In vitro presentation of gliadin-derived peptides by different cell lines.
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.
Chirdo
Fernando Gabriel
FG
Cátedra de Inmunología, Facultad de Ciencias Exactas, UNLP (1900), cc 116, La Plata, Argentina. fchirdo@biol.unlp.edu.ar
Zwirner
Norberto Walter
NW
Rumbo
Martín
M
Fossati
Carlos Alberto
CA
D001710:Biotin / Q000737:chemistry; D002462:Cell Membrane / Q000187:drug effects / Q000378:metabolism; D004305:Dose-Response Relationship, Drug; D004797:Enzyme-Linked Immunosorbent Assay / Q000379:methods; D005903:Gliadin / Q000737:chemistry / Q000378:metabolism* / Q000494:pharmacology; D000949:Histocompatibility Antigens Class II / Q000187:drug effects / Q000378:metabolism*; D006801:Humans; D007371:Interferon-gamma / Q000494:pharmacology; D007413:Intestinal Mucosa / Q000378:metabolism; D007422:Intestines / Q000166:cytology; D010446:Peptide Fragments / Q000032:analysis / Q000378:metabolism*; D013997:Time Factors; D014407:Tumor Cells, Cultured
D000949:Histocompatibility Antigens Class II; D010446:Peptide Fragments; D001710:Biotin; D007371:Interferon-gamma; D005903:Gliadin