Protein Profiling of the Secretome of FcεRI Activated RBL-2H3.1 Cells

Background: Secretory proteins of IgE receptor activated mast cells and basophils play a pivotal role in the generation of immediate and long term immune responses in allergy and type I hypersensitivity. Objective: The present study aims to generate a 2-D map and profile of proteins secreted from a high secretory variant of the rat basophilic leukemia cell line, RBL-2H3.1, which in view of the difficulty associated with gaining adequate numbers of pure primary mast cell and basophiles, represents an accepted model system for the study and standardization of the methodology to characterize the secretome of these cell types. Methods: A 2-D map of secretory proteins was generated by 2-D PAGE and a shotgun mass spectrometric approach carried out for protein identi fication. Results: Study resulted into identification of 299 proteins released from resting and IgE receptor activated RBL-2H3.1 cells after 90 s, 30 min and 3 h antigen challenge. Further sequence analysis identified ~53% of total proteins as secretory proteins which could be attributed to classical and non-classical secretory pathways. Additionally, functional classification of classic secretory proteins verified the presence of proteins belonged to cytokines, receptors, membrane proteins, lysosomal proteins and proteins associated with specific sub-cellular localizations such as endoplasmic reticulum, mitochondria, nucleus, cytoplasm and ribosome. According to this data the presence of some secretory proteins such as cytokines (e.g. MCP-2, PF-4, CSF-1 and TGF-β1) are all subject to Ag challenge which may point to their importance toward pathogenesis in allergic diseases. Conclusion: In view of both a beneficial and adverse role of mast cell mediators in health and disease, an identification of temporal changes in the secretory pattern may form the basis for future tailor made intervention strategies that may enable us to harvest the therapeutic potential inherent in mast cell exocytosis while inhibiting/attenuating negative outcomes.