Abstract

Most studies of injury and repair to mucosal tissue have used nonspecific mediators to induce injury. Damage to the mucosal epithelium resulting from chemical or radiation treatment associated with cancer therapy may fall into this category of injury. When such treatments are applied, it is generally not possible to predict or control the extent of possible injury. This fact makes analysis of inductive and reparative processes difficult. In addition, the role of the immune system in the etiology and subsequent healing of mucosal tissue following cancer therapy with or without bone marrow transplantation remains unclear. To study tissue- and antigen-specific immune damage of intestinal mucosal tissue, we generated transgenic mice that express a nominal antigen exclusively in intestinal epithelial cells. The transfer of antigen-specific CD8 T cells with concomitant virus infection resulted in the destruction of intestinal epithelial cells and disease. The destructive phase in some cases was followed by complete recovery and tolerance induction. This model will provide a system that can be regulated for analysis of the mediators of mucosa-specific tissue damage and repair.

The intestinal mucosal immune system is composed of inductive and effector sites. Peyer's patches (PPs) and mesenteric lymph nodes (MLNs) serve as sites for antigen presentation and primary activation of naive T and B cells. After activation in these sites, some lymphocytes then migrate to tertiary tissue sites, including the epithelium, the lamina propria (LP), and the effector sites of the intestinal mucosa (1,2). Numerous activated CD4 and CD8 T cells, as well as B and plasma cells, can be found within the LP (3–7). Plasma cells in the LP mainly produce immunoglobulin A, which is subsequently actively transcytosed across the epithelium by a receptor-mediated process (8). The majority of this antibody is presumed to be specific for bacterial antigens. Similarly, in normal situations, the activated and memory T cells residing in the LP and the epithelium may have been initially primed by antigens derived from normal flora. Indeed, in germ-free mice, the intestinal immune system is poorly developed and contains severely reduced numbers of lymphocytes with reduced effector function (6,9). Thus, colonization of the gut with normal flora is, in part, responsible for the formation of the mucosal immune system tissue. This process is, in effect, a symbiotic relationship between bacteria and the host in which the latter gains considerable benefit through the formation of a normal mucosal immune system poised to respond to pathogenic insult.

The intestinal epithelium is home to a substantial population of T lymphocytes termed “intraepithelial lymphocytes” (IELs). IELs are a complex population of cells that contain several lymphocyte subsets (10). Most small intestinal IELs express CD8, while large intestinal IELs contain larger populations of CD4 and CD4-8-T-cell receptor (TCR) αβ subsets (11,12). TCR usage is also distinct for IELs as compared with lymphocytes in secondary lymphoid tissue, such as that found in the spleen and lymph nodes. Small intestinal IELs in most species examined contain an appreciable population of cells that express TCRγδ [≥50% in some mouse strains (13–15)]. This enigmatic population of lymphocytes has been proposed to be involved in early responses to bacterial infection, perhaps through recognition of antigens presented by nonclassic major histocompatibility complex (MHC) molecules. The precise function of TCRγδ IELs remains elusive, although these cells have been shown to produce keratinocyte growth factor (KGF) when activated, suggesting that they may play a role in homeostasis or repair of the intestinal epithelium (16).

Mucosal epithelial cells are far from being simply a quiescent physical barrier. Rather, intestinal epithelial cells (IECs) are active participants in innate as well as adaptive immune responses (17,18). IECs produce, as well as respond to, a variety of cytokines, including factors involved in lymphocyte development, such as stem-cell factor and interleukin 7 (19,20). IECs also produce chemokines that are likely essential for mucosal lymphoid organ formation and for mounting effective immune responses (21–23). In inflammatory bowel disease, IECs can become targets of a dysregulated immune system (24). It remains unclear whether IECs are victims of direct antigen-specific immune destruction or whether inflammatory cytokines induce damage in a bystander-like fashion. To determine the consequences of antigen-specific T-cell interactions with IECs, we established the model presented here.

