Strain and age-related changes in the localization of intestinal CD161+ natural killer cells and CD8+ intraepithelial lymphocytes along the longitudinal crypt axis in inbred rats

Using a cell position approach, this study indicates that the frequency of CD161(+) natural killer (NK) cells in the epithelia of DA rats was greater than that of WKAH and F344 rats. We further divided the epithelia into proliferating and differentiated regions according to the localization of BrdU-incorporated cells. Comparison between the different regions indicates that a majority of CD161(+) NK cells were located in the proliferating region. With age, a decline in the number of CD161(+) NK cells and CD8(+) intraepithelial lymphocytes (IELs) was observed in the distal colon, especially in the proliferating region of all three strains. Taken together with our previous report that DA rats have far stronger resistance in the colon to preneoplastic lesion than do other strains, these results indicate that CD161(+) NK cells play an important role in immune-surveillance at the bottom of the crypt.     

Modulation of CD8+ intraepithelial lymphocyte distribution by dietary fiber in the rat large intestine

We studied whether ingestion of dietary fiber modifies the distribution of intraepithelial lymphocytes (IEL) in a physiological condition. Male WKAH rats were fed diets either with fiber (sugar beet fiber or crystalline cellulose, 100 g/kg diet each) or without fiber for 3 weeks. The number of CD8(+), CD4(+), and NKR-P1(+) IEL per epithelial layer in the crypt section of the cecum, proximal colon, and distal colon were scored by immunohistochemical staining. We found that the proportion of CD8(+) IEL was greater in the cecal mucosa and was gradually reduced toward the distal large intestine in general. In contrast, there was no difference in the proportion of CD4(+) and NKR-P1(+) IEL in the large intestine. Dietary sugar beet fiber, but not crystalline cellulose, increased the proportion of CD8(+) IEL, especially in the cecal mucosa, but not the CD4(+) and NKR-P1(+) IEL. Analysis of cecal organic acid concentration confirmed higher concentrations of acetate and butyrate, and lower concentration of succinate and isovalerate, in the cecum of the rats fed sugar beet fiber than other diets. These results indicate that ingestion of some dietary fiber modulates local cell proliferation of a progenitor of CD8(+) IEL or promotes homing of CD8(+) T cells into the large intestinal epithelium, most likely via the fermentation in the luminal contents.     

Treatment of Brucella-susceptible mice with IL-12 increases primary and secondary immunity

Celiac disease is a gluten-induced T-cell mediated autoimmune process that results in the destruction of the intestinal mucosa and is associated with an expansion of CD8(+) CD103(+) TCRalphabeta intraepithelial lymphocytes (IELs) in the damaged epithelium. The role of this IEL population in the pathology is unknown. The aim of this work was to compare the cytokine profile and the cytotoxicity pattern from CD8(+) IEL clones isolated from celiac (CD) and non-celiac (NCD) biopsies. We report that the number of IL-10 producing CD clones was significantly lower (26%) than that obtained from the NCD sample (62%). Instead, IL-2 was produced by more CD (44%) than NCD clones (26%). Cytotoxicity patterns against intestinal epithelial cell lines suggest different functional subsets of CD8(+) IELs. CD clones capable of high cytotoxicity produced IL-2 whereas most cytotoxic NCD IELs produced IL-10. This clonal analysis indicates that an impaired immune regulation in celiac mucosa may be partially attributed to the low generation of regulatory CD8(+) IELs that produce IL-10.     

CD8 alpha alpha-mediated intraepithelial lymphocyte snatching of thymic leukemia MHC class Ib molecules in vitro and in vivo

Thymic leukemia (TL) is a MHC class Ib molecule that interacts with CD8alphaalpha homodimers. CD8alphaalpha is abundantly expressed by intraepithelial T lymphocytes (IELs) located in close proximity to TL-expressing intestinal epithelial cells. In this study, we show that CD8alphaalpha(+) IELs "snatch" TL from the plasma membrane of TL-expressing cells and express TL in its proper orientation on their own cell surface. TL snatching is enhanced by cross-linking of IEL TCRs in a phosphatidylinositol kinase-dependent manner, and results in overall alterations to the IEL cell surface detected by enhanced binding of peanut agglutinin lectin. Induction of bowel inflammation results in the presence of TL on IELs, probably via in vivo snatching, providing the initial evidence for the interaction of CD8alphaalpha IELs with intestinal cells.     

