Non-inflammatory involution of the mammary gland is critical for subsequent normal lactation cycles. Involution is mediated in part by apoptosis, a tightly regulated program leading to individual cell death. Regardless of the signals inducing apoptosis and the pathways mediating it, recognition and uptake of apoptosing cells by professional and amateur phagocytes is the final common event. Phagocytosis occurs prior to cell lysis, thereby preventing the release of pro-inflammatory and immunogenic intracellular contents, and promoting restoration of normal tissue structure and function in the face of considerable cell death. In spite of its biological importance, clearance of apoptotic cells in vivo is poorly understood. The involuting mammary gland offers a unique opportunity to study the mechanisms mediating clearance of apoptotic cells by amateur phagocytes (mammary epithelial cells), compared to the more well-known professional phagocytes (macrophages). While macrophages are known to mitigate into the gland over the first few days, it is the alveolar epithelial cells engulf apoptotic bodies in early involution. 2. Uptake of apoptosing mammary cells in vitro by mammary epithelial cells, compared to mammary macrophages, will be examined using standard phagocytosis assays in the presence or absence of inhibitors which block known receptors for apoptotic cells. In addition, we will try to identify novel recognition mechanisms used by mammary epithelial cells. 2. Uptake of cells undergoing apoptosis in vivo will be examined by immunohistochemistry using morphometry techniques for data analysis. Specific inhibitors in vivo will be delivered by intra mammary implants. 3. The ability of mammary epithelial cells to carry out involution without macrophages will be studied by clodronate depletion of macrophages and epithelial cells, will be studied in C1q knockout mice. In addition, we will determine whether mammary epithelial cells which have phagocytosed apoptotic cells produce TGFbeta, like macrophages, and/or other mediators to contribute to anti- inflammatory tone during involution. These studies will provide important information about how apoptotic cell clearance, particularly by mammary epithelial cells, contributes to non-inflammatory mammary gland involution.
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