Expression of CCL5 is induced by polyinosinic : polycytidylic acid in cultured hCMEC/D3 human brain microvascular endothelial cells

Abstract

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.

Induction of C‐C motif chemokine ligand 2 through Toll‐like receptor 3 signaling in human cerebral microvascular endothelial cell/D3 cells: possible regulation by nuclear factor‐κB

“…19 We have previously reported that the treatment of hCMEC/D3 cells with poly IC induces the expression of CCL5 in an IFN-b-independent manner, 15 whereas CXCL10 20 and ISG60 17 are induced by poly IC in an IFN-b-dependent manner. 19 We have previously reported that the treatment of hCMEC/D3 cells with poly IC induces the expression of CCL5 in an IFN-b-independent manner, 15 whereas CXCL10 20 and ISG60 17 are induced by poly IC in an IFN-b-dependent manner.…”

“…Briefly, the cells were transfected with siRNA and were incubated for 48 h. The transfected cells were treated with poly IC for the indicated period and Figure 2 RNA interference against Toll-like receptor (TLR3) results in the decrease in polyinosinic-polycytidylic acid (poly IC)-induced expression of C-C motif chemokine ligand 2 (CCL2). 15 Data are shown as the mean AE SD (n = 3; *P < 0.05, **P < 0.01 by t-test). After 48 h treatment, the cells were treated with 30 lg/mL poly IC.…”

“…15 In the experiments using SN50 (10 lmol/L), MR67307 (1 lmol/L) and human type I IFN neutralizing antibody mixture (1:20), the cells were pretreated with these reagents 1 h before the addition of poly IC (30 lg/mL). Effective knockdown of TLR3 and p65 were confirmed in a previous study.…”

“…15 The newly synthesized IFN-b induces the expression of IFN-stimulated genes (ISG) in an autocrine and/or a paracrine manner. 15 The newly synthesized IFN-b induces the expression of IFN-stimulated genes (ISG) in an autocrine and/or a paracrine manner.…”

“…Stimulation of hCMEC/D3 cells, a human brain microvascular endothelial cell line, with a TLR3 agonist is known to activate IFN regulatory factor (IRF)3 and nuclear factor (NF)-jB, and induce the expression of IFN-b. 15 The newly synthesized IFN-b induces the expression of IFN-stimulated genes (ISG) in an autocrine and/or a paracrine manner. ISG play an important role in the inhibition of viral infections by inhibiting various stages of the viral life cycle.…”