Expression of MICA in the small bowel mucosa

ALLEGRETTI, YESSICA; GUZMÁN, LUCIANA; CUETO RUA, EDUARDO; DRUT, RICARDO; ZWIRNER, NORBERTO WALTER; CHIRDO, FERNANDO GABRIEL

Trieste, Italia

Workshop; 21st Meeting of the European Working Group on Prolamin Analysis and Toxicity; 2006

European Working Group on Prolamin Analysis and Toxicity

MICA (MHC class I chain-related A) is a non-classical MHC class I polymorphic protein, that is not associated with 2-microglobulin and is not an antigen-presenting molecule. MICA has been shown to be expressed upon infection with intracellular pathogens and as a consequence of malign transformation, a process that has been associated with the DNA damage response. In addition, MICA is expressed in the intestinal epithelium, and also in fibroblasts, endothelial cells, dendritic cells, and activated T cells. MICA has an overall homology with conventional MHC class I molecules of only 30%. The a1 and a2 domains of MICA interact with the activating NKG2D receptor, a molecule that is expressed by CD8+ ab T cells, gd T cells and NK cells. NKG2D, a C-type lectin molecule, is expressed as homodimers that are non-covalently associated with the transmembrane adaptor protein DAP10. This association is critical for the activation of the phosphatidylinositol 3 kinase (PI3K) pathway upon recognition of different ligands of NKG2D. Such engagement triggers cellular cytotoxicity and IFN-g secretion. Previously, quantitative PCR analysis in intestinal biopsy samples failed to demonstrate an increase in the expression of MICA in the intestinal mucosa of coeliac patients ascompared to controls. However, MICA was upregulated when intestinal biopsy samples from treated patients were incubated with gliadin peptides (a p31-49). Additional experimental evidence demonstrated that IL-15 plays an important role in MICA induction by these gliadin peptides and a concomitant stimulation of a cellular cytotoxicity mediated by intraepithelial T lymphocytes. There is increasing experimental evidence for the role of the gluten-specific CD4+ T-cell response in the pathogenesis of coeliac disease. More recently, the participation of innate mechanisms in the intestinal mucosa, involving intraepithelial lymphocytes (IEL) stimulated by gliadin peptides, has also been highlighted. Hue et al. and previous results from our laboratory showed that MICA expression in the enterocytes is mainly intracellular, where MICA appears as aggregates, suggesting a localization in a vacuolar/endosomic compartment. In addition, we observed a high number of MICA positive cells in the lamina propria in our previous study. The aim of this study was to investigate the expression of MICA in enterocytes and cells from the intraepithelial and lamina propria compartments in intestinal biopsy specimens from paediatric patients. Immunohistochemical analysis showed that MICA was expressed in enterocytes as well as lamina propria cells. Surprisingly, in all the cases, MICA seemed to be expressed mainly in the cytoplasm rather than on the cellular surface. In this study, multicolour confocal microscopy was used to assess the pattern of expression of MICA. With this approach, we identified the MICA+ cells in the intestinal mucosa (due to printing limitations, results were described depiction of the images). In normal tissue, the intensity of MICA expression was very low or even negative. Samples with inflammatory signs showed the highest MICA expression in the enterocytes. In patients with atrophy, a long-lasting condition, enterocytes were not labelled at all in some of the tested samples. There were also differences in the intensity of the staining along the villi; however, the most intensively stained cells were commonly found at the top of the villi. Cells from the crypts were also stained in tissues with signs of inflammation. The analysis of the MICA expression in the enterocytes showed that this molecule mainly expressed as large aggregates in an intracellular compartment, in a pattern resembling the endoplasmic reticulum. The intracellular location of MICA in enterocytes remains to be elucidated. Furthermore, a pool of intensively stained MICA+ cells was detected in both the intraepithelial compartment and the lamina propria. To analyze the MICA expression in the intestinal mucosa, biopsy sections were costained with antibodies specific for different lineage markers. The IEL compartment consists of different lymphocytes, the vast majority expressing CD7+. We found that the majority of MICA positive cells from the IEL compartment were CD7+ cells, with only a few of them being CD3+. The number of CD7+ MICA+ cells was high in specimens with evident signs of inflammation. We did not detect MICA expression in NK cells (in the present study we did not analyze gd T cells). Remarkably, lamina propria MICA+ cells exhibited the highest intensity of staining. Labelled cells were observed even in the absence of MICA+ enterocytes. These MICA+ cells were heterogeneous in morphology. In the lamina propria we found a high number of CD3+ cells expressing MICA. The number of these cells was increased dramatically in tissues histologically classified as type I enteropathy or atrophy. According to recent reports [19], lamina propria antigen presenting cells (MHC class II+) comprise two non-overlapping populations: the majority (80%) expresses CD68, and corresponds to macrophages, while the second (20%) are CD11c+ cells that correspond to dendritic cells. Taking this into account, we analyzed the expression of MICA in CD68+ cells and CD1a (a dendritic cell marker). In very few cases, we observed MICA expression in CD68+ or CD1a+ cells. However, due to the very low number of cells detected, this finding requires further analysis using a higher number of samples. Also, we failed to detect B cells (CD20+ cells), but we observed a massive infiltration of plasma cells identified as CD138+ cells, which showed MICA staining. The number of CD138+ MICA+ cells was high in inflamed tissue and atrophy, and the MICA staining in these cells was homogeneously distributed in the cytoplasm. In conclusion it can be stated that there are distinct MICA+ cell populations in the small intestinal mucosa. Enterocytes express MICA mainly in the cytoplasm at very low levels in normal tissue. However, the expression of MICA is high in enterocytes of coeliac patients that show signs of an inflammatory process. However, the mostsignificant finding was the presence of intensively stained cells in both the intraepithelial and lamina propria compartments. Among IELs, the majority of MICA positive cells were CD7+ cells, while in the lamina propria the most relevant MICA positive populations were CD3+ T cells and CD138+ plasma cells. In mucosa with severe enteropathy, the number of MICA+ cells was dramatically high in the intraepithelial as well as lamina propria compartments. The functional role of the MICA expression in these cells remains to be elucidated.