CELLULAR BIOLOGY OF MURINE MAST CELL DEVELOPMENT

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Mast cells are key initiators of inflammatory reactions in allergic diseases and normal hot defense. Circulating progenitor mast cells (PrMC) migrate to tissues where they mature into potent effector cells with considerable inter tissue heterogeneity. This heterogeneity is likely determined both by cognitive and soluble microenvironmental factors, and by developmentally regulated PrMC characteristics, which are not well understood. This project compares PrMC developed (PrMC/Triad), with replicates grown in IL-3 (PrMC/IL-3). Based on a relative ack of granule associated neural proteases and requirements for both SCF and IL-3 for maximal thymidine uptake, PrMC/Triad represent a more primitive PrMC than previously recognized in vitro. Because granule protease content partly determines mast cell effector capability. Specific Aim 1 focuses on the mechanistic basis for the differences in granule protease content between the two PrMC, and explores the basis for cytokine-induced maturation and protease acquisition as determined by steady-state RNA expression for murine mast cell proteases 1,2,4,5,6,7, and 9 *by RNA blot analysis), measurement of the corresponding proteins (by SDS-PAGE immunoblot), and nuclear run-on with measurement of the half-life of each RNA species in each respective PrMC population before and after week-long changes in cytokine provision, including culture in the presence of SCF alone, SCF + IL-10, and the triad of SCF/6/10. Preliminary studies also suggest a novel inductive effect of IL-6 on 5-LO pathway activity, another key mast cell effector system with special relevance to asthma, and suggest a concomitant IL-6 induced suppression of PGD2 generation. Specific Aim 2 therefore aims to establish the basis for these IL-6-mediated effects by comparing PrMC/Triad with PrMC/IL-3 for their IgE-dependent generation of 5-LO pathway products and PGD2, comparing the two PrMC for their content of each constituent 5- LO or PGHS/PGD2S pathway protein by SDS-PAGE immunoblot, RNA blot, and cell-free enzymatic assays, and exploring the mechanistic basis for any alterations in either pathway occurring in response to the above noted changes in cytokine supplementation by the same analyses. Finally, because PrMC characteristics may influence their tissue homing properties in vivo. Specific Aim 3 compares intravenous infusion of PrMC/triad with PrMC/IL-3 for reconstruction of the tissue mast cells mast cell-deficient c-kit w/wv mice, and examines the restoration of jejunal reactive mast cell hyperplasia in response to infection with Trichinella spiralis and the changes in pulmonary function immediately following inhalation challenge of ovalbumin-sensitized reconstituted mice.