Uptake and presentation of antigen to T cells by primary colonic epithelial cells in normal and diseased states  Grzegorz W. Telega, *,‡, Daniel C. Baumgart, *, Simon R. Carding, *  Gastroenterology  Volume 119, Issue 6, Pages 1548-1559 (December 2000) DOI: 10.1053/gast.2000.20168 Copyright © 2000 American Gastroenterological Association Terms and Conditions

Fig. 1 Microscopic appearance of murine CECs. (A) The morphology and purity of CECs isolated from C57BL/6 mice were evaluated by bright-field illumination. Detection of alkaline phosphatase activity by (B) incubation with a phosphatase substrate, and immunohistochemical staining with (C) a rabbit antidesmin antibody and (D) a monoclonal anti-CD45 antibody. More than 95% of the isolated cells were positive for cytokeratin, 60% for alkaline phosphatase, and fewer than 5% for CD45. The results are representative of those obtained from more then 10 experiments. (Original magnification 250×.) Gastroenterology 2000 119, 1548-1559DOI: (10.1053/gast.2000.20168) Copyright © 2000 American Gastroenterological Association Terms and Conditions

Fig. 2 Flow-cytometric analysis of freshly isolated CECs. Cells from (A–C) C57BL/6-IL-2+/+ or (D) C57BL/6-IL-2−/− mice were stained with (A) control antibodies of irrelevant specificity, (B) FITC- anticytokeratin and PE–anti-CD45 antibodies, or (C and D) FITC-anticytokeratin and PE–anti-MHC II antibodies. The percentage values shown in the quadrants of each plot represent the frequency of cells staining positive with the antibodies. The results are representative of more than 10 independent experiments. Gastroenterology 2000 119, 1548-1559DOI: (10.1053/gast.2000.20168) Copyright © 2000 American Gastroenterological Association Terms and Conditions

Fig. 3 Uptake of FITC-OVA by freshly isolated murine C57BL/6 CECs. CECs were pulsed with FITC-OVA (30 μmol/L) for (C) 1 or (A and B) 4 hours, washed extensively, and evaluated microscopically under (A) phase-contrast or (B and C) UV illumination. The results are representative of those obtained from more than 5 experiments. Original magnifications: A and B, 250×; C, 600×. Gastroenterology 2000 119, 1548-1559DOI: (10.1053/gast.2000.20168) Copyright © 2000 American Gastroenterological Association Terms and Conditions

Fig. 4 Release of intracellular FITC by CECs pulsed with FITC-OVA. CECs from C57BL/6 mice were incubated with either media alone or media containing 1000 μmol/L FITC-OVA at 37°C or 4°C for 1 hour. The cells were then washed, pelleted, and resuspended in either isotonic (ISO-PBS) or hypotonic (HYPO-PBS) saline, and cell-free supernatants were analyzed for the presence of FITC. Values represent the MFI ± SEM as determined by flow-cytometric analysis (see Material and Methods). The results are representative of those obtained from 3 independent experiments. Gastroenterology 2000 119, 1548-1559DOI: (10.1053/gast.2000.20168) Copyright © 2000 American Gastroenterological Association Terms and Conditions

Fig. 5 Flow-cytometric analysis of antigen uptake by murine CECs. CECs freshly isolated from adult C57BL/6 mice were pulsed with FITC-OVA for 16 hours and analyzed by flow cytometry. (A) Light-scatter profile of CECs. (B) Fluorescence profile of all viable CECs. The shaded curve represents CECs cultured with media alone, and the open curves represent CECs pulsed with 10 μmol/L (solid line) and 1000 μmol/L (dashed line) FITC-OVA. The results are representative of those obtained from more than 9 experiments. Gastroenterology 2000 119, 1548-1559DOI: (10.1053/gast.2000.20168) Copyright © 2000 American Gastroenterological Association Terms and Conditions

Fig. 6 Requirements for antigen uptake by IECs. The effect of (A) fixation, (B) incubation time, (C) temperature, and treatment with (D) colchicine or (E) cytochalasin B on the uptake of FITC-OVA by CECs from adult C57BL/6 mice was determined by flow cytometry as described in Materials and Methods; values represent MFI ± SEM. (F) Antigen uptakes by CECs and SIECs from the same animal were also compared. The results are representative of at least 3 independent experiments each. Gastroenterology 2000 119, 1548-1559DOI: (10.1053/gast.2000.20168) Copyright © 2000 American Gastroenterological Association Terms and Conditions

Fig. 7 Antigen uptake by IECs from IL-2−/− mice and the effect of IL-2. (A) CECs and (B) SIECs isolated from C57BL/6-IL-2−/− mice with mild or moderate colitis (see Materials and Methods) were pulsed with FITC-OVA for 16 hours, and uptake was determined as described in Materials and Methods. As a control, CECs from wild-type (C57BL/6-IL-2+/+) littermates were also used. (C) CECs from IL-2−/− mice with moderate colitis were incubated with FITC-OVA for 16 hours at 37°C in the presence or absence of recombinant IL-2. The results are representative of those obtained from 3 independent experiments. (A) *P < 0.05 comparing values obtained for CECs from IL-2+/+and both groups of IL-2−/− mice; **P < 0.05 comparing values obtained for CECs from IL-2−/− mice with mild vs. moderate colitis. (B) *P < 0.05 comparing values obtained for SIECs from IL-2+/+and both groups of IL-2−/− mice. (C) *P < 0.05 comparing values obtained for control media vs. IL-2 (30 and 100 U/mL)–treated cultures pulsed with 1000 μmol/L of antigen. Gastroenterology 2000 119, 1548-1559DOI: (10.1053/gast.2000.20168) Copyright © 2000 American Gastroenterological Association Terms and Conditions

Fig. 8 Presentation of OVA to an OVA/I-Ab specific to CD4+ T-cell hybridoma by CECs and SIECs. IECs obtained from adult C57BL/6 mice were cultured for 6 days in Matrigel-coated wells of 24-well plates before being pulsed with FITC-OVA. Some preparations of IECs were depleted of CD45+ cells before culture (CD45− EC). (A and B) Cultures were pulsed with increasing amounts of OVA for 16 hours, washed, and then incubated for 24 hours at 37°C with the OVA/I-Ab specific to CD4+ T-cell hybridoma, KZO, transfected with an NFAT-lacZ reporter gene construct. Control cultures contained fixed IECs, IECs, or T cells alone; IECs and T cells in the absence of antigen; or T cells plus antigen in the absence of IECs. The results are representative of 3 independent experiments. (C) CEC cultures pulsed with different concentrations of OVA were incubated with T cells and either an anti–MHC class II or control antibody for 24 hours. (D) CEC cultures established from colonic mucosal samples of C57BL/6-IL-2−/− mice with mild or moderate colitis or from C57BL/6-IL-2+/+ littermates were pulsed with varying concentrations of OVA at 37°C for 16 hours before coculture with T-cell hybridoma for 24 hours. T-cell hybridoma activation and lacZ activity were determined spectrophotometrically using the chromogenic substrate and measuring absorbance at 595 nm (OD 595). *P < 0.05, **P < 0.01 comparing CECs and SIECs at OVA concentrations of 30 and 100 μmol/L, respectively. Gastroenterology 2000 119, 1548-1559DOI: (10.1053/gast.2000.20168) Copyright © 2000 American Gastroenterological Association Terms and Conditions