Mouse TCRalphabeta+CD8alphaalpha intraepithelial lymphocytes express genes that down-regulate their antigen reactivity and suppress immune responses

Mouse small intestine intraepithelial lymphocytes (IEL) that express alphabetaTCR and CD8alphaalpha homodimers are an enigmatic T cell subset, as their specificity and in vivo function remain to be defined. To gain insight into the nature of these cells, we performed global gene expression profiling using microarray analysis combined with real-time quantitative PCR and flow cytometry. Using these methods, TCRalphabeta(+)CD8alphaalpha IEL were compared with their TCRalphabeta(+)CD8beta(+) and TCRgammadelta(+) counterparts. Interestingly, TCRalphabeta(+)CD8alphaalpha IEL were found to preferentially express genes that would be expected to down-modulate their reactivity. They have a unique expression pattern of members of the Ly49 family of NK receptors and tend to express inhibitory receptors, along with some activating receptors. The signaling machinery of both TCRalphabeta(+)CD8alphaalpha and TCRgammadelta(+) IEL is constructed differently than other IEL and peripheral T cells, as evidenced by their low-level expression of the linker for activation of T cells and high expression of the non-T cell activation linker, which suppresses T cell activation. The TCRalphabeta(+)CD8alphaalpha IEL subset also has increased expression of genes that could be involved in immune regulation, including TGF-beta(3) and lymphocyte activation gene-3. Collectively, these data underscore the fact that, while TCRalphabeta(+)CD8alphaalpha IEL resemble TCRgammadelta(+) IEL, they are a unique population of cells with regulated Ag reactivity that could have regulatory function.     

Bivariate flow cytometric analysis of murine intestinal epithelial cells for cytokinetic studies

The heterogeneous nature of the small intestine and the lack of methods to obtain pure crypt populations has, in the past, limited the application of standard flow cytometric analysis for cytokinetic studies of the proliferating crypts. We describe a flow cytometric technique to discriminate crypt and villus cells in an epithelial cell suspension on the basis of cell length, and to measure the DNA content of the discriminated subpopulations. Our data indicate that bivariate analysis of a mixed epithelial cell suspension can be used to distinguish mature villus cells, G1 crypt cells, and S-phase crypt cells. In addition, continuous labeling studies suggest that the position of a cell on the cell length axis reflects epithelial cell maturity. We applied this flow cytometric technique to determine the cytokinetic nature of epithelial cells obtained by sequential digestion of the small intestine.     

Smad3 contributes to positioning of proliferating cells in colonic crypts by inducing EphB receptor protein expression

Deficiency of Smad3, an intracellular mediator of TGF-β, was shown to significantly accelerate re-epithelialization of the colonic mucosa. This study was performed to investigate the molecular mechanisms by which Smad3 controls colonic epithelial cell proliferation and crypt formation. Smad3^ex8/ex8 C57BL/6 mice were used in this study and wild-type littermates served as controls. The number of proliferating cells in the isolated colonic epithelium of Smad3^−/− mice was significantly increased compared to that in wild-type littermates. Protein levels of the cell cycle inhibitors p21 and p27 were significantly decreased, while that of c-Myc was increased in the isolated colonic epithelium from Smad3^−/− mice. In the colonic tissue of wild-type mice, cell proliferation was restricted to the bottom of the crypts in accordance with nuclear β-catenin staining, whereas proliferating cells were located throughout the crypts in Smad3^−/− mice in accordance with nuclear β-catenin staining, suggesting that Smad3 is essential for locating proliferating cells at the bottom of the colonic crypts. Notably, in Smad3^−/− mice, there was loss of EphB2 and EphB3 receptor protein expression, critical regulators of proliferating cell positioning, while EphB receptor protein expression was confirmed at the bottom of the colonic crypts in wild-type mice. These observations indicated that disturbance of the EphB/ephrin B system brings about mispositioning of proliferating cells in the colonic crypts of Smad3^−/− mice. In conclusion, Smad3 is essential for controlling number and positioning of proliferating cells in the colonic crypts and contributes to formation of a “proliferative zone” at the bottom of colonic crypts in the normal colon.     

DNAM-1 mediates epithelial cell-specific cytotoxicity of aberrant intraepithelial lymphocyte lines from refractory celiac disease type II patients

In refractory celiac disease (RCD), intestinal epithelial damage persists despite a gluten-free diet. Characteristic for RCD type II (RCD II) is the presence of aberrant surface TCR-CD3(-) intraepithelial lymphocytes (IELs) that can progressively replace normal IELs and eventually give rise to overt lymphoma. Therefore, RCD II is considered a malignant condition that forms an intermediate stage between celiac disease (CD) and overt lymphoma. We demonstrate in this study that surface TCR-CD3(-) IEL lines isolated from three RCD II patients preferentially lyse epithelial cell lines. FACS analysis revealed that DNAM-1 was strongly expressed on the three RCD cell lines, whereas other activating NK cell receptors were not expressed on all three RCD cell lines. Consistent with this finding, cytotoxicity of the RCD cell lines was mediated mainly by DNAM-1 with only a minor role for other activating NK cell receptors. Furthermore, enterocytes isolated from duodenal biopsies expressed DNAM-1 ligands and were lysed by the RCD cell lines ex vivo. Although DNAM-1 on CD8(+) T cells and NK cells is known to mediate lysis of tumor cells, this study provides, to our knowledge, the first evidence that (pre)malignant cells themselves can acquire the ability to lyse epithelial cells via DNAM-1. This study confirms previous work on epithelial lysis by RCD cell lines and identifies a novel mechanism that potentially contributes to the gluten-independent tissue damage in RCD II and RCD-associated lymphoma.     

