Continual surveillance of tissues by lymphocytes is necessary to control and meditate systemic immune responses. However, aberrant trafficking of lymphocytes, and other leukocytes, results in inflammation. Understanding lymphocyte extravasation at the molecular level may lead to new therapies for inflammatory diseases. Lymphocyte extravasation and migration to selected microenvironments requires the concerted efforts of adhesion molecules and metalloproteases. MDCs (metalloprotease disintegrin cysteine-rich) are a family of cell surface proteins recently identified in several mammalian tissues. Members of this protein family possess domains that are predicted to have both adhesive and proteolytic functions. We hypothesize that members of the MDC protein family function in leukocyte extravasation. To investigate this, lymphocytes were initially examined for the expression of MDC proteins. We have isolated a novel member of this family, MDC-L, that appears to be specifically expressed by lymphocytes as both a transmembrane and secreted form.
The aims of this project are to examine the expression and function of MDC-L. Both immunohistochemical and flow cytometric approaches will be used to examine the phenotype of MDC-L expressing lymphocytes and the microenvironments each MDC-L form are found. Integrin recognition of the MDC-L disintegrin domain will be investigated using a combination of biochemical methods. Once the identity of the integrin that recognizes MDC-L is determined, a site directed mutagenesis, monoclonal antibody (mAb), and synthetic peptide approach will be used to determine the molecular aspect of MDC-L recognition by that integrin. The mAbs and peptides produced that inhibit integrin binding will be used to investigate the role of MDC-L as an adhesion molecule in in vitro lymphocyte transmigration assays. The substrate specificity of the metalloprotease domain will be determined. Anti-MDC-L mAbs produced during the course of this project and existing inhibitors of metalloproteases will be assessed for MDC-L inhibitory activity. These protease inhibitors will be used to assess the function of the MDC-L metalloprotease in in vitro lymphocyte transmigration assays. Like many matrix metalloproteases, MDC-L may be synthesized as a zymogen that is activated upon removal of the prodomain. The regulation of the metalloprotease will be investigated by recombinant expression of mutant MDC-L constructs and assaying for metalloprotease activity. The results obtained from this project may identify a new class of potential therapeutic targets, such as MDC-L, for intervention of lymphocyte extravasation.
