Chemokine and chemikine receptor expression by human brain endothelium :an in vitro and in situ study

Subileau, Eva-Anne (2006). Chemokine and chemikine receptor expression by human brain endothelium :an in vitro and in situ study. PhD thesis The Open University.



The infiltration of leukocytes across the BBB is thought to be mediated by chemokines released in the CNS. This study investigated the production of chemokines and expression of chemokine receptors by human brain endothelial cells (HBEC) as well as the consequences of CXCL10 stimulation on brain endothelial function in terms of leukocyte migration and endothelial permeability.
Purified primary HBEC as well as a novel human brain endothelial cell line, called hCEMC/D3 and generated in collaboration with the Institute Cochin, were used in the present study. Characterisation of hCMEC/D3 cells was effected by determining the expression and localisation of HBEC markers such as tight junction and adherens junction proteins. Both primary and immortalized HBEC were tested for the production of four chemokines (CCL2, CCL5, CXCL8 and CXCL10) considered to play a role in multiple sclerosis. CXCL8 and CCL2 were constitutively released, whereas CXCL10 and CCL5 could not be detected at basal levels. By contrast, all these chemokines were up-regulated in response to either TNF-a or IFN-y or a combination of both. TNF-a had the most striking effect, up-regulating the production of CCL2, CCL5 and CXCL8, while IFN-y up-regulated CXCL10 exclusively. The chemokine receptors CXCR1 and CXCR3, whose ligands include CXCL8 and CXCL10, were expressed constitutively by HBEC both in vitro and in vivo, and only CXCR3 was up-regulated in response to cytokine stimulation in vitro. Furthermore, CXCR3 on endothelial cells is functional as CXCL10 induces phosphorylation of p42/44 MAP kinase, p38 MAP kinase and JNK/SAPK. However, activation of HBEC by CXCL10 did not induce any permeability change nor did it modulate leukocyte adhesion.
These results demonstrate that endothelial cells could play an important role in chemokine production and hence leukocyte infiltration during inflammatory pathologies such as MS. The role of chemokine receptors requires further investigation.

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