin Y and methyl green. Bar, 25 µm. B, qRT-PCR showing that forced expression of TAgWt in villus enterocytes is associated with a reduction in IL-7 mRNA levels. Mean values ± S.E. are plotted. Transcript levels were first normalized to glyceraldehyde-3-phosphate dehydrogenase mRNA. The normalized values were then referenced to levels of IL-7 mRNA (arbitrarily set at 1) in LCM jejunal villus epithelial RNA obtained from normal littermate controls. C, qRT-PCR analysis of the effects of TAgWt expression on levels of TCRdelta , TGF-beta 3, Flt3 ligand, and Flt3 receptor mRNAs in LCM villus epithelium (n = 3 mice/experiment; two independent experiments in both B and C).

To address the question of whether the TAgWt-induced proliferative abnormality in villus enterocytes produced changes in gamma delta TCR+ IEL gene expression, we purified these cells, using flow cytometry, from the jejunums of 6-8-week-old germ-free male Fabpi-TAgWt and normal mice (n = 50 mice/group). qRT-PCR studies indicated that the IELs from transgenic mice did not contain detectable levels of TAg mRNA, in agreement with the results of our immunohistochemical studies (see above). IL-7Ralpha mRNA levels were significantly decreased in gamma delta TCR+ IELs from transgenic compared with normal mice (5.5-fold; p < 0.05; Fig. 7).


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  		Fig. 7.   qRT-PCR analysis of IL-7Ralpha , TGF-beta 3, and Flt3 ligand expression in gamma delta TCR+ IELs purified from 6-week-old germ-free Fabpi-TAgWt male transgenic mice and their normal FVB/N littermates. IELs were harvested from 50 mice/group. Each IEL RNA preparation was assayed in triplicate. Mean values ± S.D. are plotted.

Fujihashi et al. (26) used IL-7 knockout mice to show that IL-7 signaling is necessary for IL-7R expression in gamma delta TCR+ IELs and for their subsequent activation and cell division. Based on this observation and the findings described above, we concluded that TAgWt expression in villus enterocytes results in decreased epithelial expression of IL-7, leading to a concomitant decrease in expression of the IL-7 receptor in gamma delta TCR+ IELs, and impeded intestinal development of gamma delta TCR+ CD8alpha alpha IELs.

TAgWt Expression in Enterocytes Is Associated with Reduced Levels of Other gamma delta TCR+ IEL-derived Factors That May Affect IEL:Epithelial Cross-talk-- Analysis of published DNA microarray-based expression profiles of gamma delta TCR+ IELs purified from conventionally raised C57Bl/6 mice (33) allowed us to identify factors whose expression is enriched in gamma delta TCR+ IELs relative to alpha beta TCR+ cells and that may affect epithelial barrier functions and/or important interactions between the epithelium and its population of IELs.

TGF-beta 3-- Recent reports have shown that IL-7 regulates TGF-beta 3 production in fibroblasts (49). Increased expression of TGF-beta 3 leads to enhanced intestinal epithelial cell migration across wound edges in an in vitro model. Neutralizing antibodies to TGF-beta 3 inhibit this process (50). TGF-beta 3 also functions as a signaling factor that induces apoptotic cell death during involution of the mammary epithelium (51), suggesting that it may help regulate epithelial cell census.

qRT-PCR studies disclosed a 10-fold decrease in the steady state level of TGF-beta 3 mRNA in LCM villus epithelium from germ-free Fabpi-TAgWt compared with germ-free normal littermates (Fig. 6C). Furthermore, enterocytic expression of TAgWt is associated with a 262-fold decrease in the concentration of gamma delta TCR+ IEL TGF-beta 3 mRNA (Fig. 7). Together, these results indicate that one consequence of reduced enterocytic IL-7 expression is reduced gamma delta TCR+ IEL-derived TGF-beta 3. Loss of TGF-beta 3 may alter the integrity of the epithelial barrier, contribute to the observed influx of thymically derived alpha beta TCR+ IELs, or help regulate the extent of the p53-independent apoptotic response that occurs in villus enterocytes undergoing unscheduled, TAgWt-induced reentry into the cell cycle.

Flt3L-- The DNA microarray studies revealed that the mRNA encoding the ligand for Fms-like tyrosine kinase-3 (Flt3) is enriched in gamma delta TCR+ IELs compared with alpha beta TCR+ cells (33). The function of Flt3L in gamma delta TCR+ IELs is not known. Flt3 was initially identified in hematopoietic stem cells (52). It is a member of the class III receptor tyrosine kinases that includes c-Kit. Flt3 ligand stimulates proliferation of quiescent as well as cytokine-stimulated hematopoietic progenitors (e.g. see Refs. 53-55). However, this proliferative response is not shared by other progenitors; Flt3 ligand inhibits EGF- and FGF2-stimulated division of neuronal stem cells (56). There is very little information about the regulation of expression of Flt3 ligand and its receptor or their functions in epithelia. One report indicated that Flt3 mRNA is present in mouse bile duct epithelium (57), whereas another identified the transcript in dividing neuroepithelial cells (56).

