In the present study, we demonstrated that EGCG, a bioactive polyphenol in green tea, suppressed the expression of LPS-induced inflammatory cytokines in brain endothelial cells. This finding supports a previous report showing that EGCG attenuated LPS-mediated inflammation by suppressing the TNF-α and IL-1β expression in macrophages, leading to the downregulation of inflammatory responses [32
EGCG pretreatment also inhibited the monocyte adhesion to the brain endothelial cell monolayer and blocked the negative impact of LPS on the tight junctional protein expression in brain endothelial cells. MCP-1/CCL2 plays a critical role in the recruitment of leukocytes to the site of inflammation elicited by LPS [33
]. It has been reported that EGCG could decrease the MCP-1 and CCR2 gene expression, together with MCP-1 secretion and CCR2 expression at the cell surface in THP-1 monocytes, thus preventing the migration and adhesion of monocytes to fibronectin [34
]. Our data showed that in unstimulated hCMEC/D3 cells, the basal expression of MCP-1/CCL2 was low but measurable, while LPS treatment greatly induced the expression of MCP-1/CCL2. This is consistent with a previous report [35
] showing that hCMEC/D3 cells express lower levels of MCP-1/CCL2 than two primary human brain endothelial cells generated from multiple sclerosis brain tissue or from temporal lobe resections from epileptic patients.
In addition, we found that LPS treatment of hCMECs induced the expression of ICAMs and VCAMs, the key ligands for the beta2 integrin molecules present on leukocytes [36
]. The expression of ICAM-1 in hCMEC/D3 cells is higher than that of VCAM-1 [17
]. ICAM-1 and VCAM-1 facilitate the adhesion of monocytes to the endothelium. Endothelial ICAM-1 was shown to be essential for T cell diapedesis across the BBB in vitro
under static conditions [37
]. Adamson and colleagues [38
] revealed that endothelial ICAM-1 was essentially involved in lymphocyte migration through brain endothelial cell monolayers by rearrangement of the endothelial actin cytoskeleton and functional endothelial cell Rho proteins. Treatment of HBMEC with TNF-α resulted in increased polymorphonuclear leukocyte adhesion that was significantly inhibited by blocking antibodies to E-selectin and ICAM-1, but not VCAM-1 [39
It is well known that NF-κB activation is required for the LPS-induced inflammatory cytokine production [40
]. Endothelial-selective blockade of NF-κB activation repressed expression of multiple endothelial adhesion molecules and reduced neutrophil infiltration into multiple organs [41
]. EGCG was found to have the ability to block NF-κB activation in the intestinal epithelial cell line IEC-6 [42
]. In addition, theaflavin-3,3’-digallate (another polyphenol) from black tea was also reported to have even stronger suppression of LPS-induced NF-κB activity than other polyphenols through downregulation of IκB kinase activity in macrophages [43
]. Thus, the suppression of NF-κB activation by EGCG justifies the inhibitory effect of EGCG on LPS-mediated endothelial inflammation.
It is known that TLR4 is involved in LPS-mediated inflammation. Sheth and colleagues [44
] showed that LPS disrupts tight junctions in cholangiocyte monolayers by a c-Scr-, TLR4- and LBP-dependent mechanism. A recent report [32
] showed that EGCG downregulated inflammatory responses by directly suppressing TLR4 mRNA and protein expression. In addition, EGCG treatment of macrophages was found to upregulate the expression of Tollip [32
], a negative regulator of the TLR signaling pathway. However, our data demonstrated that EGCG had little effect on TLR4 expression in brain endothelial cells. Youn and colleagues [45
] reported that EGCG could inhibit LPS- or PolyI:C-mediated activation of interferon regulatory factor 3. EGCG could modulate both MyD88- and TIR-domain-containing adapter-inducing interferon-(TRIF)-dependent signaling pathways of TLR3 and the subsequent inflammatory target gene expression in macrophages. However, we did not observe a modulatory effect of EGCG on the expression of MyD88, a key adaptor to mediate the TLR-MyD88-dependent signaling pathway. These discrepancies may be due to the different types of cells used to investigate EGCG activity, suggesting that other mechanisms are likely to be involved in EGCG-mediated inhibition of endothelial inflammation.
67LR is a receptor that presents on the eukaryotic cell membrane for the cellular prion proteins and also interacts with extracellular matrix components [46
]. 67LR was also found to express on both rodent and human cerebral endothelial cells [47
]. It has been suggested that 67LR serves as a co-receptor for bacterial pathogens that target the BBB. 67LR on brain endothelial cells interacts with three most commonly neuroinvasive bacteria: pneumococcus, H. influenzae
, and meningococcus. The binding of 67LR to these pathogens initiates the bacterial interaction with the BBB and promote their CNS tropism, inducing cell signaling through the other receptors, such as the TLRs. The interaction of TLRs with 67LR may synergistically promote bacterial adherence and invasion of BBB [47
]. Interestingly, 67LR was shown to be involved in the inhibitory effect of EGCG on the TLR4 signaling pathway in macrophages [32
]. These findings promoted our interest in examining the role of 67LR in the anti-inflammatory effect of EGCG in brain endothelial cells. Our data that pretreatment of hCMEC cells with anti-67LR antibody significantly blocked the EGCG effect on LPS-mediated TNF-α and IL-1β induction, as well as on NF-κB activation, indicate that EGCG exerts its anti-inflammatory effect in endothelial cells at least partially through 67LR. It is likely that pretreatment of hCMEC/D3 cells with EGCG enables the binding of 67LR to EGCG and disrupts or modulates LPS interaction with 67LR. It has been suggested this disruption or modulation might engender unexpectedly broad protection against systemic infections [47
]. Thus, our data support the notion that EGCG can be used as a potential therapeutic compound to treat CNS inflammation related to the BBB.