Objective Brain microvascular endothelial cells are one of the cell types that form the blood-brain barrier, and play an important role in the defense system of the brain. Toll-like receptor 3 (TLR3) is a pattern-recognition receptor against double-stranded RNA, and TLR3 signaling is important in antiviral innate immune reactions. However, TLR3 signaling in brain microvascular endothelial cells is not well understood. We aimed to investigate the role of TLR3 signaling in chemokine CCL5 production in human brain microvascular endothelial cells. Methods The hCMEC/D3 human brain microvascular endothelial cells were cultured, and treated with an authentic TLR3 agonist polyinosinic : polycytidylic acid. The expression of CCL5 was examined using quantitative realtime reverse transcription polymerase chain reaction and enzyme-linked immunosorbent assay. Involvement of TLR3, interferon (IFN)-b, or IFN-k1, IFN-regulatory factor 3 or nuclear factor-jB p65 in this reaction was examined using RNA interference. Translocation of p65 into the nucleus was examined using immunofluorescence staining. Results Treatment of cells with polyinosinic : polycytidylic acid induced the expression of CCL5, IFN-b and IFN-k1, and also the translocation of p65 into the nucleus. Knockdown of TLR3, IFN-regulatory factor 3 or p65 inhibited the induction of these molecules, while knockdown of neither IFN-b nor IFN-k1 affected the expression of CCL5. Conclusions TLR3 activation by polyinosinic : polycytidylic acid induces the expression of CCL5 in cultured hCMEC/D3 cells, and IFN-regulatory factor 3 and p65 are involved in this reaction. CCL5 induced by TLR3 signaling in brain microvascular endothelial cells might contribute to antiviral protective reactions and/or detrimental responses associated with viral infection in the brain.

Objective Brain microvascular endothelial cells (BMEC) are the major component of the blood-brain barrier, and play a critical role in protecting the brain from pathogens. Although Toll-like receptor 3 (TLR3) is known as an important pattern-recognition receptor against viral double-stranded RNA, the downstream reactions of TLR3 in human BMEC are not fully characterized. The purpose of the present study was to investigate the role of TLR3 signaling in the expression of retinoic acid-inducible gene-I (RIG-I), melanoma differentiation-associated gene 5 (MDA5) and C-X-C motif chemokine ligand 10 (CXCL10) in human BMEC. Methods The hCMEC/D3 cells, a human BMEC cell line, were cultured and stimulated with polyinosinic-polycytidylic acid, a synthetic TLR3 agonist. Quantitative real-time reverse transcription polymerase chain reaction, western blotting and enzyme-linked immunosorbent assay were used to examine the expression of interferon-b, RIG-I and CXCL10. RNA interference against TLR3, interferon-b, RIG-I or MDA5 was also carried out. Results Expression of RIG-I, MDA5 and CXCL10 were induced by polyinosinic-polycytidylic acid, and this was inhibited by knockdown of either TLR3 or interferon-b. Knockdown of RIG-I or MDA5 did not affect the CXCL10 expression in the early phase (4 h), but decreased the CXCL10 induction in the late phase (8 or 24 h). Conclusions RIG-I, MDA5 and CXCL10 are induced through TLR3 activation in hCMEC/D3 cells, and RIG-I and MDA5 might function to enhance and/or prolong the reactions mediated by CXCL10. These molecules might play a distinct role in innate immune reactions against double-stranded RNA viruses in human BMEC.