IgE-activated mast cells generate a large number of cytokines that have proinflammatory and immunoregulatory capabilities that are likely to affect processes that occur in bronchial asthma. However, little is known about the regulation of cytokine production in mast cells. Therefore, the first Specific Aim of this Project will determine the effects of mast cell-regulatory cytokines such as KL, IL-4, IL-9, and IL- 10 on basal and IgE-elicited increases in mast cell-produced cytokines. Immature mast cells cultured from mouse bone marrow in recombinant IL-3 will be exposed to mast cell-regulatory cytokines and their effects on mast cell-produced cytokine transcripts and proteins will be assessed by RNA blots and ELISA analyses, respectively. The contributions of de novo transcription and changes in transcript stability to the steady-state levels of mast cell-produced cytokine transcripts will be assessed by nuclear run-on and Actinomycin D mRNA half-life studies, respectively. The fraction of each mast cell population producing a particular cytokine and the spectrum of cytokines that individual mast cells can make will be assessed by in situ hybridization and immunocytochemistry, respectively. Moreover, the latter two technologies will also be used to assess cytokine production by mature serosal mast cells isolated from the mouse peritoneal cavity, and to study the effects of mast cell regulatory-cytokines on these cells ex vivo. The second Specific Aim of this Project is to determine whether the ability of the immunosuppressive agent FK506 to inhibit cytokine production in IgE-activated mast cells is influenced by the stage of mast cell development and the expression of an intracellular FK506-binding protein, FKBP12. FK506 prevents cytokine transcription in T cells, but immature IL-3-dependent bone marrow culture-derived mast cells are resistant to FK506 and are concomitantly deficient in FKBP12. Using RNA and protein immunoblot techniques, we will measure the expression of FKBP12 and other FK506- binding proteins in mature serosal mast cells and in bone marrow-derived mast cells cultured in mast cell-regulatory cytokines. The ability of FK506 to inhibit IgE-mediated increases in steady-state levels of cytokine transcripts and FKBP12-dependent inhibition of calcineurin phosphatase activity (a target of the FK506-FKBP12 complex) will be measured as functional readouts to be related to the expression of FKBP12 in the mast cells. Finally, mast cell lines transfected to overexpress particular FKBPs will be assessed functionally to link definitively FKBP12 expression with FK506-mediated inhibition of cytokine production in activated mast cells. Overall, the studies in this project are expected to reveal new aspects about the regulation of the production and pharmacologic inhibition of mast cell-produced cytokines.
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