Correlationoftightjunctionmorphologywiththe expressionoftightjunctionproteinsinblood-brain barrierendothelialcells Stefan Liebner a2) , Uwe Kniesel ab2) , Hubert Kalbacher c , Hartwig Wolburg a1) a Institute of Pathology, University of Tübingen, Tübingen/Germany b Institute of Zoology, University of Hohenheim, Stuttgart/Germany c Medical and Natural Sciences Research Center, Tübingen/Germany Received May 15, 2000 Received in revised version June 30, 2000 Accepted July 2, 2000 Rat ± chicken ± tight junction ± blood-brain barrier ± claudin-1 ± claudin-5 ± occludin Endothelial cells of the blood-brain barrier form complex tight junctions, which are more frequently associated with the protoplasmic (P-face) than with the exocytoplasmic (E-face) membrane leaflet. The association of tight junctional particles with either membrane leaflet is a result of the expression of various claudins, which are transmembrane constituents of tight junction strands. Mammalian brain endothelial tight junctions exhibit an almost balanced distribution of particles and lose this morphology and barrier function in vitro. Since it was shown that the brain endothelial tight junctions of submammalian species form P-face-associated tight junctions of the epithelial type, the question of which molecular composition underlies the morphological differences and how do these brain endothelial cells behave in vitro arose. Therefore, rat and chicken brain endothelial cells were investigated for the expression of junctional proteins in vivo and in vitro and for the morphology of the tight junctions. In order to visualize morphological differences, the complexity and the P-face association of tight junctions were quantified. Rat and chicken brain endothelial cells form tight junctions which are positive for claudin-1, claudin-5, occludin and ZO-1. In agreement with the higher P-face association of tight junctions in vivo, chicken brain endothelia exhibited a slightly stronger labeling for claudin-1 at membrane contacts. Brain endothelial cells of both species showed a significant alteration of tight junctions in vitro, indicating a loss of barrier function. Rat endothelial cells showed a characteristic switch of tight junction particles from the P-face to the E-face, accompanied by the loss of claudin-1 in immunofluorescence labeling. In contrast, chicken brain endothelial cells did not show such a switch of particles, although they also lost claudin-1 in culture. These results demonstrate that the maintenance of rat and chicken endothelial barrier function depends on the brain microenvironment. Interestingly, the alteration of tight junc- tions is different in rat and chicken. This implies that the rat and chicken brain endothelial tight junctions are regulated differently. Abbreviations. CBE Chicken brain endothelial cell. ± E-face Exocytoplas- mic fracture face. ± EFA E-face association. ± P-face Protoplasmic fracture face. ± PFA P-face association. ± RBE Rat brain endothelial cell. ± TJ Tight junction. ± ZO-1 Zonula occludens protein 1. Introduction The blood-brain barrier (BBB) of higher vertebrates is located in the endothelial cells of the brain microvessels. On the one hand, its function is to protect the fragile physiological equilibrium of the brain from blood-borne substances, and on the other to ensure the supply of nutrients via specific transport systems. It is known that the BBB changed phylo- genetically from a glial (invertebrates, elasmobranchs) to an endothelial (teleost and higher vertebrates) barrier (Abbott, 1991), which gradually develops during ontogeny under the influence of the neuroectodermal milieu (Engelhardt and Risau, 1995). An intact microenvironment is essential for the maintenance of the endothelial barrier, as was demonstrated by transplantation experiments using chick-quail-chimeras (Stewart and Wiley, 1981). The basis for the mature BBB are elaborate tight junctions (TJs), which continuously connect the endothelial cells, there- by separating the extracellular compartments of the neural paremchyma and the microvasculature. In freeze-fracture EJCB 707 European Journal of Cell Biology 79, 707 ± 717 (2000, October) ´  Urban & Fischer Verlag ´ Jena http://www.urbanfischer.de/journals/ejcb 0171-9335/00/79/10-707$15.00/0 1) Dr. Hartwig Wolburg, Institute of Pathology, University of Tübin- gen, Liebmeisterstraûe 8, D-72076 Tübingen/Germany, e-mail: hgwol- bur@med.uni-tuebingen.de 2) Both authors contributed equally to this work.