Cytokine signaling in the human brain capillary endothelial cell line hCMEC/D3

Abstract

Brain microvascular endothelial cells are part of the blood–brain barrier and participate actively in immunological processes including cytokine-mediated inflammatory reactions. Using the human brain capillary endothelial cell line hCMEC/D3, activation of JAK/STAT signaling pathways were studied in response to stimulation by cytokines. The phenotype of hCMEC/D3 cells was confirmed by flow cytometry analysis of cell adhesion factors (cluster of differentiation molecules CD31 and CD34) and the von Willebrand factor endothelial marker was detected by immunofluorescence. Strong STAT1, STAT6 and STAT3 activation was observed in response to interferon-gamma (IFN-γ), interleukin 4 (IL-4) and interleukin 6 (IL-6), respectively. Nuclear translocation of phosphorylated STAT proteins was visualized by confocal microscopy. Treatment of hCMEC/D3 cells with IFN-γ resulted in interferon-induced upregulation of major histocompatibility complex (MHC) class I within 48 h. Interferon-alpha (IFN-α) did not activate STAT1 or STAT3 nor did it induce MHC class I upregulation. Therefore, hCMEC/D3 cells were judged to be non-responsive to IFN-α. We also observed that hCMEC/D3 cells exhibit functional expression of alternative cytokine signal transduction pathways (i.e. TNF-α mediated activation of NF-κB). Together these results indicate that human blood–brain barrier hCMEC/D3 cells are responsive towards stimulation with various cytokines. We conclude that this unique cell line can be used to explore in vitro human blood–brain barrier functionality under proinflammatory conditions.