Characterization of the cell adhesion molecules L1, N-CAM and J1 in the mouse intestine.

To gain insight into the cellular and molecular mechanisms underlying epithelial cell surface interactions in the adult mouse intestine, we have characterized the cell adhesion molecules L1, N-CAM and J1 by immunocytological, biochemical and cell biological methods. Whereas N-CAM and J1 expression was found to be confined to the mesenchymal and neuroectodermally-derived parts of the intestine, L1 was localized in the proliferating epithelial progenitor cells of crypts, but not in the more differentiated epithelial cells of villi. L1 was detected in crypt cells by Western blot analysis in the molecular forms characteristic of peripheral neural cells, with apparent mol. wts of 230, 180 and 150 kd. Aggregation of single, enriched crypt, but not villus cells, was strongly inhibited in the presence of Fab fragments of polyclonal L1 antibodies. These observations show that L1 is not confined to the nervous system and that it may play a functional role in the histogenesis of the intestine in the adult animal.     

Intestinal intraepithelial lymphocyte gamma delta-T cell-derived keratinocyte growth factor modulates epithelial growth in the mouse

Keratinocyte growth factor (KGF) promotes intestinal epithelial growth. To understand the relevance of intraepithelial lymphocyte (IEL)-derived KGF expression on epithelial growth, we used a mouse model of villus atrophy by the administration of total parenteral nutrition, and a model of villus hypertrophy by the creation of a short bowel syndrome. KGF expression was confined to gammadelta-TCR(+) IELs. IEL-derived KGF expression was highest in the crypts, somewhat less in the lower portion of villi, and markedly lower in the upper portion of villi. Total parenteral nutrition administration was associated with a down-regulation of IEL-derived KGF expression, and short bowel syndrome was associated with an up-regulation of IEL-derived KGF expression. In the absence of gammadelta-TCR(+) IEL, using gammadelta(-/-) mice, intestinal epithelial cell proliferation decreased in control, and in both mucosal atrophy (22% decline) and mucosal hypertrophy (14%) models. These results show that KGF from IELs is an important factor for maintenance of intestinal epithelial cell proliferation and villus growth.     

Intestinal CD8 alpha alpha and CD8 alpha beta intraepithelial lymphocytes are thymus derived and exhibit subtle differences in TCR beta repertoires

Intraepithelial lymphocytes (IEL) of the small intestine are anatomically positioned to be in the first line of cellular defense against enteric pathogens. Therefore, determining the origin of these cells has important implications for the mechanisms of T cell maturation and repertoire selection. Recent evidence suggests that murine CD8 alpha alpha intestinal IELs (iIELs) can mature and undergo selection in the absence of a thymus. We analyzed IEL origin by cell transfer, using two congenic chicken strains. Embryonic day 14 and adult thymocytes did not contain any detectable CD8 alpha alpha T cells. However, when TCR(+) thymocytes were injected into congenic animals, they migrated to the gut and developed into CD8alphaalpha iIELs, while TCR(-) T cell progenitors did not. The TCR V beta 1 repertoire of CD8 alpha alpha(+) TCR V beta 1(+) iIELs contained only part of the TCR V beta 1 repertoire of total iIELs, and it exhibited no new members compared with CD8(+) T cells in the thymus. This indicated that these T cells emigrated from the thymus at an early stage in their developmental process. In conclusion, we show that while CD8 alpha alpha iIELs originate in the thymus, T cells acquire the expression of CD8 alpha alpha homodimers in the gut microenvironment.     

Recognition of double-stranded RNA by TLR3 induces severe small intestinal injury in mice

The role of TLRs on intestinal epithelial cells (IECs) is controversial, and the mechanisms by which TLRs influence mucosal homeostasis are obscure. In this study, we report that genomic dsRNA from rotavirus, and its synthetic analog polyinosinic-polycytidylic acid (poly(I:C)), induce severe mucosal injury in the small intestine. Upon engaging TLR3 on IECs, dsRNA triggers IECs to secrete IL-15, which functions to increase the percentage of CD3+NK1.1+ intestinal intraepithelial lymphocytes (IELs) and enhances the cytotoxicity of IELs. Moreover, The CD3+NK1.1+ IELs are proved as CD8alphaalpha+ IELs. These results provide direct evidence that abnormal TLR3 signaling contributes to breaking down mucosal homeostasis and the first evidence of pathogenic effects mediated by CD8alphaalpha+ IELs. The data also suggest that genomic dsRNA may be involved in the pathogenesis of acute rotavirus gastroenteritis.     

