This application requests continuation of support for R37 HL24066 first funded in 1979. The research has focused on proteinases and their regulation throughout the course of this program. Over the last ten years, the work has been narrowed to the study of alpha2-macroglobulin (alpha2M) a broad specificity plasma proteinase """"""""inhibitor"""""""". It is the central tenet of this research that alpha2M should be viewed more as a sensor for proteinase activation than as an inhibitor. Alpha2M-proteinase complexes bind to a number of cell types, activating signaling cascades. Moreover, during proteolytic activation alpha2M may bind a number of non-proteolytic proteins. In 1993 and 1994 we first demonstrated that alpha2M-antigen complexes are taken up by macrophages and presented to T cells. The efficiency of antigen presentation is several hundred to 10,000-fold greater than administering free antigen in complete Freund's adjuvant. This work has significant practical implications in vaccine development. Our ongoing research in this area is focused on HIV and several other key targets. In the current application we will pursue three highly related aims, which will provide the underpinning for such translational applications. Specifically, we propose to: 1) study the role of alpha2M in intracellular antigen trafficking. These studies employ confocal microscopy and FAX as tools for analysis; 2) study the role of alpha2M as a DNA binding protein in immune responsiveness. Recent observations in our laboratory demonstrate for the first time that alpha2M binds to immunoregulatory oligonucleotides in a manner analogous to proteins and these complexes are taken up by macrophages; 3) probe the role of signal transduction by alpha2M in antigen presentation. Other antigen presenting systems require specific patterns of signal transduction similar to those triggered by receptor-recognized forms of alpha2M. We will specifically demonstrate that the activation of these cascades by alpha2M-antigen complexes is required for antigen processing by macrophages. In particular, the PI 3-kinase signaling pathway is critical for antigen presentation. Our previous studies demonstrate that this pathway is activated when receptor-recognized forms of alpha2M bind to macrophage receptors. Future studies will determine whether this pathway also is essential for alpha2M-antigen processing and presentation.
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