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In vitro presentation of gliadin-derived peptides by different cell lines
Fernando Gabriel Chirdo1, Norberto Walter Zwirner, Martín Rumbo
1Cátedra de Inmunología, Facultad de Ciencias Exactas, UNLP (1900), cc 116, La Plata, Argentina. fchirdo@biol.unlp.edu.ar
Background:
Gliadin peptide presentation and T-cell activation are critical events in the pathogenesis of celiac disease. Several studies have been performed to identify the toxic gliadin peptides but the complexity of the antigenic fraction makes this analysis difficult. In this work, an in vitro model for the analysis of gliadin peptide presentation is studied.
Methods:
The human cell lines U937 and THP-1 (monocytic), DUCAF and VAVY (immortalised B cells) and HT-29 and Caco-2 (intestinal epithelial cells) were incubated with biotin-labelled gliadin (bG). FITC-labelled streptavidin was used to detect biotinylated peptides at the cell surface by flow cytometry.
Results:
All cell lines tested showed a fluorescence signal derived from bG, that was highest when cells were stimulated with IFN-gamma for 48 h. Time course experiments performed using THP-1 cells showed that after 4-h incubation, almost a maximal signal can be reached. THP-1 cells incubated at 4 degrees C or after paraformaldehyde fixation showed a substantial signal reduction, suggesting that metabolic activity was necessary for the detection of the maximal fluorescence signal at the cell surface. The presence of HLA class II-bound biotinylated peptides was observed in cell lysates of THP-1 cells incubated with bG.
Conclusions:
In all cell lines tested, a specific biotin-peptide-derived signal was observed. This was increased after IFN-gamma treatment and decreased after fixation or incubation at low temperature. The signal was higher in monocytic and B-cell lines than in the epithelial cell lines. The use of this procedure could be a useful tool to study the in vitro processing and presentation of naturally gliadin-derived peptides.