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Beta Chemokine expression in an in vitro model of the human blood-brain barrier Chui, Ray

Abstract

It is now well established that the interactions between endothelium and circulating white blood cells are of critical importance in the evolution of an inflammatory response. Chemokines are small chemoattractant cytokines with the unique ability to stimulate and guide the movement of specific classes of inflammatory cells to sites of inflammation. The role of chemokines in CNS inflammation has not been fully elucidated. Over the last decade, various studies have suggested that CCL2(MCP-1) and CCL3 (MIP-1α) are major players in multiple sclerosis (MS) and its animal model, experimental autoimmune encephalitis (EAE). The aim of the present study is to investigate the expression and cytokine upregulation of CCL2 and CCL3 by human brain microvessel endothelial cells (HBMEC), an in vitro model of the human bloodbrain barrier. Primary cultures of HBMEC grown to confluence were stimulated with TNFα, IFNγ, IL-1β or LPS for 24-72 h. RNA expression was confirmed by RT-PCR and intracellular protein expression was detected with immunogold silver staining (IGSS). Protein release was quantitated with ELISA. CCL2 RNA levels were similar in both unstimulated and cytokine-treated cells. CCL3 RNA was upregulated upon cytokine stimulation. IGSS confirmed chemokine expression in both unstimulated and cytokinetreated cells. The basal release of CCL2 by resting HBMEC was significantly upregulated after cytokine treatment in a concentration and time-dependent manner. ELISA of the supernatants showed CCL3 release only after stimulation. Immunoelectron microscopy studies show chemokine binding to the apical and basal surfaces of HBMEC. These studies suggest that the cerebral endothelium may play an active role in the initiation, selective recruitment and activation of circulating lymphocytes and monocytes during CNS inflammation.

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