The proposed work aims to determine how mononuclear phagocytes (MP) are cleaved from tissues during the resolution of inflammation in vivo (using a murine dermal wound healing model) ant to define the molecules that mediate their clearance. Determining how MP leave sites of inflammation is an important issue as evidence suggests that the inability of MP to be cleared from tissues may contribute to the progress of chronic inflammatory diseases, including atherosclerosis. MP are cleared from in vitro constructs of a human blood vessel wall by migration across endothelium in the basal-to-apical direction. To determine if a similar mechanism operates in vivo, phagocytic retrieval of fluorescent beads applied to wounds will be used to label Mp that enter the wounds. Wounds, blood, draining lymph nodes, and selected organs from mice sacrificed at daily intervals will be analyzed for the presence of labeled MP. Identification of MP will be determined by immunostaining with the monoclonal antibody (mAb) F4/80, a marker for murine macrophages. Using stereology, labeled Mp that leave the wounds by migration into blood or lymph nodes will be quantitated and compared to the number of labeled, apoptotic MP remaining in the dermis (assessed by TUNEL-labeling technique). To search for adhesion molecules required for basal-to-apical transmigration, mAbs will be generated from mice immunized against human endothelial cells. mAbs will be screened for their ability to inhibit basal-to-apical transmigration using an established in vitro assay. Such mAbs will allow cloning and biochemical characterization of the antigen.
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| Muller, W A; Randolph, G J (1999) Migration of leukocytes across endothelium and beyond: molecules involved in the transmigration and fate of monocytes. J Leukoc Biol 66:698-704 |
| Randolph, G J; Luther, T; Albrecht, S et al. (1998) Role of tissue factor in adhesion of mononuclear phagocytes to and trafficking through endothelium in vitro. Blood 92:4167-77 |
