Background The emerging role of IL-17A in immune-mediated inflammatory diseases urges characterisation of the cellular source. Numerous recent studies have described the presence of IL-17A-positive mast cells in inflamed target tissues. Since mouse studies have not indicated mast cells as IL-17A producers, we investigated the mechanism of IL-17A expression by human mast cells.
Methods Quantitative PCR and Western blot analysis was performed on mast cells and CD4+ T-cells FACS-purified from human tonsils, ex vivoand after stimulation with PMA/ionomycin. Uptake of exogenous protein was analysed by microscopy, imaging-flowcytometry, live imaging and Western blot. DynaminII-inhibitor was used to block endocytosis.
Results Strikingly, gene expression analysis of highly purified primary mast cells failed to detect both IL-17A, IL-17F and their transcription factor RAR-related orphan receptor C (RORC), even after stimulation. Although RORC was also absent on the protein level, IL-17A was abundantly detected, conflicting with the mRNA data. Subsequent analysis of subcellular location showed the presence of IL-17A in lysosomal storage granules and in early endosomes, suggesting a role for endocytosis. We investigated whether mast cells are capable of internalising exogenous IL-17A in vitro. Microscopy, imaging-flowcytometry and Western blot analysis indicated that LAD2 mast cell line as well as primary mast cells are able to engulf exogenous IL-17A, but not TNFa. Live imaging revealed further that internalisation of IL-17A is specific and can be halted by blocking clathrin-mediated endocytosis.
Conclusion This data strongly suggest that human mast cells do not produce IL-17A, but rather engulf exogenous IL-17A from the inflamed milieu and store it in granules. Molecular mechanisms of IL-17A uptake and the contribution of this process to disease are currently under investigation.
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