Attenuation of immune responses, caused by infection, age or genetic abnormality, leads to increased susceptibility to normally harmless pathogens. Conversely, aberrant activation of immune responses, such as in autoimmune diseases, is also severely detrimental to health. Thus, the balance between activation and inactivation of lymphocytes is essential for human well-being. Our long-term objective is to understand the molecular mechanisms that achieve this balance, and thereby design rational strategies to intervene in these processes. In this application we propose to investigate how NF-KB proteins participate in lymphocyte activation and inactivation.
The Specific Aims are as follows: 1) to study for the molecular basis of down-regulation and cytoplasmic tethering of NF-kB proteins upon cessation of an activating signal. These studies will provide insights into the re-establishment of a resting slate after an immune response; 2) to study the mechanisms that govern the timing of activation-induced cell death (AICD) in T cells. We will examine the role of NF-KB in priming cells for AICD, and maintaining cell viability during the effector phase; 3) to characterize tissue-specific modifications of IkBa and the basis for differential association with Rel family members. These objectives will be accomplished by a combination of biochemical and genetic methods. Nuclear import and export of NF-KB will be assayed in yeast and mammalian cells using fluorescence microscopy; mutant yeast strains, and pharmacologic inhibitors will be used to study the role of IKB proteins in cytoplasmic retention and down-regulation of NF-kB. The role of NF-KB in AICD will be examined by uncoupling FasL induction from the induction of death. These studies will also seek a mechanism for the inhibition of AICD by the drug pentoxifylline. Lastly, phosphorylation of IK-Ba and its effects on c-Rel versus p65 association will be analyzed by mutagenesis of IkBa; putative kinases and phosphatase will be identified in vitro.
