Antigen Up-take by Intestinal Epithelial Cells in IBD

Abstract

Table 1. Percentage of the cells stained with CD45 (leukocytes) and EpCAM (epithelial cells) antibodies for flow cytometry analysis of the mucosal fractions obtained from surgical specimens from UC inflamed. UC non-inflamed. CD and NL patients. Statistic comparisons performed between UC I, CD and NL (***p<0.001. **p<0.01 and *p<0.05). Previous work of our group showed that inflammatory bowel disease (IBD) patients could not be orally tolerized against keyhole limpet hemocyanin (KLH) [1]. Our aim is to test in vitro the antigen up-take ability of the same antigen in isolated IECs from IBD patients and non-inflammatory controls. 1. Kraus TA et al. Failure to induce oral tolerance to a soluble protein in patients with inflammatory bowel disease. Gastroenterology (2004) 126: 1771-1778. Surgical specimens were obtained from intestinal resections performed in Mount Sinai Medical Center. Five ulcerative colitis (with inflamed tissue (UC I) and four out of them also with non-inflamed tissue (UC NI)), and five Crohn's disease (CD) patients and 8 normal mucosal samples (NL) were included. Briefly, the protocol of IECs isolation consisted in intense cleaning of surgical pieces with PBS; the mucosa was stripped from the submucosa in small pieces (3-5 mm). The pieces were incubated in DTT (1 mM in RPMI medium) for 15 min followed by 2 incubations of 30 min each in 25 mL of Dispase II (1.5 mg/mL in RPMI) at 37 °C. After washing and centrifugation, IECs were recovered from the supernatant and were counted and adjusted to a final concentration of 106 cells/mL in RPMI (plus antibiotics and Fungizone). 105 cells/well were plated in 96 wells plates in a total volume of 200 μl/well of RPMI with or without 100 μg of KLH. At different time points (0.5, 1, 2, 4 and 18 h) IECs were surface stained for flow cytometry analysis with CD45 (as a marker of leukocytes) and Ep-CAM (as a marker of IECs) antibodies, fixed and permeabilized and intra-cellular stained with an anti-KLH antibody. In 4 UC inflamed, 4 CD and 7 NL samples from same patients MHCII expression was analyzed by flow cytometry. In other set of experiments, after overnight (ON) incubation with or without KLH, MHCII expression was analyzed by flow cytometry in Ep-CAM + cells from 3 NL and 3 UC inflamed samples. Comparisons between groups were statistically analyzed by Mann-Whitney test for unpaired samples. As showed in Table 1, the proportion of leukocytes and IECs in mucosa reflected the inflammatory state of the tissue with increased proportion of leukocytes and decreased levels of IECs in IBD samples. IECs from UC inflamed showed an increased earlier up-take of KLH compared to CD, however CD IECs had larger overnight uptake of KLH than UC inflamed (Fig. 1). The percentage of IECs that are MHCII + is significantly higher in inflamed UC patients compared to NL, although KLH incubation does not have any effect on% of IECs expressing MHCII+ (Fig. 2). The different kinetics of KLH up-take by IECs from CD and UC patients can affect the way through which KLH can be presented to immune cells. Although KLH presence does not seem to be important to IECs expression of MHCII molecules, differences in MHCII expression suggests a difference in the ability of IECs from both types of IBD to stimulate different immune cells subsets.