Project Summary Monocytes have been shown to normally circulate through the trabecular meshwork (TM) and to be recruited in increased numbers to the TM following the application of laser irradiation. In addition, monocytes have been shown to enhance aqueous outflow in an animal model in vivo and also to increase the permeability of cultured Schlemm's canal endothelial (SCE) cells in vitro. The organizing hypothesis of this project is that in the human eye monocytes interact with SCE cells in a paracrine manner, thus forming a cellular module that regulates the outflow of aqueous humor. The specific aims are to test particular predictions of this hypothesis about the roles of monocytes by: [1] Determining the types of monocytes that modulate the permeability and intercellular junction status of Schlemm's canal (SC) lining endothelial cells in situ. Human peripheral blood monocytes will be separated into three types and then these groups of monocytes, and media conditioned by these cell preparations, will be compared in their ability to influence the time-course behavior of SC permeability, as well as in terms of their ability to influence the junction assembly/disassembly process in the outflow tissues from perfused eyes. [2] Testing the prediction that individual cytokines secreted by monocytes bind onto SC endothelial cells that can induce pronounced changes in outflow facility. [3] Identifying intracellular signaling pathways regulating SCE-permeability via the junction-disassembly process. The secretion by monocytes of several cytokines will be assessed using ELISA. The results will provide a model to envision the role of intracellular signaling pathways, and important mediators including cytokines and intercellular junctions. The model specifically considers the role of monocytes and monocyte secreted cytokines. These cytokines are proposed to bind onto the cells lining SC and to modulate candidate intracellular signaling pathways including RhoA GTPase and the myosin-light chain kinase. These signaling molecules in turn induce a sequence of intracellular events that include alternating the state of cortical cytoskeletal elements, inducing the disassembly of intercellular junctions and increasing the egress of aqueous from the eye. The identification of key cytokines, which function by increasing the permeability properties of cellular barriers, will open up a new therapeutic approach to treat glaucoma by promoting the outflow of aqueous through the trabecular or conventional aqueous outflow pathway. Therefore, subsequent studies would translate our theoretical model into clinically relevant approaches for glaucoma treatments, and including cytokine-based or cell-based treatment modalities.