Variation in crypt size and its influence on the analysis of epithelial cell proliferation in the intestinal crypt.

The standard model of epithelial cell renewal in the intestine proposes a gradual transition between the region of the crypt containing actively proliferating cells and that containing solely terminally differentiating cells (Cairnie, Lamerton and Steel, 1965 a, b). The experimental justification for this conclusion was the gradual decrease towards the crypt top of the measured labeling and mitotic indices. Recently, however, we have proposed that intestinal crypts normally undergo a replicative cycle so that at any time in any region of the intestine, crypts will be found to have a wide range of sizes. We show here that if this intrinsic size variation is taken into account, then a sharp transition between the proliferative and nonproliferative compartments of individual intestinal crypts is consistent with the labeling and mitotic index distributions of mouse and rat jejunal crypts. Thus there is no need to invoke the region of gradual transition from proliferating to nonproliferating cells as is done in the standard model. The position of this sharp transition is estimated for both the mouse and rat. Experiments to further test our model are suggested and the significance of the results discussed.     

Endoplasmic reticulum stress response promotes cytotoxic phenotype of CD8αβ+ intraepithelial lymphocytes in a mouse model for Crohn's disease-like ileitis

Endoplasmic reticulum (ER) unfolded protein responses (UPR) are implicated in the pathogenesis of inflammatory bowel disease. Cytotoxic CD8αβ(+) intraepithelial lymphocytes (IEL) contribute to the development of Crohn's disease-like ileitis in TNF(ΔARE/+) mice. In this study, we characterized the role of ER-UPR mechanisms in contributing to the disease-associated phenotype of cytotoxic IEL under conditions of chronic inflammation. Inflamed TNF(ΔARE/+) mice exhibited increased expression of Grp78, ATF6, ATF4, and spliced XBP1 in CD8αβ(+) IEL but not in CD8αα(+) IEL or in lamina propria lymphocytes. Chromatin immunoprecipitation analysis in CD8αβ(+) T cells showed selective recruitment of ER-UPR transducers to the granzyme B gene promoter. Heterozygous Grp78(-/+) mice exhibited an attenuated granzyme B-dependent cytotoxicity of CD8αβ(+) T cells against intestinal epithelial cells, suggesting a critical activity of this ER-associated chaperone in maintaining a cytotoxic T cell phenotype. Granzyme B-deficient CD8αβ(+) T cells showed a defect in IL-2-mediated proliferation in Grp78(-/+) mice. Adoptively transferred Grp78(-/+) CD8αβ(+) T cells had a decreased frequency of accumulation in the intestine of RAG2(-/-) recipient mice. The tissue pathology in TNF(ΔARE/+) × Grp78(-/+) mice was similar to TNF(ΔARE/+) mice, even though the cytotoxic effector functions of CD8αβ(+) T cells were significantly reduced. In conclusion, ER stress-associated UPR mechanisms promote the development and maintenance of the pathogenic cytotoxic CD8αβ(+) IEL phenotype in the mouse model of Crohn's disease-like ileitis.     

Oligoclonality of rat intestinal intraepithelial T lymphocytes: overlapping TCR beta-chain repertoires in the CD4 single-positive and CD4/CD8 double-positive subsets

Previous studies in humans and mice have shown that gut intraepithelial lymphocytes (IELs) express oligoclonal TCR beta-chain repertoires. These studies have either employed unseparated IEL preparations or focused on the CD8+ subsets. Here, we have analyzed the TCR beta-chain repertoire of small intestinal IELs in PVG rats, in sorted CD4+ as well as CD8+ subpopulations, and important differences were noted. CD8alphaalpha and CD8alphabeta single-positive (SP) IELs used most Vbeta genes, but relative Vbeta usage as determined by quantitative PCR analysis differed markedly between the two subsets and among individual rats. By contrast, CD4+ IELs showed consistent skewing toward Vbeta17 and Vbeta19; these two genes accounted collectively for more than half the Vbeta repertoire in the CD4/CD8 double-positive (DP) subset and were likewise predominant in CD4 SP IELs. Complementarity-determining region 3 length displays and TCR sequencing demonstrated oligoclonal expansions in both the CD4+ and CD8+ IEL subpopulations. These studies also revealed that the CD4 SP and CD4/CD8 DP IEL subsets expressed overlapping beta-chain repertoires. In conclusion, our results show that rat TCR-alphabeta+ IELs of both the CD8+ and CD4+ subpopulations are oligoclonal. The limited Vbeta usage and overlapping TCR repertoire expressed by CD4 SP and CD4/CD8 DP cells suggest that these two IEL populations recognize restricted intestinal ligands and are developmentally and functionally related.