Our LCM/qRT-PCR studies revealed that the receptor is expressed in normal jejunal villus epithelium. Moreover, expression is down-regulated by the engineered proliferative abnormality; mRNA levels are reduced an average of 7.5-fold in LCM TAgWt compared with nontransgenic epithelium (Fig. 6C).

The qRT-PCR/LCM analysis indicated that the mRNA encoding Flt3 ligand is also reduced in TAgWt epithelium (Fig. 6C). qRT-PCR assays disclosed that TAgWt expression in enterocytes does not have a discernible effect on IEL Flt3 ligand expression (Fig. 7). Since the extent of the reduction in Flt3 ligand mRNA in TAgWt epithelium was severalfold greater than the reduction of gamma delta TCR+ IEL number (5- versus 2-fold), and since IEL Flt3 ligand expression is unaffected by enterocytic TAgWt expression, we concluded that the proliferative abnormality reduces epithelial expression of the ligand. The response of Flt3 and its ligand to changes in the proliferative status of enterocytes raises the possibility that signaling through this system may normally serve to help suppress cell division as members of this lineage execute their terminal differentiation program.

Prospectus-- These studies reveal that an engineered proliferative abnormality in postmitotic enterocytes impedes intestinal development of gamma delta TCR+ CD8alpha alpha IELs and promotes accumulation of thymically educated CD4 and CD8alpha beta subsets of alpha beta TCR+ IELs. Our findings highlight the interdependent contributions of enterocytes and gamma delta TCR+ IELs to intestinal mucosal biology, a point illustrated by the diminution in enterocytic IL-7 expression associated with TAgWt production. The resulting diminution in intestinal maturation of gamma delta TCR+ IELs "robs" the epithelium of IEL-derived factors known or postulated to support epithelial barrier function (e.g. TGF-beta 3). Gnotobiotic FVB/N Fabpi-TAgWt mice provide an environmentally and genetically defined, "sensitized" model system for genetic or pharmacologic tests of the role of enterocyte-derived factors postulated to promote maturation of gamma delta TCR+ IELs, of IEL-derived factors that may affect epithelial barrier function, and/or of microbes or microbially derived products that may influence mucosal biology.

    ACKNOWLEDGEMENTS

We are indebted to Aude Fahrer and co-workers for generously providing the Mu6K GeneChip data sets of IEL gene expression, our colleague Jason Mills for help in analyzing the data sets, and David O' Donnell and Maria Karlsson for superb technical assistance in generating and maintaining the germ-free mice used in this study.

    FOOTNOTES

* This work was supported in part by National Institutes of Health Grant DK30292.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

The on-line version of this article (available at http://www.jbc.org) contains one table.

Present address: Dept. of Pathology, University of Alabama at Birmingham, Birmingham, AL 35233.

|| To whom correspondence should be addressed: Dept. of Molecular Biology and Pharmacology, Box 8103, Washington University School of Medicine, 660 S. Euclid Ave., St. Louis, MO 63110. Tel.: 314-362-7243; Fax: 314-362-7047; E-mail: jgordon@molecool.wustl.edu.

Published, JBC Papers in Press, July 22, 2002, DOI 10.1074/jbc.M205300200

    ABBREVIATIONS

The abbreviations used are: IEL, intraepithelial lymphocyte; TCR, T cell receptor; IL-7, interleukin-7; IL-7R, interleukin-7 receptor; TAg, SV40 large T antigen; TAgK107/8, mutant TAg with Glu right-arrow Lys substitution at positions 107 and 108; FACS, fluorescence activated cell sorting; LCM, laser capture microdissection; qRT-PCR, real time quantitative reverse transcriptase-PCR; Flt3, Fms-like tyrosine kinase 3 receptor; Flt3L, ligand for Fms-like tyrosine kinase 3 receptor; BrdUrd, bromodeoxyuridine; PBS, phosphate-buffered saline; FITC, fluorescein isothiocyanate; PE, phycoerythrin; TGF, transforming growth factor.

    REFERENCES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS AND DISCUSSION
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Z. Sun, P. W. Denton, J. D. Estes, F. A. Othieno, B. L. Wei, A. K. Wege, M. W. Melkus, A. Padgett-Thomas, M. Zupancic, A. T. Haase, et al.
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S. Ishizuka, S. Tanaka, H. Xu, and H. Hara
Fermentable Dietary Fiber Potentiates the Localization of Immune Cells in the Rat Large Intestinal Crypts
Experimental Biology and Medicine, October 1, 2004; 229(9): 876 - 884.
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T. S. Stappenbeck, J. C. Mills, and J. I. Gordon
Molecular features of adult mouse small intestinal epithelial progenitors
PNAS, February 4, 2003; 100(3): 1004 - 1009.
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