Immunohistochemical Demonstration of Nitrotyrosine, a Biomarker of Oxidative Stress, in Islet Cells of the NOD Mouse

Abstract

Reactive oxygen species and nitric oxide generated within the beta cell or by intraislet immune cells may be major mediators of beta cell death during insulin-dependent diabetes mellitus. Here, the intraislet expression of nitrotyrosine, a biomarker of oxidative damage, and its cellular sources were examined in the islets of NOD mice at various stages of spontaneous and cyclophosphamide-accelerated diabetes. At day 30, nitrotyrosine-positive cells were undetectable; they were rare at day 40, being expressed in only a few beta cells and in macrophages located in the periphery of some islets. At day 90 and at onset of diabetes, an increasing number of macrophages and beta cells were nitrotyrosine positive. In the cyclophosphamide group at day 4, the number of beta cells and macrophages with positive immunolabeling declined slightly from day 0 (day of cyclophosphamide injection = day 90). This pattern of immunolabeling increased gradually by day 7 but increased markedly by days 11 and 14 after cyclophosphamide administration. In the spontaneous and cyclophosphamide groups, macrophages were the predominant source of nitrotyrosine and were present within the intraislet and periislet regions. Nitrotyrosine labeling was observed in a significant number of beta cells but less than in immunolabeled macrophages. We conclude that protein nitration in beta cells and macrophages is an important feature of the diabetogenic process in the NOD mouse. Whether this deleterious process also occurs during the very early stages of the disease and preceding insulitis is unclear.