ABSTRACT. The blood-nerve barrier (BNB) is a highly specialized unit that maintains the microenvironments of the peripheral nervous system. Since the breakdown of the BNB has been considered a key step in autoimmune neuropathies such as Guillain-Barré syndrome and chronic inflammatory demyelinating polyraduculoneuropathy, it is important to understand the cellular properties of the peripheral nerve microvascular endothelial cells (PnMECs) which constitute the BNB. For this purpose, we established an immortalized cell line derived from human PnMECs. The human PnMECs were transduced with retroviral vectors encoding the temperaturesensitive SV40 large T antigen and human telomerase. This cell line, termed FH-BNB, showed a spindle fibershaped morphology, expression of von Willebrand factor and uptake of acetylated low density lipoprotein. These cells expressed tight junction proteins including occludin, claudin-5, ZO-1 and ZO-2 at the cell-cell boundaries. P-glycoprotein and GLUT-1 were also detected by a Western blot analysis and the cells exhibited the functional expression of p-glycoprotein. In addition, transendothelial electrical resistance experiments and paracellular permeabilities of sodium fluorescein and fluorescein isothiocyanate-labeled dextran of molecular weight 4 kDa across these cells demonstrated that FH-BNBs had functional tight junctions. These results indicated that FHBNBs had highly specialized barrier properties and they might therefore be a useful tool to analyze the pathophysiology of various neuropathies.

Objectives: The breakdown of the blood-brain barrier (BBB) has been considered to be a key step in the disease process of a number of neuroimmunological disorders. Although several in vitro BBB models derived from human tissues have been established, no human conditionally immortalized in vitro BBB models using a temperature-sensitive SV40-T antigen (tsA58) and human telomerase reverse transcriptase (hTERT) have ever been reported. In the present study, we established a new human brain microvascular endothelial cell line harboring tsA58 and hTERT genes, and extensively characterized this new model. Methods: TY08 cells, derived from the human BBB and harboring tsA58, were infected with retroviruses possessing hTERT genes. We examined whether this new model retains its barrier-specific nature, independent of the passage number. Results: The obtained endothelial cell line, termed TY09, proliferated well under the permissive temperature and stopped growing under the nonpermissive temperature, despite the acquisition of hTERT as an additional immortalizing gene. Even with a high-passage number, the cells maintained a spindle-shaped morphology, the expression of the von Willebrand factor, tight junction proteins and transporters. Furthermore, we carried out a transendothelial transport study for TY09 cells and hCMEC/D3 cells, thereby proving that both cell lines have almost the same nature with respect to transcellular permeability of various hydrophilic and hydrophobic substances. Conclusions: The new stable conditionally immortalized TY09 cells, retaining the in vivo BBB functions, should facilitate the performance of future studies for determining the pathophysiology of various neuroimmunological diseases. (Clin Exp Neuroimmunol

The autocrine secretion of MMP-2/9 by BMECs induced by humoral factors, other than IgG, in sera obtained from NMOSD patients potentially increases BBB permeability. IgG obtained from NMOSD sera, apart from anti-AQP4 antibodies, affect the BBB by upregulating VCAM, thereby facilitating the entry of inflammatory cells into the central nervous system.

BackgroundPathological destruction of blood-brain barrier (BBB) has been thought to be the initial key event in the process of developing multiple sclerosis (MS). The purpose of the present study was to clarify the possible molecular mechanisms responsible for the malfunction of BBB by sera from relapse-remitting MS (RRMS) and secondary progressive MS (SPMS) patients.MethodsWe evaluated the effects of sera from the patients in the relapse phase of RRMS (RRMS-R), stable phase of RRMS (RRMS-S) and SPMS on the expression of tight junction proteins and vascular cell adhesion protein-1 (VCAM-1), and on the transendothelial electrical resistance (TEER) in human brain microvascular endothelial cells (BMECs).ResultsSera from the RRMS-R or SPMS patients decreased the claudin-5 protein expression and the TEER in BMECs. In RRMS-R, this effect was restored after adding an MMP inhibitor, and the MMP-2/9 secretion by BMECs was significantly increased after the application of patients' sera. In SPMS, the immunoglobulin G (IgG) purified from patients' sera also decreased the claudin-5 protein expression and the TEER in BMECs. The sera and purified IgG from all MS patients increased the VCAM-1 protein expression in BMECs.ConclusionsThe up-regulation of autocrine MMP-2/9 by BMECs after exposure to sera from RRMS-R patients or the autoantibodies against BMECs from SPMS patients can compromise the BBB. Both RRMS-S and SPMS sera increased the VCAM-1 expression in the BBB, thus indicating that targeting the VCAM-1 in the BBB could represent a possible therapeutic strategy for even the stable phase of MS and SPMS.

The sera from MMN patients may disrupt the BNB function via the autocrine secretion of VEGF in PnMECs, or the exposure to autoantibodies against PnMECs that are contained in the MMN sera. Autoantibodies against PnMECs in MMN sera may activate the BNB by upregulating the VCAM-1 expression, thereby allowing for the entry of a large number of circulating inflammatory cells into the peripheral nervous system.