) The cross-linking of immunoglobulin E (IgE) bound to high affinity Fc receptors on the surface of mast cells or basophils triggers much of the inflammatory responses and symptomatology in allergic reactions and extrinsic asthma. The cytokine interleukin 4 (IL-4) mediates primary IgE responses by inducing class switching to IgE, via the transcription factor Stat6. The binding of IL-4 to its receptor on B cells initiates the dimerization, activation, and nuclear translocation of Stat6 so that it binds to IL-4 response elements in the promoter of IL-4 inducible genes. Germline E transcription follows and permits deletional recombination upstream of the heavy chains that encode for IgE. Knockout models have confirmed in vivo that primary IgE class switching depends upon the activation of the transcription factor Stat6. However, the mechanism for IL-4-mediated secondary or memory IgE remains obscured. Understanding this mechanism is important to comprehending the pathophysiology of human clinical allergic disease because it is characterized by repeated exposures to allergens, resulting in even greater IgE production. The overall aim is to determine whether or not the role of IL-4 signaling though Stat6 is an established or memory IgE allergic immune response. The applicant proposes to develop an inducible dominant negative system in order to study the importance of Stat6 the memory IgE response. Dominant negative Stat6 molecules will be generated by either a carboxy terminus truncated Stat6 mutant molecule or a chimeric Stat6/KRAB protein each possessing the hCMV regulatory minimal promoter with upstream tet-operators regulated by the rtTA-encoding gene that becomes activated in the presence of doxycycline. The efficacy of the system will be evaluated in vitro and in vivo in transgenic mice and employed to determine if the expression and induction of dominant negative Stat6 can alter a secondary IgE response. Both the memory allergic response to antigenic stimulation and the polyclonal IgE response will be studied. In addition, the components of the memory response that are affected by Stat6 activity will be analyzed.