Materials and Methods

Mice. C57BL/6J (Ly5.1) mice were purchased from The Jackson Laboratory, Bar Harbor, ME. The OT-I mouse line (25) was from W. R. Heath (Walter and Eliza Hall Institute, Parkville, Australia) and F. Carbone (Monash Medical School, Prahan, Victoria, Australia) and was maintained as a C57BL/6-Ly5.2 line on a RAG–/– background. Intestinal fatty acid-binding protein promoter-truncated ovalbumin (IFABP-tOVA) transgenic mice were created by use of a construct containing the long form of the IFABP promoter (nucleotides –1178 to +28; a gift of J. I. Gordon, Washington University School of Medicine, St. Louis, MO) (26,27), tOVA cDNA (encoding amino acids 138–386), which does not include the signal sequence so the protein remains cytosolic (28), and human growth hormone (hGH) (nucleotides 498–2652; from R. M. Perlmutter, University of Washington, Seattle) (29). An SalI fragment containing these elements was microinjected into C57BL/6-Ly5.1 fertilized eggs by the Transgenic Animal Facility at the University of Connecticut Health Center. To detect the transgene, genomic DNA was analyzed by polymerase chain reaction to identify a 608-base- pair band by using an IFABP-specific 5` primer (5`-GCCATCACACTTGACCCTAA-3`) and an OVA-specific 3` primer (5`-TCAGGCAACAGCACCAACAT-3`). Mice were kept in specific pathogen-free housing and were analyzed between 8 and 10 weeks of age.

RNA analysis. Total RNA from the indicated tissues was isolated by cell lysis with guanidine isothiocyanate followed by 16 hours of centrifugation at 22 °C over a cesium chloride cushion (30). Purification of poly (A) RNA was accomplished by using a Poly(A) Quik messenger RNA (mRNA) Isolation Kit from Stratagene (La Jolla, CA), according to the manufacturer's instructions. One microgram of poly (A) RNA was dot blotted onto a nylon membrane, which was then hybridized with a 32P-labeled OVA cDNA fragment. The blot was stripped and reprobed with a glyceraldehye-3-phosphate dehydrogenase-specific cDNA probe to allow for mRNA quantification. A Molecular Dynamics (Sunnyvale, CA) PhosphorImager was used to quantitate hybridization.

Isolation of lymphocyte populations and adoptive transfer of OVA-specific CD8 T cells. IEL and LP cells were isolated as described previously (12,13). Lymph nodes (LN) and spleens were removed and single-cell suspensions were prepared. Peripheral LN included brachial, axillary, and superficial inguinal lymph nodes. The resulting preparation was filtered through Nitex, and the filtrate was centrifuged at 200g for 5 minutes at 4 °C to pellet the cells. For adoptive transfer, 5 × 105 OT–I/RAG–/–/Ly5.2 pooled LN cells were injected intravenously into Ly5.1 B6 or transgenic hosts. Where indicated, mice were infected 24 hours later by intravenous (IV) injection of 1 × 106 plaque-forming units (pfu) vesicular stomatitis virus (VSV)-encoding OVA (31).

Detection of antigen-specific CD8 T cells with MHC tetramers. Mice were infected by injection of 1 × 106 pfu of VSV-OVA. Six days later, lymphocytes were isolated and VSV nucleoprotein (N)-specific or OVA-specific CD8 T cells were detected by using H-2Kb tetramers containing the N protein–derived peptide RGYVYQGL (32) or the OVA-derived peptide SIINFEKL (33). Peptides were purchased from Research Genetics, Huntsville, AL. Tetramers were produced essentially as described previously (34,35). In brief, H-2Kb containing the BirA-dependent biotinylation substrate sequence (the construct was provided by J. Altman, Emory University, Atlanta, GA) was folded in the presence of human β2-microglobulin and the N or OVA peptide. Biotinylation was performed with biotin–protein ligase (Avidity, Denver, CO). Tetramers were then produced from biotinylated high-pressure liquid chromatography–purified monomers by the addition of streptavidin–allophycocyanin (APC) (Molecular Probes, Eugene, OR).

