Under physiological conditions, the blood–brain barrier (BBB) maintains homeostasis of the central nervous system (CNS) by restricting uncontrolled diffusion of water soluble molecules from the blood into the CNS and by limiting immune cell trafficking into the CNS. The BBB is established at the level of CNS microvascular endothelial cells, which are highly specialized to achieve precise control over the substances that leave or enter the brain. Lack of fenestrae and an extremely low pinocytotic activity of BBB endothelial cells inhibit the transcellular passage of molecules across the barrier whereas tight junctions (TJ) between the brain endothelial cells restrict the paracellular diffusion of solutes and ions across the BBB [
47]. BBB TJs are unique as in a belt-like region the opposing membranes of adjacent brain endothelial cells form continuous and highly interconnected TJ strands, which appear as a chain of fusion points at the ultrastructural level [
10]. In freeze-fracture electron microscopy analysis the fusion points are visualized as strands of TJ particles [
46]. At the BBB TJ particles are predominantly associated with the protoplasmic membrane leaflets (P-face) as usually observed in epithelial cells [
46], whereas strands of TJs particles of peripheral endothelial cells are rather found associated with the extracellular fracture face (E-face) of the membrane bilayer. TJ strands or particles are composed of integral transmembrane proteins [
16], of which occludin and the claudins have been identified to date [
40,
41]. Although structurally unrelated, occludin and the claudins are both type II transmembrane proteins bearing 4 transmembrane domains. They are linked to the actin cytoskeleton by TJ associated cytoplasmic peripheral membrane proteins of the MAGUK (membrane associated with a guanylyl kinase-like domain) family, such as zonula occludens (ZO)-1, ZO-2 and ZO-3 [
41,
45]. Occludin is highly expressed in TJ of the BBB but not in endothelial TJs of non-neuronal tissues [
4,
22], while intact BBB TJs can form in the absence of occludin in vivo [
38]. In contrast to occludin, claudins have been shown to be sufficient to induce TJ strands when transfected into fibroblasts [
15]. The claudin family is comprised of 24 members, which form the backbone of TJs by establishing homophilic and heterophilic interactions via their extracellular loops [
26]. The individual claudins display tissue specific expression patterns and differ in their individual properties in forming dynamic aqueous pores that allow size- and charge-selective paracellular diffusion [
17]. Within each tissue, expression of a unique combination of claudins, therefore, determines the respective paracellular tightness of the TJs for different solutes. Claudin-5 has been demonstrated to be the endothelial cell-specific component of TJs strands [
34] and is also expressed in BBB TJs [
35]. Upon transfection into fibroblasts claudin-5 induces E-face associated TJs [
34], whereas claudin-3, which is specifically expressed in TJ of CNS but not other endothelial cells [
44,
47], induces P-face associated TJs [
16]. This suggests a specific function for claudin-3 in BBB TJs, which is supported further by the recent observation that expression of claudin-3 correlates with BBB maturation in response to Wnt/β-catenin signaling during development [
30]. Whereas the exact function of claudin-3 in BBB TJs remains to be investigated, an essential function for claudin-5 in sealing BBB TJs against small molecules up to 800 Da has been demonstrated by genetic deletion of claudin-5 in mice [
35]. In addition to claudin-5 and claudin-3, expression of claudin-12 has also been described at the BBB [
35]. Previous studies have shown immunostaining for claudin-1 in brain endothelium [
31]. Later studies, however, which specifically excluded cross-reactivity of anti-claudin-1 antibodies with claudin-3 failed to detect any immunostaining for claudin-1 in CNS parenchymal microvessels or in primary mouse or human brain endothelial cells [
2,
20,
43,
47]. Thus, the combined homophilic and heterophilic interactions between claudin-3, claudin-5, and probably also claudin-12, at the BBB seem to be responsible for the unique barrier function of the BBB TJs [
26].
During neurological disorders such as multiple sclerosis (MS) or its animal correlate experimental autoimmune encephalomyelitis (EAE), focal loss of BBB integrity is observed. MS is an inflammatory demyelinating disease of the CNS, in which a large number of inflammatory cells gains access to the CNS parenchyma forming perivascular inflammatory lesions. Lesion formation in MS is known to be associated with focal BBB dysfunction as visualized by gadolinium-enhanced magnetic resonance imaging and occurs early in the pathogenesis of new lesions in MS [
24]. Immunohistological analysis of
post mortem brain samples of MS patients has identified BBB TJs as the anatomical route for BBB leakiness in MS [
29]. In these studies, abnormal distributions of TJ proteins such as occludin and ZO-1 in brain vessels were found to correlate with enhanced BBB leakiness for serum proteins [
25,
37]. EAE reliably models the inflammatory phase of MS and BBB alterations observed in EAE resemble those observed in MS [
1]. We have previously observed the specific loss of claudin-3 immunostaining from those brain microvessels that were surrounded by inflammatory infiltrates [
47], suggesting a direct role for inflammatory cells in disrupting BBB TJs. While much has been learned regarding the sequential steps of immune cell rolling or capture, adhesion and crawling on the BBB, the cellular pathways and the molecular cues mediating immune cell diapedesis across the BBB are only just being unraveled (summarized in [
6,
19]). BBB TJs could be disrupted by the immune cells penetrating the BBB on a paracellular route through the endothelial cell–cell contacts or alternatively, immune cells might traverse the BBB on a transcellular route through the brain endothelial cell itself and thus indirectly alter BBB TJ architecture (summarized in [
9]).
In the present study, we therefore aimed to delineate the role of BBB TJs in immune cell infiltration and focal BBB leakiness during EAE. Based on our previous observation of the specific loss of claudin-3 from BBB TJs, we hypothesized that in brain endothelial cells ectopic expression of claudin-1, which like claudin-3 induces P-face associated TJs upon transfection into fibroblasts [
15], might seal BBB TJs and therefore reduce the paracellular component of immune cell diapedesis across the BBB and/or inflammation-induced BBB leakiness. This notion is supported by previous findings demonstrating that claudin-1 seals TJs in skin epithelial and lung endothelial cells [
13,
14]. Furthermore, TJ strands induced by claudin-3 associate with those induced by claudin-1 suggesting that ectopic expression of claudin-1 in BBB TJs would productively integrate into the BBB TJ strands [
16]. We, therefore, established transgenic mouse lines with tetracycline (TET)-regulated endothelial cell-specific expression of claudin-1. In two independent transgenic mouse lines, we observed TET-induced expression of claudin-1 in 30–50% of PECAM-1
+ CNS microvessels. This partial expression of claudin-1 in BBB TJs sufficed to lead to a significant amelioration of chronic but not acute EAE in double transgenic Tie-2 tTA//TRE–claudin-1 C57BL/6 mice compared to single transgenic littermates.