PhD

dissertationThe mast cell is one of die major cellular mediators in allergic responses. There are two different types of tissue mast cells in the mouse, the mucosal mast cell and the connective tissue mast cell. With an in vitro culturing system, murine bone marrow mast cells were analyzed for the expression of cytokines, transcription factors, and novel genes. IL-3 derived mast cells (MMC) preferentially express IL-4, IL-10, and EL-13, whereas c-kit ligand derived mast cells (CTMC) do not express these transcripts on a constitutive basis. However, when CTMC were either treated with IL-3 or stimulated by IgE-mediated crosslinking, they were able to upregulate the transcription of these genes. Furthermore, IL-10 protein was shown stored in mast cell granules in preformed state. A lymphoid lineage specific transcription factor, Ikaros, was shown to be constitutively expressed by cultured mast cells. Unlike the T cells which express up to six different forms of Ikaros protein, mast cells only express the protein for form V and VI. However, nuclear extracts from these cells failed to show DNA binding activity when incubated with a DNA binding site from CD3 delta promoter. One the other hand, when increasing mast cell extracts were added to suboptimal amount of T cell nuclear extracts, a DNA binding activity was evident, suggesting that other T cell factors may be required for the complex formation. A differential screening experiment based on rapid PCR amplification was set up to identify genes that are differentially expressed during mast cell differentiation. One gene, Pactolus, was found to be preferentially expressed by CTMC. Two forms of transcripts have been identified, which are generated through alternative splicing. The Pactolus gene is highly homologous to murine P2 integrin subunit. The two isoforms predict two different proteins, with the shorter one encoding a soluble protein and the longer one encoding a transmembrane protein. Polyclonal antisera against a peptide in the cytoplasmic tail of the full length Pactolus protein, but not the preimmune serum, recognized a prominent protein band about 95kD. This band was not present in the spleen despite prolonged exposure. Interestingly, no associating protein could be identified through co-immunoprecipitation, raising the possibility that Pactolus may function alone instead of forming heteroduplex with other adhesion molecules