Flow cytometric analysis. Lymphocytes were resuspended in 0.2% phosphate-buffered saline (PBS), 0.1% bovine serum albumin (BSA), and NaN3 at a concentration of 1 × 106 to 1 × 107 cells/mL followed by incubation at 4 °C for 30 minutes with 100 μL of properly diluted monoclonal antibody (MAb). The MAbs either were directly labeled with fluorescein isothiocyanate (FITC), phycoerythrin (PE), Cy5, APC or were biotinylated. For the latter, avidin-PE-Cy7 (Caltag Laboratories, Burlingame, CA) was used as a secondary reagent for detection. For tetramer staining, cells were first reacted with PE-labeled anti-CD8 (Caltag Laboratories) and FITC-labeled anti-CD11a at 4 °C followed by staining for 1 hour at room temperature with APC-coupled MHC tetramers. After staining, the cells were washed twice with PBS/BSA/NaN3 and fixed in 3% paraformaldehyde in PBS. Relative fluorescence intensities were then measured with a FACSCalibur (Becton-Dickinson, San Jose, CA). Data were analyzed by using WinMDI software (J. Trotter, Scripps Clinic, La Jolla, CA).

Histologic analysis. Duodenum, jejunum, and ileum from experimental animals were fixed in 10% formalin (Fisher Scientific, Pittsburgh, PA). Paraffin-embedded tissue was sectioned and then stained with hematoxylin–eosin. All images were magnified × 200.

Results

With the goal of testing the impact of T-cell reactivity with an IEC-specific antigen, we generated transgenic mice expressing a chicken tOVA gene under control of an IEC-specific promoter (Fig. 1). The IFABP directs protein expression to mature enterocytes but not to crypt epithelial cells (26,27). In addition, IFABP is expressed primarily in the small intestine and is expressed weakly or not at all in the stomach or the large bowel. The hGH gene that contains introns and exons was used to provide signals for the poly (A) addition and to increase in vivo transgene expression (29). When expressed, the OVA lacks a signal sequence, so that the protein remains cytosolic (28). Because of this, IECs should effectively process and present OVA-derived peptides in the context of MHC class I. The mouse strain used for transgenic production was C57BL/6J, an H-2b haplotype strain. The OVA contains an eight amino acid peptide, SIINFEKL, which has been shown to bind to H-2Kb(33). Thus, MHC class I-restricted CD8 T cells of the appropriate specificity should recognize IECs expressing the tOVA.

We examined the OVA mRNA levels in two lines of IFABP-tOVA transgenic mice. There was a striking difference in mRNA levels between the two lines, with the 232–4 line expressing approximately 10-fold more mRNA than the 232–6 line. The expression patterns were similar in the two lines, with the highest concentrations of OVA mRNA present in the ileum. Fig. 2 depicts the relative mRNA levels detected in sections of the intestine of IFABP-tOVA mice. We were unable to detect OVA by immunohistochemistry or western blotting, suggesting that low levels of protein were expressed or the truncated protein was rapidly degraded in the cytoplasm of IECs. We first tested whether CD8 T cells of the transgenic mice were tolerant to OVA. This test was performed by infecting the mice with a recombinant virus containing the OVA gene (VSV-OVA). In normal mice, a robust OVA-specific CD8 T-cell response can be visualized by use of MHC class I tetramers that contain the SIINFEKL peptide. In contrast, few if any OVA-specific CD8 T cells could be found after VSV-OVA infection of IFABP-tOVA mice (36). This result indicated that IEC-expressed antigen had gained access to the systemic immune system. The mechanism by which this occurred is unknown but may be mediated by dendritic cells carrying IEC-derived proteins to the draining lymph nodes and beyond (37). At that point, cross tolerance would be induced to this “self-antigen” via interaction of T cells with dendritic cells that had not been activated by inflammatory signals (see Fig. 4). Further experiments will be necessary to delineate this important pathway for maintenance of self-tolerance to gut-specific protein.

