Regulation of chemokine receptor expression and function on CD4+T lymphocytes during central nervous system inflammation.

Chemokines are a family of cytokines that exhibit selective chemoattractant properties for target leukocfles, including CD4* T l¡rmphocfes, and play a significant role leukocyte migration. However, atarget leukocyte can only respond to a chemokine if it expresses the cognate receptor(s). Recent studies have demonstrated alterations in both chemokine and chemokine receptor expression patterns in the CNS during experimental autoimmune encephalomyelitis (EAE), a model for Multiple Sclerosis. Accordingly, the aim of the research presented in this thesis was to investigate chemokine receptor regulation and function on CD4* T lymphocytes during T cellmediated central nervous system (CNS) inflammation in vivo. In the proteolipid protein (PlP)-induced model of EAE, two inflammatory (CXCR3 and CCR5) and one supposedly homeostatic (CXCR4) chemokine receptors were upregulated on CD4* T cells during antigen-dependent clonal selection in the draining ly-ph nodes. As the CD4* T cells migrated through the blood and into the CNS tissue, expression of these receptors remained elevated such that, at the peak of clinical disease, the majority of neuroantigen-specific CD4" T cells in the CNS expressed elevated levels of CXCR4, CXCR3 and CCR5. Detailed characterisation of these receptors revealed that upregulation occurred in co-ordination with cellular division. Subsequent experiments were performed in order to determine the biological conssquences of specific chemokinelreceptor interactions during EAE. Amino terminal modifications of chemokines, which convert agonists to antagonists, have previously been shown to interfere with ligand/receptor interactions during inflammation. Therefore, a series of synthetic N-terminal chemokine mutants were initially tested in vitro for their ability to act as antagonists in preventing the migration of neuroantigen-activated lymphocytes to ligands of the receptors CXCR4, CXCR3 and CCR5. These analyses revealed that the synthetic mutants SDF-1 PzG,I-TAC 479 and RANTES 9-68 possessed potent antagonistic capacities. Following EAE induction, treatment every second day with the antagonists until day 15 resulted in a significant decrease in the severity of the neurological syrnptoms of EAE. Histological analyses demonstrated that the reduction in disease severity corresponded with a reduced number of inflammatory infiltrates in the spinal cords of antagonist-treated mice at peak clinical disease compared -',,jth control-treated mice. The ability to separate the disease process into two separate phases (sensitisation and effector) using adoptive transfer experiments provided a means to investigate the temporal and spatial control that specific chemokine/receptor interactions exerted during the pathogenesis of EAE. Accordingly, a series of ex vivo proliferation assays and adoptive transfer experiments were conducted. From these experiments, a potential role for the SDF-I/CXCR4 interaction was identified in the sensistisation phase of the disease. These results indicated that SDF-I|CXCLI2 and CXCR4 interactions not only play a role in homeostasis, but may also provide costimulatory signals to antigen-stimulated CD4+ T cells. Converely, roles for I-TAC/CXCR3 and RANTES/CCR5 interactions, but not SDF-I/CXCR4 interactions were identified in the effector phase of EAE. Collectively, the results generated in the present thesis, together with those from other studies, enabled the construction of a model detailing the temporal and spatial parameters of chemokine/chemokine receptor regulation of CD4* T cell activation and migration during a CD4* T cell-mediated immune response in the CNS.