Cells of the monocyte/macrophage lineage are now thought to be prominent effector cells that leave the circulation and play a pathogenetic role in several neurologic disorders that affect mental health status, including AIDS dementia complex, multiple sclerosis and Alzheimer's disease. However, little is known of the signals that promote the extravasation of monocytes through the restrictive blood-brain barrier (BBB), or the cells with which they interact once they enter the brain. To address these issues, a cell culture model of the human BBB will be used to analyze - for the first time - the distinct phases of monocyte migration through the BBB in response to a variety of stimuli. The long-term objective of this proposal is to test the hypothesis that monocyte migration across the BBB can be promoted by a variety of factors, e.g., specific cytokines, chemotaxins and other substances, which affect the activation state of either monocytes or brain microvessel endothelial cells (BMEC). Initial studies will assess the effects of monocyte stimulation/activation with lipopolysaccharide (LPS), phorbol myristate acetate (PMA) and interferon- gamma (IFN-gamma). These agents have been shown to promote monocyte adhesion to/migration across peripheral vasculature, but have yet to be analyzed with respect to monocyte interaction with the BBB. This is critical, as BBB endothelium may behave distinctively from that of peripheral vessels. Subsequent studies will analyze the role of endothelial stimulation/activation by LPS and cytokines tumor necrosis factor-alpha (TNF-alpha), IFN-gamma and interleukins-l and -2 (IL-1, IL-2) - substances again known to foster monocyte migration across the peripheral circulation, and which, in the case of the cytokines, have been shown to be up-regulated in the brain concurrent with monocytic infiltration. Combinations of monocyte/BMEC stimulation will also be performed to see if this further enhances monocyte migration. The effect of BMEC infection with cytomegalovirus (CMV) will be evaluated as well, as CMV infects BMEC in AIDS, and has been shown to augment monocyte adhesion to infected peripheral vessel endothelial cells. CMV may thus aid in recruiting monocytes to the brain. The effects of monocyte chemotactic peptides MCP-1 and TGF-beta will additionally be analyzed, as these may be released by astrocytes during CNS inflammatory disease, and could also foster monocyte trafficking to the brain. These studies will be performed in the presence and absence of astrocyte-conditioned media, as such media has been shown to influence BBB permeability and, consequently, glial- derived factors might influence monocyte migration. BBB integrity will be analyzed during the course of these studies by both electron and fluorescence microscopy, to determine whether monocyte migration requires or induces endothelial cell damage. Antibody """"""""blocking"""""""" experiments will be also be performed to identify cell adhesion molecule:ligand pathways operant in monocyte migration across the BBB. Lastly, the effects of monocyte:astroglial interactions on monocyte migration across the BBB model will be evaluated as well. These experiments will illuminate factors that regulate monocyte entry into the brain, and thus identify potential targets for therapeutic intervention in a variety of crippling CNS diseases.
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