Studies in the past decade have resulted in an increased appreciation for the inflammatory underpinnings of asthma. These studies show that asthmatics often have tissue and peripheral blood eosinophilia. T cells play an important role in the generation of eosinophil abnormalities, largely due to their ability to produce interleukin-5 (IL-5). T cells of the helper 2 subtype (Th2) produce IL-5 in response to stimulation by antigens, mitogens and specific cytokines. Among these stimulants, those that elevate intracellular cAMP levels induce IL-5 production by Th2 cells but inhibit cytokine (IL-2 and interferon-gamma) production by Th1 cells. The molecular events involved in the activation of IL-5 gene transcription in Th2 cells and those that prevent its production in Th1 cells are poorly understood. We have demonstrated that: (a) The CLE0 site and the GATA-3 binding sequence in the IL-5 promoter are both necessary to mediate cAMP mediated genetranscription. This is the first demonstration of the importance of the GATA-3 site in any cytokine gene. (b) The NF-AT site does not play a crucial role in the stimulation of IL-5 gene transcription. (c) Antigens and mitogens stimulate IL-5 gene expression via different cis-response elements in the IL-5 promoter. This has led us to hypothesize that: 1. IL-5 gene activation is controlled by combinations of regulatory sequences in the IL-5 gene that are activated in a stimulus-specific fashion and 2. IL-5 gene expression is regulated differently in Th1 and Th2 cells, explaining at least, in part, the different cytokine profiles of these cells. To test this hypothesis, we will use both ex vivo and in vivo approaches. Specifically, we will:
Aim # I: Characterize the Key regulatory DNA Sequences that mediate regulation of IL-5 gene expression in murine Th1 and Th2 cell clones. DNA sequences that mediate regulation of IL-5 gene expression in murine Th1 and Th2 cell clones. DNASE I hypersensitivity assays, footprinting assays, electrophoretic mobility shift assays, and transfection experiments will be performed to characterize the molecular mechanisms that permit IL-5 gene expression in Th2 cells but not in Th1 cells.
Aim # II: Investigate the expression of IL-5 promoter-reporter constructs in T cells isolated from TCR (Beta) transgenic mice from different genetic backgrounds. Data derived from Aim #1 will be compared to similar experiments performed with T cells purified from TCR (beta) transgenic mice from BALB/c (Th2 phenotype) and B10.D2 (Th1 phenotype) backgrounds.
Aim # III: Determine the in vivo importance of the cis-elements defined in Aim # 1. Transgenic mice harboring wild-type or mutated IL-5 promoter sequences driving a reporter (luciferase) gene will be generated. The expression of this gene will be determined in 3 models of airway inflammation and hyperresponsiveness: antigen (ovalbumin)-, hapten (picrylchloride)- or virus-induced. The in vivo role of IL-4 in driving IL-5 gene expression will be determined in IL-4-/- mice. A direct gene transfer method will be used in parallel to characterize the cis-elements that are operative in vivo.
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