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OP0226 Human Mast Cells Engulf and Store Exogenous IL-17A

Background IL-17A plays an important role in rheumatic diseases, like rheumatoid arthritis and spondyloarthritis. Direct analysis of inflamed synovial tissue revealed an abundant presence of IL-17A-positive mast cells. Objectives As mast cells are not known to produce IL-17A in mice, we aimed to investigate the mechanism of IL-17A expression by human mast cells. Methods IL-17A, IL-17F and RORC mRNA and protein expression was assessed ex vivo and after PMA/ionomycine stimulation in primary human mast cells sorted from tonsils. Internalization of exogenous IL-17A was assessed by Western blot, imagestream, live imaging and confocal microscopy. Results Immunohistochemistry and western blot analysis confirmed the presence of IL-17A protein in primary human mast cells. In contrast to T cells, however, mast cells did not express RORC protein, the exclusive transcriptional factor controlling IL-17A expression. Accordingly, IL17A, IL17F, and RORC gene expression was readily detectable in sorted T lymphocytes but not in mast cells, even after ex vivo stimulation. Given the discrepancy between the presence of IL-17A protein and absence of its transcriptional machinery, we investigated the uptake of GFP-fused or 6xhistidin-tagged recombinant IL-17A. Imagestream and Western blot indicated that both primary mast cells and the LAD2 mast cell line engulf and store exogenous IL-17A. Live imaging and confocal microscopy revealed that internalized IL-17A is stored in endocytic vesicles and that this uptake can be blocked by inhibiting receptor-mediated endocytosis. Conclusions Human mast cells do not produce IL-17A but engulf and store exogenous IL-17A from the inflamed milieu. Molecular pathways of IL-17A uptake and eventually release are under investigation. Disclosure of Interest None declared DOI 10.1136/annrheumdis-2014-eular.2566