Our laboratory has concentrated on the purification and characterization of a unique 85 kilodalton immunosuppressive glycoprotein termed uromodulin which was originally isolated from human pregnancy urine. This material blocks antigen specific proliferative assays which are dependent upon T cell proliferation at concentrations of 10-10M. Uromodulin is also a specific inhibitor of interleukin 1 and blocks both thymocyte and IL-1 dependent T cell lines at concentrations of 10-10M. Interestingly Uromodulin is a specific and high affinity ligand for both recombinant IL-1 alpha and IL-1 beta, and it appears that this is the mechanism by which uromodulin is able to regulate the activity of IL-1. We have expanded these studies and recently have been able to sequence over 200 amino acids of native uromodulin. Using this sequence data we have recently isolated what we believe is a full length message for uromodulin. This message has been inserted into several different vectors and is in the process of being expressed in several transient expression systems. Preliminary data suggest that a subline of COS-7 is able to synthesize uromodulin. We have also been characterizing the mechanism of binding of IL-1 to uromodulin and find that IL-1 actually recognizes carbohydrate sequences expressed by uromodulin. A number of studies have been utilized including digestion with endoglucoaminidase F, pronase, and isolation of released fragments by HPLC and high performance thin layer chromatography to further characterize the carbohydrate sequence responsible for binding to IL-1. Clinical studies have also been instituted. We have developed a number of ELISA assays based on several monoclonal antibodies generated in our laboratory and preliminary evidence suggests that uromodulin is elevated in a number of clinical conditions which are associated with elevations of IL-1.
