Leukocyte adhesion to endothelium is a central event in the immune system's response to injury, inflammation and infection. It is the first step in the well-established consensus model of leukocyte trafficking. Adherence to selectins expressed on the endothelium initiates rolling along the vessel wall, followed by sticking and diapedesis across the vascular barrier. This model functions in many tissues, and key molecules involved in the migration of lymphocytes to skin are currently being elucidated. For example, T cells infiltrate the skin in psoriasis, cutaneous T-cell lymphoma and allergic contact dermatitis using skin- related homing molecules, such as cutaneous lymphocyte-associated antigen (CLA) and E-selectin. By day 16 of gestation, T-cells known as dendritic epidermal cells, have already migrated from the thymus to the epidermis, via poorly understood molecular events. Our primary goal is to define when cutaneous lymphocytes and inflammatory responses in the fetal mouse skin develop, using a novel model of intravital fetal microscopy. In collaboration with Dr. Ulrich von Andrian, we have visualized the intact microcirculation of murine fetal skin and identified a compound, acridine orange, that crosses the placenta and labels fetal circulating cells, in situ. To date, no model of fetal microcirculation has been reported and we have the unique, technical expertise to optimally establish such a system. In the first year of this proposal, we will focus on optimizing technical parameters, including: l). the maximum time that blood flow can be maintained in exteriorized fetuses, 2). the minimum gestational age that can be tested, 3). the optimal conditions for maintaining fetal viability during the time of analysis, 4). the ability of reagents (chemokines, immunoglobulins and inflammatory mediators) to cross the placenta when injected into the maternal circulation, and 5). the ability to elicit a fetal skin inflammatory response with topically-applied mediators of inflammation. This model will subsequently be used with genetically-engineered mice whose cells lack key molecules important in trafficking to inflamed skin, such as E-selectin, P-selectin and 1,4- fucosyltransferases (enzymes responsible for the expression of carbohydrate ligands for the selectins). We will establish the time of gestation when circulating cells first interact with cutaneous vessels, the normal anatomy of fetal vessels and the role of molecules known to regulate migration into the adult skin. We believe that these studies will help us to further understand how congenital skin disorders, such as hemangiomas, infantile atopic dermatitis and seborrheic dermatitis, arise.
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