(Applicant?s Abstract) There is now strong evidence that the Th2 cytokines IL-4 and IL-13 are important in allergic asthma. It is our hypothesis that airway smooth muscle cells may be important targets of these cytokines. Our preliminary data indicate that IL-4 and IL-13 receptors are present on cultured human airway smooth muscle (HASM) cells and that the JAK/STAT, ERK, and PI-3-kinase pathways are each activated by IL-13 and IL-4 in these cells. Furthermore, IL-4 and IL-13 each induce changes in the function of HASM cells, including reductions in the response to beta-agonists, and increases in VCAM and eotaxin expression, although the effects of IL-4 and IL-13 and their signaling patterns differ. The goal of this proposal is to study the biological functions of IL-13 and IL-4 on HASM cells and to test the potential impact of known and new polymorphisms on these functions.
In Aim 1, we will test the hypothesis that IL-4 and IL-13 induce different biological responses in HASM cells. To do so, we will examine the dose and time related effects of U-4 and IL-13 on HASM cell responses to beta-agonists, as well as their effects on VCAM and eotaxin expression.
In aim 2, we will test the hypothesis that IL-4 and IL-13 signaling differ in HASM cells. To do so, we will examine the dose and time related effects of IL-4 and IL-13 on STAT-6, STAT-3, ERK and PI-3-kinase activation using Western blotting and in vitro kinase assays.
In aim 3, we will test the hypothesis that differences in IL-4 and IL-13 signaling mediate the differences in biological responses. We will use an approach combining selective ERK and PI-3-kinase inhibitors, as well as transfection of HASM cells with dominant negative forms of MEK, PI-3 kinase, or STAT-6. Outcome indicators will include isoproterenol (ISO) induced CRE-luciferase activity, HASM cell stiffness responses to ISO, VCAM expression, as well as beta2 receptor, VCAM, and eotaxin promoter activity.
In aim 4, we will test the hypothesis that polymorphisms of the EL-4 receptor which are associated with asthma influence responses of HASM to IL-4 and IL-13. Genornic DNA from HASM cells derived from multiple donors will be genotyped for 6 polymorphisms in the coding region of IL-4Ra. In cells from donors with informative genotypes, we will examine the effects of IL-13 and IL-4 on ISO induced changes in HASM cell stiffness and cAMP formation, VCAM and eotaxin expression, as well as IL-4/IL-13 signal transduction and stratify the results by genotype and haplotype. Understanding the effects and mechanisms of action of IL-13 and IL-4 on HASM cells may lead to new modalities for the treatment of asthma.
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