In vitro presentation of gliadin-derived peptides by different cell lines (1)
The amount and complexity of dietary antigens and microorganisms that challenge the surface of the intestinal epithelia make it as one of the most important tissues in mucosal immunity. Some sequences have been identified as putatively relevant peptides because they are toxic by organ culture studies or they are able to bind to HLA-DQ2 or -DQ8 molecules or to induce specific T- cell proliferation. Antigen transport routes in the intestinal epithelium consist of trafficking inside the enterocyte and on passage through the tight junctions, both of which routes are enhanced by IFN-g. The use of biotin-labelled gliadin as an intact dietary antigen was tested for in vitro analysis of antigen presentation in different cell lines.
Little HLA class II was expressed by unstimulated cells and this was upregulated by IFN- g in THP-1, HT-29 cells and U937. IFN-g had no effect on MHC class II expression by Caco-2, VAVY and DUCAF cells. Incubation for 16 h in medium containing 500 mg/ml of gliadin had no effect on HLA class II expression by any of the cell lines tested.
All cell lines produced a fluorescence signal, presumably due to biotinylated gliadin-derived peptide presentation. In all cases, a higher fluorescence signal was observed in IFN-g-treated cells.
Time course experiments on THP-1 cells incubated with bG (500 ug/ml) showed that detection of biotinylated peptides was time-dependent. Incubation of THP-1 cells for 16 h with different bG concentrations showed that the fluorescence signal follows a dose-dependent response. A strong fluorescence signal was produced by THP-1 cells incubated with bG at 500 ug/ml. This concentration was selected for the subsequent experiments.
THP-1 cells incubated with bG at 4 °C showed a lower fluorescence signal compared to that obtained after incubation with bG at 37 °C
Unfixed THP- 1 cells presented biotinylated compounds in a dose- dependent manner. In THP-1 cells incubated with bG at 500 ug/ml, fixation resulted in a reduction of 60% of the signal observed.
Biotinylated peptides bound to HLA class II could be detected in cell lysates. The signal derived from biotinylated peptides was concentration-dependent.
The transcellular passage of luminal antigens results mainly, in a breakdown of polypeptide antigens, but a minor fraction could pass through the cells intact. Gliadins enter into endocytic compartments and can be processed in the intestinal mucosa of celiac patients. Using purified biotin-labelled a-, b-, g- and w-gliadins, similar results were observed, suggesting that gliadin from different groups were processed in the same manner. Gliadins bind to the IgG Fc fragment.