Establishment of a human model of the blood-brain barrier has proven to be a difficult goal. To accomplish this, normal human brain endothelial cells were transduced by lentiviral vectors incorporating human telomerase or SV40 T antigen. Among the many stable immortalized clones obtained by sequential limiting dilution cloning of the transduced cells, one was selected for expression of normal endothelial markers, including CD31, VE cadherin, and von Willebrand factor. This cell line, termed hCMEC/D3, showed a stable normal karyotype, maintained contact-inhibited monolayers in tissue culture, exhibited robust proliferation in response to endothelial growth factors, and formed capillary tubes in matrix but no colonies in soft agar. hCMEC/D3 cells expressed telomerase and grew indefinitely without phenotypic dedifferentiation. These cells expressed chemokine receptors, up-regulated adhesion molecules in response to inflammatory cytokines, and demonstrated blood-brain barrier characteristics, including tight junctional proteins and the capacity to actively exclude drugs. hCMEC/D3 are excellent candidates for studies of blood-brain barrier function, the responses of brain endothelium to inflammatory and infectious stimuli, and the interaction of brain endothelium with lymphocytes or tumor cells. Thus, hCMEC/D3 represents the first stable, fully characterized, well-differentiated human brain endothelial cell line and should serve as a widely usable research tool.

Leukocyte migration into the central nervous system (CNS) is mediated by chemokines expressed on CNS endothelial cell surfaces. This study investigated the production of chemokines and expression of chemokine receptors by human brain endothelial cells (HBECs) in vitro and in situ. Four chemokines (CCL2, CCL5, CXCL8, and CXCL10) were demonstrated by immunohistochemistry in endothelial cells in brain samples from patients with multiple sclerosis. CXCL8 and CCL2 were constitutively released and increased by primary HBECs and the brain endothelial cell line hCEMC/D3 in response to tumor necrosis factor and/or interferon gamma. CXCL10 and CCL5 were undetectable in resting endothelial cells but were secreted in response to these proinflammatory cytokines. Tumor necrosis factor strongly increased the production of CCL2, CCL5, and CXCL8; interferon gamma upregulated CXCL10 exclusively. CCL3 was not secreted by HBECs and seemed to be confined to astrocytes in situ. The chemokine receptors CXCR1 and CXCR3 were expressed by HBECs both in vitro and in situ; CXCR3 was upregulated in response to cytokine stimulation in vitro. In contrast, CXCR3 expression was reduced in noninflammatory (silent) multiple sclerosis lesions. The particularly high levels of CXCL10 and CXCL8 expressed by brain endothelium may contribute to the predominant TH1-type inflammatory response observed in chronic inflammatory conditions such as multiple sclerosis.