Lymphocyte infiltrates in the islets of Langerhans, "insulitis", is a characteristic hallmark of immune-mediated diabetes in rodent models and human beings. Strategies to prevent invading lymphocytes from reaching the pancreatic beta-cells have been effective in preventing diabetes in non-obese diabetic (NOD) mice. Recently, much attention has been focused on the regulation of matrix metalloproteinases (MMPs) in diseases ranging from metastatic breast cancer to rheumatoid arthritis because these key enzymes regulate tumor cell migration and lymphocyte migration to sites of inflammation. At present, several broad-spectrum MMP inhibitors are being tested in human phase II and Ill trials to determine whether these drugs can prevent tumor metastasis and rheumatoid arthritis. Similarly, we postulated that MMPs play a role in insulitis in NOD mice and possibly human type 1 diabetes. The studies presented here will determine whether a novel broad-spectrum MMP inhibitor can prevent insulitis and diabetes in NOD mice. Six week-old and eighteen week-old NOD mice will be treated with intraperitoneal injections of an MMP inhibitor and followed for development of diabetes. We will evaluate an early treatment protocol (6 weeks of age) to see if MMP inhibition can prevent insulitis and a late treatment protocol (18 weeks of age) to see if MMP inhibition can prevent diabetes once insulitis has progressed. Furthermore, splenic and pancreatic lymph node T cells will be obtained from NOD mice and challenged in vitro with glutamic acid decarboxylase 65. We will assess the Thl/Th2 profile of lymphocytes isolated from these two anatomic sites and whether MMP inhibition can alter the cytokine profile. In collaboration with the University of Arizona, we will evaluate the role of MMPs and their inhibitors in T-lymphocyte development and migration using an in vitro fetal thymic organ culture (FTOC) model. This system has been called "in vitro IDDM" because NOD thymic T lymphocytes can target and infiltrate NOD pancreatic islets when the two organs are co-cultured. Since MMP inhibition not only regulates cell migration, but apoptosis, we will perform dose and time courses with MMP inhibitors to determine whether this strategy can alter thymic T-lymphocyte selection and maturation. Finally, TNF-alpha can regulate the production of diabetogenic T-cells and regulatory NKT cells bearing both natural killer and T-cell phenotypes (DX5+ cells) in the FTOC model. Cell membrane bound metalloproteinases called ADAMs (A Disintegrin And Metalloproteinase) process membrane-bound pro-TNF-a to the biologically active cytokine. Thus, we will determine whether MMP inhibition can downregulate intrathymic TNF-a production and increase the levels of NKT cells. We will determine levels of CD4+,CD8+, and NKT cells using flow cytometry. We anticipate that these studies will give us supporting data to initiate a human trial to treat pre-type 1 diabetic patients with a broad-spectrum MMP inhibitor.
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