Interleukin-3 (IL-3) is a colony-stimulating factor which supports the growth land development of hematopoietic cells. Its expression is limited to activated but not resting T lymphocytes, NK cells, mast cells, and thymic epithelial cells. We propose to continue the characterization of the expression and repression in HTLV-uninfected and -infected T-cells and non T-cells by molecular biology techniques. Those sequences important for IL-3 expression will be determined by transient transfection of IL-3 promoter-reporter gene constructs in HTLV-uninfected and infected T cells and in non-T-cell lines. IL-3 promoter constructs will consist !of 5' upstream sequences subcloned in front of the CAT reporter gene, and oligonucleotide site-directed mutants of promoter sequences of these constructs. Enhancer and repressor sequences will be identified by subcloning suspected regulatory regions into a vector containing a heterologous promoter linked to a ,reporter gene. To look for promoter-protein interactions electrophoretic mobility shift assays (EMSA) will be performed using nuclear extracts from T cells, unstimulated and stimulated T cells, HTLV-infected T cells and non-T cells. To differentiate the protein species by molecular weight, UV cross-linking will be performed on retarded bands followed by SDS-PAGE analysis. In order to obtain more 'purified protein fractions, column chromatography using heparin-agarose or oligonucleotide affinity columns will be used, and these fractions will be tested for activity by EMSA. We will continue to characterize IL-3 expression in T cells activated by elevated intracellular cyclic AMP. We have previously shown that increases in intracellular cyclic AMP induce expression of IL-3 in T cells. Using the cAMP analogue, 8-Br-cAMP, dibutyryl cAMP as well as the adenylate cyclase activators PGE(2) and forskolin a dose response curve and a time course of cyclic AMP will on T cells will be developed. IL-3 promoter-reporter gene constructs will be transfected into T cells and stimulated with the cAMP analogue to map the cyclic AMP responsive regions of the IL-3 promoter. In this manner we will characterize the expression of IL-3 in cells in response to conventional T-cell activators and to increases in the intracellular second messenger, cyclic AMP.
