Mast cells initiate inflammatory reactions in asthma and allergic diseases. Circulating human progenitor mast cells (hPrMC) migrate to tissues where they mature into mature mast cells (hMC). The effector capabilities of hMC show inter-tissue heterogeneity, reflecting the microenvironmental regulation. Cord blood mononuclear cells cultured in the triad of recombinant human stem cell factor (rhSCF), rhIL-6, and rhIL-10 (SCR/IL-6/IL- 10) develop into hPrMC characterized cytofluorographically by monophasic expression of c-kit and an IL-3 receptor (IL-3R); greater than 70 percent of the hPrMC are immunoreactive for chymase and carboxypeptidase A (CPA) expression. SCF-driven proliferation of hPrMC is augmented by rhIL-6 or rhIL-3, but no rhIL-2, rhG-CSF or rhM-CSF, supporting their commitment to the mast cell lineage. rhIL-3 strikingly and reversibly attenuates the rhSCF-dependent steady-state expression of tryptase mRNA and protein by hPrMC independent of proliferation. An additional attenuating effect of rhGM-CSF on rhSCF-dependent tryptase expression indicates a functional GM-CSFR on hPrMC, while rhIL-9 counteracts the suppressive effects of rhIL-3. The IL-3R is lost with progressive maturation of hPrMC to hMC, which are characterized by uniform, strong metachromasia with toluidine blue dye, and strong immunoreactivity for tryptase and chymase. hMC heterogeneity likely reflects the interactions between cytokine receptors with their respective ligands at critical phases of hPrMC development.
Specific Aim 1 focuses on the surface expression of receptors for T cell-derived cytokines on hPrMC and hMC, including the alpha subunits of IL-3R, IL-4R, IL- 5R, IL-6R, IL-9R, IL-13R, and GM-CSFR; the betac of IL-3R, GM- CSFR and IL-5R; the common gamma subunit of the IL-4R and IL-9R; and c-kit at 3 and 9 wk of development (reflecting homogeneous hPrMC and hMC, respectively) and at a transitional point of 6 wk. The dose-dependent effects of the corresponding cytokines will be examined on cell viability, proliferation, ultrastructural phenotype, and FcepsilonRI-mediated exocytosis.
Specific Aim 2 focuses on the regulation of protease expression in hPrMC and hMC by rhSCF, which induces tryptase, and explores the counteractive effects of rhIL-3 and rhGM-CSF at transcriptional and post- transcriptional levels, with additional studies of rhIL-9 and other T cell-derived growth factors previously implicated in the regulation of mast cell proteases.
Specific Aim 3 focuses on the roles of these growth factors in determining the arachidonic acid metabolism phenotype of hPrMC and hMC for their generation of leukotriene (LT)C4 and prostaglandin (PG)D2 in response to IgE- dependent stimulation. Cytokine-induced alterations in LTC4 or PGD2 generation will be further studied in terms of changes in the level(s) of the functional and immunodetectable proteins and corresponding mRNA transcripts of the 5-lipoxygenase (LO)/LTC4 synthase and the PGH synthase/PGD2 synthase pathways, respectively, of hPrMC and hMC.
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