There are phenotypic and functional differences between vascular endothelium from different tissues and between microvascular and macrovascular endothelial cells (ECs) from the same tissue. Relatively little is known about the human blood-nerve barrier (BNB). We report the development of an in vitro BNB model using primary human endoneurial ECs freshly isolated and purified from decedent sciatic nerves via endoneurial stripping, connective tissue enzymatic digestion, and density centrifugation. Primary human endoneurial ECs are spindle shaped and contact inhibited. They rapidly differentiate to form capillary-like networks and microvessels, bind Ulex Europaeus Agglutinin 1 lectin, express von Willebrand factor, and endocytose acetylated low-density lipoprotein. They also express specific transport and cellular adhesion molecules and tight junction proteins, consistent with cells that form a highly restrictive endothelial barrier similar to the blood-brain barrier. When cultured on collagen-coated transwell inserts, the primary human endoneurial ECs develop an in vitro BNB with high transendothelial electrical resistances (160 Omega x cm(2); maximal 12 days after seeding) and low solute permeability coefficient to fluoresceinated high-molecular weight (70 kDa) dextran (2.75 x 10(-3) cm/minute). This in vitro BNB model retains essential known or expected characteristics of the human BNB and has many potential applications for studies of solute, macromolecule, microbial, virus, and leukocyte interactions with this highly specialized endothelial barrier.

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The mechanisms of hematogenous leukocyte trafficking at the human blood-nerve barrier (BNB) are largely unknown. Intercellular adhesion molecule-1 (ICAM-1) has been implicated in the pathogenesis of Guillain-Barré syndrome (GBS). We developed a cytokine-activated human in vitro BNB model using primary endoneurial endothelial cells. Endothelial treatment with 10 U/mL tissue necrosis factor-α and 20 U/mL interferon-γ resulted in de novo expression of proinflammatory chemokines CCL2, CXCL9, CXCL11 and CCL20, with increased expression of CXCL2-3, CXCL8 and CXCL10 relative to basal levels. Cytokine treatment induced/ enhanced ICAM-1, E- and P-selectin, vascular cell adhesion molecule-1 and the alternatively spliced pro-adhesive fibronectin variant, fibronectin connecting segment-1 expression in a time-dependent manner, without alterations in junctional adhesion molecule-A expression. Lymphocytes and monocytes from untreated GBS patients express ICAM-1 counterligands, αM- and αL-integrin, with differential regulation of αM-integrin expression compared to healthy controls. Under flow conditions that mimic capillary hemodynamics in vivo, there was a >3-fold increase in total GBS patient and healthy control mononuclear leukocyte adhesion/ migration at the BNB following cytokine treatment relative to the untreated state. Function neutralizing monoclonal antibodies against human αM-integrin (CD11b) and ICAM-1 reduced untreated GBS patient mononuclear leukocyte trafficking at the BNB by 59% and 64.2% respectively. Monoclonal antibodies against αL-integrin (CD11a) and human intravenous immunoglobulin reduced total leukocyte adhesion/migration by 22.8% and 17.6% respectively. This study demonstrates differential regulation of αM-integrin on circulating mononuclear cells in GBS, as well as an important role for αM-integrin-ICAM-1 interactions in pathogenic GBS patient leukocyte trafficking at the human BNB in vitro.

This study suggests that CCL2 expressed by Schwann cells and CXCL10 expressed by endoneurial endothelial cells may induce F4/80+ macrophage and CD3+ T cell-mediated inflammation and demyelination in sm-EAN. CCL2-CCR2 and CXCL10-CXCR3 signalling pathways are potential targets for therapeutic intervention in peripheral nerve inflammation.

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