Estevao, Ana Carolina; (2019) The effect of chemokines on brain endothelial cell function. Doctoral thesis (Ph.D), UCL (University College London).

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Abstract

The blood-brain barrier (BBB) and the blood-retinal barrier (BRB) are two closely related tight endothelial cell barriers that control the movement of molecules and cells between the blood and the central nervous system (CNS). This feature is partly due to the presence of tight intercellular junctions between opposing endothelial cells (ECs) that limit the diffusion of molecules and restrict the passage of immune cells. This latter phenomenon is the primary driver for the observed immune privilege of the brain and retina. The breakdown of these barriers is a feature of many disorders of the CNS including Multiple Sclerosis, Alzheimer's disease, and stroke. At a molecular level, the breakdown of the BBB and BRB involves disengagement of intercellular junctions and a consequent increase in barrier permeability. Increased entry of macromolecules and/or immune cells through the BBB in pathological states have been extensively described in the literature. These events can be mediated by a paracellular route or a transcellular route of entry. Leukocyte migration from the blood, across the vascular wall and to the underlying tissue, occurs both in inflammation and immunosurveilance. The multi-step paradigm that explains this process, involves a series of interactions between leukocytes and endothelial cells with the involvement of chemokines. Chemokines were originally discovered as mediators of immune cell chemoattraction to sites of injury or inflammation. Immune cells are known to express receptors for chemokines and their behavior can be governed by receptor engagement with the various chemokine ligands. ECs express chemokine receptors raising the possibility that these cells can respond following exposure to chemokines. 5 Since chemokines are heavily involved in inflammatory processes, we have investigated the role of the chemokines CCL4, CXCL8 and CXCL10 on BBB endothelial cell function. We observed that the chemokines studied induced signaling events in human umbilical vein endothelial cells (HUVEC) and in an immortalised human brain endothelial cell line (hCMEC/D3). All chemokine treatments resulted in phosphorylation of the MAPKs and CCL4 resulted in a significant translocation of these proteins to the nucleus of hCMEC/D3. The distribution of junctional proteins ZO-1 and VE-cadherin were altered in response to chemokine treatments. VE-cadherin total protein was not affected by chemokines but there was internalization of the protein following treatments. There was evidence that the cytoskeleton, in the form of stress fibers, was also modulated by these chemokines. In hCMEC/D3 treatment with the chemokines CCL4 and CXCL10 resulted in an increased transendothelial cell monolayer permeability and an increase in the rate of migration of CD4+ T lymphocytes across the monolayer. In vivo there was also evidence that CCL4 induced increased permeability in the BBB. Taken together, these results indicate that that these chemokines may contribute to the opening of the BBB and the pathogenesis of neuroinflammatory diseases.

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