The purpose of this proposal is to study the regulation of macrophage antigen-presenting function from the perspective of IL-1. Defining the function of IL-1 in inflammation and lymphocyte activation requires an in-depth understanding of both its mode of action and the regulation of its expression. The broad, long-term goals of this project are (1) to understand how T cells regulate macrophage function, and in particular, how they initiate the activation of IL-1 gene expression in macrophage, as well as to define transcriptional and post-transcriptional events, and (2) to determine the role(s) for IL-1 in T cell activation, and how the response to IL-1 may be modulated. In this context, IL-1 regulation is important not only in its own right, but additionally as it serves as a model system for following T cell-macrophage communication.
One specific aim of the current proposal is to characterize regulatory pathways leading to the differential expression of two physically-distinct forms of IL-1, the integral membrane protein (mIL-1) and the secreted or soluble form (sIL-1). Experiments are proposed to define the possible transcriptional and translational controls that regulate IL-1 alpha and beta expression. Autoreactive helper T cells and LPS will be compared for induction of IL-1 alpha and beta expression. IL-1 RNA synthesis will be evaluated by dot blotting and Northern analysis, including evaluation of kinetics, rate of transcription (nuclear runoff), half-life (Actinomycin D sensitivity and 3H Uridine incorporation) and efficiency of RNA translation (reticulocyte lysate system). Expression at the protein level will be evaluated by Western blotting. These experiments are designed to reveal mechanisms by which the expression of IL-1 alpha and beta may be differentially controlled, and further evaluate the proposed relationship of mIL-1 to ILL-L alpha, and sIL-1 to IL-1 beta. The goal of the second project in the proposal is to evaluate the basis for the preferential response of selected T cells to mIL-1 or to IL-1 that has been crosslinked on sepharose. These studies will include evaluation of sIL-1 responsive and non-responsive subclones for number, affinity, and turnover of IL-1 receptors (radiolabeled IL-1 binding and determination of fate of bound IL-1), evaluation of IL-1 receptor heterogeneity by affinity crosslinking, and studies on the mechanism of utilization of naturally- or artificially-crosslinked IL-1. The study of autoreactive T cell-induced IL-1 may also be of relevance in providing a model system for the evaluation of some aspects of autoimmunity.
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