Regulation of leukocyte trafficking at the BBB

Project 3: Regulation of leukocyte trafficking at the BBB: Perivascular accumulation of mononuclear cells within the white matter of the central nervous system (CNS) is a pathologic hallmark of multiple sclerosis (MS). The mechanisms involved in the selective infiltration of the CNS by circulating T cells and monocytes during the pathogenesis of MS are not completely understood. This process is dependent on expression of chemokines and their receptors, which direct leukocyte migration t9o specific sites within the vasculature, and adhesion molecules, which mediate selective leukocyte adhesion to endothelium resulting in subsequent extravasation into perivascular regions. The hypothesis to be tested is that the kinetic and region expression of chemokines, their receptors, adhesion molecules and matrix metalloproteinases (MMP's) by endothelial cells (EC) and astrocytes, components of the specialized vessels of the CNS, a swell as by T cells, monocytes and cells specific to the CNS are critical to the type of tissue mononuclear cell accumulation. Subsets of leukocytes express different chemokine receptors and adhesion proteins And transmigrate in response to specific chemoattractants. The timing and sequence of chemokine and adhesion molecule expression within the CNS may have a direct effect on the inflammatory profile and phenotype characteristic of the developing MS lesion. In the previous granting period, a tissue culture model of the human BBB was developed. The model enabled analysis of mechanisms involved in transmigration of mononuclear cells (PBMC) as they relate to the CNS-specific inflammation that occurs in MS. The major findings of these studies are: monocyte and T cell transmigration across co-cultures is chemokine and adhesion molecule dependent; astrocyte-derived factors significantly regulate transmigration; astrocyte-derived MCP-1 is a potent inducer of monocyte but not T cell migration; T cells transmigrate in response to IFN-gamma-inducer factors; treatment of T cells and monocytes with IFN-beta, an approved therapy for MS, inhibits their transmigration in the BBB model; and chemokine expression in human microglia and astrocytes is induced by TH-1-like cytokines. These novel findings form the basis for the experiments in this proposal. Specifically, the Aims are to: 1. Examine the effects of chemokines and adhesion molecules on T cell and monocyte trafficking across the BBB model. The role of the IFN- gamma induced chemokines, IP-10 and MIG, in transmigration will be assayed. SDF-1 will also be tested as it may facilitate the influx of unactivated as well as activated T cells into the CNS. The cytokine, chemokine and chemokine receptor profiles of cells subsequent to transmigration will be assayed. 2. Characterize the mechanisms by which IFN-beta inhibits transmigration across the BBB model. 3. Analyze the mechanisms by which PBMC and gamma/delta T cell clones from MS patients transmigrate across co-cultures. 4. Determine the regulation of expression of MIG, IP-1-, SDF-1 and their receptors, CXC43 and CXCR4, in astrocytes and microglia.. 5. Determine the expression in vivo of SDF-1, MIG and IP-10 and their receptors, as well as of MMP's in tissues from MS lesions of different ages.