G-protein receptor signalling in CNS endothelial cells and its role in supporting lymphocyte migration

The traffic of white blood cells (leukocytes) from the blood into the solid tissues of the body is a normal function of the immune system. This process involves a sequence of events starting with leukocyte adhesion to the blood vessel wall followed by penetration and migration into the tissue beyond. This phenomenon has been studied extensively and the main steps in the process delineated. However, the molecular basis of this event remains poorly understood. Until recently, it was believed that migration was largely determined by the leukocyte, with the endothelial cells of the blood vessel wall providing points for leukocyte attachment. This view has now been challenged by emerging evidence that the endothelial cell plays a pro-active role of in regulating leukocyte migration. This may be especially true in the brain and retina where the blood vessels form a more impermeable barrier than in other organs.
Recent data from various laboratories shows that activation of surface adhesion receptors on the endothelial cell leads to the induction of a sequence of biological signals that result in active support of leukocyte migration through the vascular barrier. Some of the endothelial signalling has been characterised, especially that mediated by the cell adhesion molecule ICAM-1 which binds circulating leukocytes. It is clear, however, that other signalling pathways must operate within the endothelial cells to regulate differential leukocyte migration across the vasculature of different tissues and at different stages of the inflaqmmatory response. We have recently reported that activation of a G-protein coupled receptor (GPCR) on the endothelial cell is necessary for successful leukocyte migration through the vascular barriers of the brain.
The aim of this project, therefore, is to gain a greater understanding of the fundamental biological events that occur as a result of activation of this GPCR and to elucidate its role in controlling leukocyte migration. Information resulting from this project will lead to a greater understanding of the cell biology underpinning leukocyte migration into the brain and retina. This is especially important as unwanted leukocyte traffic into these organs play a major role in diseases such as multiple sclerosis and retinal inflammation. Increasing our knowledge of the basic mechanisms responsible for this process will allow us to identify key targets in the biological sequence of events that would be accessible to therapeutic intervention and hence lead to improved treatment strategies for such diseases.

Leukocyte migration across the tight vascular barriers of the central nervous system (CNS) is mediated through complex interactions between the leukocyte and the vascular endothelium. Until relatively recently our understanding of the role of the endothelial cell in this phenomenon was restricted to its provision of docking molecules for the capture of circulating leukocytes. More recently, however, it has become evident that the vascular endothelium plays a central and pro-active role in facilitating the passage of leukocytes. Activation of surface adhesion receptors on the endothelial cell results in the induction of signalling cascades which, through processes that are yet to be fully elucidated, are clearly necessary for the successful penetration of leukocytes through the vascular barrier. Previous reports have shown that the immunoglobulin superfamily molecule ICAM-1 on brain endothelial cells not only serves as a leukocyte adhesion molecule, but is responsible for initiating crucial intracellular EC signalling responses. Recently, a pertussis toxin (PTX) sensitive pathway has also been identified as being necessary for leukocyte migration but the signalling pathways elicited through this G-protein coupled receptor (GPCR), and its potential cross-talk with the ICAM-1 signalling pathway, remain undefined. Furthermore, both the ligand and receptor involved in transducing the signal have yet to be identified. The purpose of this project, therefore, is to gain a detailed understanding of this EC GPCR signalling cascade. We will employ various cell and molecular biological methods to determine the activation of intracellular signalling proteins and, through the use of dominant negative signalling proteins and siRNA technology, block specific components of the EC signalling pathway to ascertain its impact on lymphocyte migration.
Our specific objectives in this project are to: 1. Characterise the brain endothelial cell signal transduction pathways triggered by the GPCR and to evaluate their potential crosstalk with well-characterized ICAM-1-mediated signalling. 2. Identify both the endothelial GPCR -that becomes activated by antigen-specific lymphocytes- and its ligand and 3. Determine the relative contribution of the identified endothelial GPCR-signalling pathways to the support leukocyte transmigration across different vascular beds.