Since the CD8 T-cell compartment of IFABP-tOVA transgenic mice was apparently tolerant to OVA, we were unable to test the reactivity of endogenous T cells with IECs. To circumvent this problem, we used an adoptive transfer system in which naive, OVA-specific TCR transgenic CD8 T cells (called OT-I) were transferred to unmanipulated IFABP-tOVA mice (Fig. 3). The transferred cells are trackable by virtue of differences between host and donor Ly5 alleles. Thus, a monoclonal antibody specific for Ly5.2 (expressed by the donor CD8 T cells but not by host cells) can be used in flow cytometry or immunohistochemistry to detect the OVA-specific CD8 T cells after transfer to the transgenic mice (31). Small numbers (5 × 105) of OT-I cells were injected by the IV route into host transgenic mice; their presence was detected 4 and 5 days later in secondary lymphoid tissues and in intestinal mucosal effector sites (LP and epithelial lymphocytes, respectively). Four days after transfer to the low antigen-expressing line 232–6, few OT-I cells were present in peripheral lymph nodes (PLNs), LP, or IELs (Table 1). In contrast, a large expansion of OT-I cells had occurred at this time point in PP and MLN. By day 5 after transfer, the MLN and PP OT-I populations had declined, whereas significant numbers of OT-I cells were now present in LP and IELs. Four days after transfer of OT-I cells to the high antigen-expressing line 232–4, many donor cells were found in MLN and PP. Unlike in the 232–6 mice, OT-I cells were also found in the LP and IELs at this time point. By day 5 after transfer to 232–4 mice, a massive expansion of OT-I cells had occurred in the LP and IEL compartments. Overall, these results suggested that IEC-derived OVA or OVA fragments are being transferred to PP and MLN, where antigen presentation to T cells can occur. The extent of activation is dependent on the antigen levels, since less activation occurred in the 232–6 (low antigen) compared with the high antigen 232–4 mice. These findings also indicated that activated CD8 T cells did not enter the intestinal mucosa until after activation in PP or MLN, since there were substantial numbers of activated OT-I cells in PP and MLN at least 1 day before their appearance in the mucosa. These data are in agreement with our previous study (2) showing that α4β7 integrins are important for migration of activated OT-I cells into the intestinal mucosa.

Despite the fact that large numbers of antigen-specific CD8 T cells entered the mucosa and apparently responded to antigen expressed by IEC, no overt damage to intestinal tissue was observed by histologic analysis. OT-I cells isolated from the epithelium of these animals exhibited potent lytic activity in a standard chromium release assay in vitro, indicating that they were functional (data not shown). Notwithstanding this fact, the apposition of cytolytic T cells with potential target cells did not result in induction of cellular damage. Therefore, functional “tolerance” had been induced in vivo but not in vitro, at least with regard to lytic activity. To determine whether the inclusion of inflammatory signals would alter the outcome of the response, mice were infected with VSV-OVA at the time of OT-I transfer. Four days after OT-I transfer and concomitant VSV-OVA infection, donor cells had expanded to a much greater extent in the MLN of IFABP-tOVA mice, as compared with their expansion in normal mice. This result showed that endogenous OVA served to potentiate the CD8 T- cell response to the VSV-OVA infection. The virus infection, along with OT-I transfer to the transgenic mice, also resulted in a transient weight loss in 232–6 mice and a sustained weight loss leading to death in most cases in 232–4 mice (36). In the absence of VSV-OVA infection, OT-I transfer did not cause weight loss (Fig. 4), and virus infection alone also had no effect on the weight of the mice or on the mucosal tissue.

Histologic examination of intestinal tissue after OT-I transfer and VSV-OVA infection revealed significant damage to the intestinal epithelium (Fig. 4). In 232–6 mice, destruction of duodenal and jejunal tissue was substantial with loss of epithelial cells, shortening of villi, and elongation of crypts (36). Crypt epithelial cells were not damaged, in keeping with the known expression pattern of IFABP. There was much less damage to ileal tissue and no effect on large intestinal tissue. In 232–4 mice, the disease was much more severe compared with that seen in 232–6 mice (Fig. 4). There was extensive destruction of epithelium in duodenal, jejunal, and ileal sections, although the latter sections were once again the least affected. The majority of these mice died of the disease within 6 days after infection. In those 232–4 mice that survived and in all 232–6 mice, intestinal tissue was histologically normal approximately 2 weeks after the destructive episode. These results clearly demonstrated that CD8 T cells are capable of antigen-specific recognition of IEC and of inducing IEC death.

