The Role of Chemokines in Leukocyte Recruitment across the Blood-Brain Barrier

Migration of autoaggressive T cells across the blood-brain barrier (BBB) is critically involved in the initiation of multiple sclerosis (MS) and its animal model experimental autoimmune encephalomyelitis (EAE). The direct involvement of chemokines in this process was suggested by our recent observat...

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Bibliographische Detailangaben
1. Verfasser: Alt, Carsten
Beteiligte: Engelhardt, Britta (Prof.) (BetreuerIn (Doktorarbeit))
Format: Dissertation
Sprache:Englisch
Veröffentlicht: Philipps-Universität Marburg 2004
Medizin
Schlagworte:
BBB
BHS
EAE
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Zusammenfassung:Migration of autoaggressive T cells across the blood-brain barrier (BBB) is critically involved in the initiation of multiple sclerosis (MS) and its animal model experimental autoimmune encephalomyelitis (EAE). The direct involvement of chemokines in this process was suggested by our recent observation that G-protein-mediated signaling is required to promote adhesion strengthening of encephalitogenic T cells on BBB endothelium in vivo. For chemokines to be involved in this process, they have to be either expressed by BBB endothelial cells themselves or would require a yet unknown transport mechanism from the central nervous system (CNS) parenchyma across the endothelial BBB to the luminal surface of the endothelial cells. To search for chemokines expressed by the endothelial BBB itself, in situ hybridizations and immunohistochemistry were performed and expression of the lymphoid chemokines CCL19 and CCL21 was found in venules surrounded by inflammatory cells. Their expression was paralleled by the presence of their common receptor CCR7 in inflammatory cells in brain and spinal cord sections of mice afflicted with EAE. Encephalitogenic T cells showed surface expression of CCR7 and the alternative receptor for CCL21, CXCR3. They specifically chemotaxed towards both CCL19 or CCL21 in a concentration dependent and pertussis toxin-sensitive manner comparable to naive lymphocytes in vitro. Functional involvement of CCL19 and CCL21 in adhesion strengthening of encephalitogenic T lymphocytes was demonstrated by binding assays on frozen brain sections of mice afflicted with EAE in vitro and preliminary by intravital fluorescence videomicroscopy in spinal cord of healthy mice in vivo. The moderate effect observed suggests additional, potentially unknown chemokines to be involved in lymphocyte recruitment across the endothelial BBB into the immunoprivileged CNS. Such chemokines, receptors as well as unknown molecules were identified at the level of the endothelial BBB by oligonucleotide microarrays, subtractive suppression hybridization (SSH) and proteomics. Besides the upregulation of expected genes and proteins described to be involved in leukocyte recruitment during EAE pathogenesis before, unexpected genes and proteins were identified. The latter included increased Duffy antigen / receptor for chemokines (DARC) expression suggesting its involvement in lymphocyte recruitment during EAE pathogenesis, which was proven as in DARC-deficient mice, disease onset was delayed, while clinical severity was increased. This may be explained by an ambiguous DARC function in EAE pathogenesis. Either endothelial cell expressed DARC "shuttles" chemokines to the luminal surface of the endothelial cells or erythrocyte expressed DARC removes chemokines by its "sink"-like function. This results in either increased or decreased chemokine concentrations accessible to encephalitogenic T lymphoblasts. Their encephalitogenicity was addressed by gene array analysis and SSH of encephalitogenic versus non-encephalitogenic T lymphoblasts identifying 79 differentially expressed genes. Based on the results obtained during this thesis, we would like to suggest the lymphoid chemokines CCL19 and CCL21 to be critically involved in lymphocyte recruitment across the endothelial BBB during EAE pathogenesis, while the chemokine receptor DARC may provide a "shuttle" mechanism for inflammatory chemokines from the CNS parenchyma across the endothelial BBB to the luminal surface of the endothelial cells during EAE. A large number of additional genes and proteins was identified to be differentially expressed in either endothelial cells or T lymphoblasts pointing to new mechanisms involved in leukocyte trafficking across the BBB.