Mast cell differentiation in vitro is accompanied by large-scale phenotypic changes as progenitors become IgE receptor-positive, granulated mast cells. Many of the phenotypic changes occur by expression of gene products unique to mast cells and basophils. Although these features make for an attractive model system for studies of cellular differentiation, the lack of a defined progenitor for mast cells (most investigators use unfractionated bone marrow) and the use of IL-3 to generate long term mast cell cultures has not allowed for a narrowly-focused window on the phenotypic changes. We have defined a late, committed stage in the differentiation of the mast cell progenitor just prior to granulation. This mast cell-committed progenitor does not require IL-3 for differentiation but does require factors provided by fibroblasts from connective tissue such as embryonic skin. This model system provides unique advantages to analyze mastocytogenesis, a process important in the development of diseases of immediate hypersensitivity. First, the in vitro differentiation system will be used to follow discrete changes in late-stage progenitor cells responding to defined factors. Preparations of isolated mast cell-committed progenitors will be phenotyped and triggered with purified fibroblast-derived factor. Cell activiation will be monitored by following the mobilization of intracellular calcium and phosphatidyl inositol turnover. Changes in expression of high affinity IgE receptors, growth factor receptors, histamine biosynthesis, and granule assembly will correlated in sequence with signal transduction. Second, the differentiation system will be used to determine if fibroblasts derived from different sites (mucosal vs. connective tissue) produce different factors affecting mast cell phenotype. Mast cell phenotype will be indicated by expression of neutral proteases and heparin proteoglycan. Third, the factors which cause the transition of the primitive bone marrow progenitor for mast cells to progress to the IL-3 independent committed progenitor will be determined by experiments designed to mimic in vitro the two most likely in vivomilieus that give rise to the committed progenitor: completely degranulated mast cells from the gut mucosa draining into the MLN, or bone marrow progenitors trafficking to the MLN as a primitive progenitor and progressing into committed progenitors in situ.