Conclusions and Recommendations for Future Research

The model described here will be invaluable for analysis of the stages of epithelial cell damage and repair inherent to the intestinal mucosal immune system. The mediators of cell death produced by CD8 T cells that act on IEC can be evaluated with the use of gene knockout mice and blocking antibodies. Moreover, the mechanisms by which tolerance versus autoimmunity are induced are readily testable in this well-defined and tissue-specific system. Future studies will focus on the interaction of CD4 T cells with IEC-expressed antigens and will provide further clues to the workings of mucosal tolerance and immunity. This model also presents a unique system in which factors influencing mucosal repair can be studied and that could have a direct impact on the repair of the epithelial damage inherent to cancer therapy. Since IEC damage can be induced selectively in enterocytes and regulated by antigen levels and, perhaps, by other factors such as T-cell number and virus dose, the system can be easily manipulated to examine the factors, immune or otherwise, of repair of mucosal tissue. By using other mucosa-specific promoters with distinct expression patterns, a further level of control can be attained. In sum, the study of immune-mediated mucosal injury and repair can be investigated in significant detail by using in vivo model systems that allow control of antigen expression and the immune response directed toward that antigen.

Table 1.

Mucosa-specific expansion of antigen-specific CD8 T cells adoptively transferred to IFABP-tOVA transgenic mice*

 Tissue 
Mouse line PLN MLN PP LP IELs 
*PLN = peripheral lymph nodes; MLN = mesenteric lymph nodes; PP = Peyer's patch; LP = lamina propria; IELs = intraepithelial lymphocytes. 5 × 105 Ly5.2 OT−I−RAG−/− T cells were transferred to Ly5.1 IFABP-tOVA transgenic mice (232–6 or 232–4). Four or five days later lymphocytes were isolated from the indicated tissues and the presence of CD8+ OT-I cells was determined by fluorescence flow cytometry. Values indicate means and standard errors of at least six determinations per tissue. 
232–6      
    Day 4 1.1 ± 0.2 11.3 ± 3.5 44.3 ± 6.5 1.8 ± 0.6 1.1 ± 0.3 
    Day 5 0.7 ± 0.3 3.8 ± 0.8 30 ± 1.0 11.4 ± 2.7 8.8 ± 1.2 
232–4      
    Day 4 5.2 ± 0.7 28.7 ± 3.5 49.3 ± 9.1 11.6 ± 1.9 11.7 ± 1.3 
    Day 5 2.6 ± 0.4 15.2 ± 1.9 57.9 ± 4.8 50.7 ± 9.6 60.9 ± 7.4 
 Tissue 
Mouse line PLN MLN PP LP IELs 
*PLN = peripheral lymph nodes; MLN = mesenteric lymph nodes; PP = Peyer's patch; LP = lamina propria; IELs = intraepithelial lymphocytes. 5 × 105 Ly5.2 OT−I−RAG−/− T cells were transferred to Ly5.1 IFABP-tOVA transgenic mice (232–6 or 232–4). Four or five days later lymphocytes were isolated from the indicated tissues and the presence of CD8+ OT-I cells was determined by fluorescence flow cytometry. Values indicate means and standard errors of at least six determinations per tissue. 
232–6      
    Day 4 1.1 ± 0.2 11.3 ± 3.5 44.3 ± 6.5 1.8 ± 0.6 1.1 ± 0.3 
    Day 5 0.7 ± 0.3 3.8 ± 0.8 30 ± 1.0 11.4 ± 2.7 8.8 ± 1.2 
232–4      
    Day 4 5.2 ± 0.7 28.7 ± 3.5 49.3 ± 9.1 11.6 ± 1.9 11.7 ± 1.3 
    Day 5 2.6 ± 0.4 15.2 ± 1.9 57.9 ± 4.8 50.7 ± 9.6 60.9 ± 7.4 
Fig. 1.

Construct used in generation of intestinal fatty acid-binding protein promoter-truncated ovalbumin (IFABP-tOVA) transgenic mice. The SAL I fragment used for production of C57BL/6J transgenic mice contained the long form of the IFABP promoter, tOVA complementary DNA (cDNA)-encoding amino acids 138–336, and the human growth hormone complementary DNA.

Fig. 1.

Construct used in generation of intestinal fatty acid-binding protein promoter-truncated ovalbumin (IFABP-tOVA) transgenic mice. The SAL I fragment used for production of C57BL/6J transgenic mice contained the long form of the IFABP promoter, tOVA complementary DNA (cDNA)-encoding amino acids 138–336, and the human growth hormone complementary DNA.

Fig. 2.

Depiction of the relative concentration of ovalbumin (OVA) messenger RNA in 232 mice along the length of the intestine. The figure is based on data from dot blot analysis of poly(A) RNA from duodenum, proximal jejunum, distal jejunum, and ileum of intestinal fatty acid binding protein promoter-truncated OVA mice by using OVA complementary DNA as a probe.

Fig. 2.

Depiction of the relative concentration of ovalbumin (OVA) messenger RNA in 232 mice along the length of the intestine. The figure is based on data from dot blot analysis of poly(A) RNA from duodenum, proximal jejunum, distal jejunum, and ileum of intestinal fatty acid binding protein promoter-truncated OVA mice by using OVA complementary DNA as a probe.

Fig. 3.

Model system for analysis of immune-mediated intestinal epithelial cell injury.

Fig. 3.

Model system for analysis of immune-mediated intestinal epithelial cell injury.

Fig. 4.

Proposed pathway of acquisition of intestine-specific antigen and induction of immune tolerance or autoreactivity. Antigen is acquired by secondary lymphoid antigen-presenting cells from ova-expressing epithelium of 232 transgenic mice either via absorption from the gut lumen as material from intestinal epithelial cell turnover or from dying epithelial cells and migrate to the Peyer's patches and mesenteric lymph nodes. In the secondary lymphoid tissue, in the absence of inflammation, tolerance induction will occur primarily through deletion for CD8 T cells. When inflammatory signals are present, as in a virus, infection, or dysfunction of regulatory cells, then the response is converted to a productive one that is potentially pathogenic. The photograph on the right shows a hematoxylin–eosin stain of a section from the ileum of a 232–4 mouse 4 days after OT-I cell transfer and concomitant virus infection.

Fig. 4.

Proposed pathway of acquisition of intestine-specific antigen and induction of immune tolerance or autoreactivity. Antigen is acquired by secondary lymphoid antigen-presenting cells from ova-expressing epithelium of 232 transgenic mice either via absorption from the gut lumen as material from intestinal epithelial cell turnover or from dying epithelial cells and migrate to the Peyer's patches and mesenteric lymph nodes. In the secondary lymphoid tissue, in the absence of inflammation, tolerance induction will occur primarily through deletion for CD8 T cells. When inflammatory signals are present, as in a virus, infection, or dysfunction of regulatory cells, then the response is converted to a productive one that is potentially pathogenic. The photograph on the right shows a hematoxylin–eosin stain of a section from the ileum of a 232–4 mouse 4 days after OT-I cell transfer and concomitant virus infection.

Supported by Public Health Service (PHS) grants DK57932 (National Institute of Diabetes and Digestive and Kidney Diseases) and AI41576 (National Institute of Allergy and Infectious Diseases [NIAID]) from the National Institutes of Health, Department of Health and Human Services. V. Vezys was supported by PHS training grant T32-AI07080 from the NIAID.

We thank Dr. Jeffrey Gordon, Washington University, St. Louis, MO, for generously providing the intestinal fatty acid binding protein promoter.

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