Abstract
BackgroundThe brain is protected by the blood-brain barrier (BBB), constituted by endothelial cells supported by pericytes and astrocytes. In Alzheimer’s disease a dysregulation of the BBB occurs since the early phases of the disease leading to an increased access of solutes and immune cells that can participate to the central inflammatory response. Here we investigated whether astrocytes may influence endothelial-leukocytes interaction in the presence of amyloid-β (Aβ).MethodsWe used an in vitro BBB model, where endothelial cells, cultured alone or with astrocytes were exposed for 5 h to Aβ, both under resting or inflammatory conditions (TNFα and IFNγ), to evaluate endothelial barrier properties, as well as transendothelial migration of peripheral blood mononuclear cells (PBMCs).ResultsIn the co-culture model, barrier permeability to solutes was increased by all treatments, but migration was only observed in inflammatory conditions and was prevented by Aβ treatment. On the contrary, in endothelial monocultures, Aβ induced leukocytes migration under resting conditions and did not modify that induced by inflammatory cytokines. In endothelial astrocyte co-cultures, a low molecular weight (MW) isoform of the adhesion molecule ICAM-1, important to allow interaction with PBMCs, was increased after 5 h exposure to inflammatory cytokines, an effect that was prevented by Aβ. This modulation by Aβ was not observed in endothelial monocultures. In addition, endothelial expression of β-1,4-N-acetylglucosaminyltransferase III (Gnt-III), responsible for the formation of the low MW ICAM-1 isoform, was enhanced in inflammatory conditions, but negatively modulated by Aβ only in the co-culture model. miR-200b, increased in astrocytes following Aβ treatment and may represent one of the factors involved in the control of Gnt-III expression.ConclusionThese data point out that, at least in the early phases of Aβ exposure, astrocytes play a role in the modulation of leukocytes migration through the endothelial layer.
Highlights
Alzheimer’s disease is a neurodegenerative disorder classically associated with amyloid β peptide (Aβ) plaques and phosphorylated τ tangles
Endothelial cell cultures were exposed to Aβ (2.5 μM), tumor necrosis factor α (TNFα) and interferon γ (IFNγ) (T&I 10 U/ml and 5 U/ml, respectively) or their association (Aβ+TNFα + IFNγ (T&I)), and viability was evaluated at different time points (5, 18 h) using MTT assay (Figure 1A)
Endothelial barrier properties were tested by evaluating permeability to FITC-conjugated dextran, an indirect measure of the tightness of tight junctions (TJ); exposure of endothelial-astrocytes co-cultures for 5 h to Aβ, T&I or their combination significantly increased dextran permeability (Figure 1C), an effect supported by the reduced expression of Claudin-5, as indicated by western blot analysis (Figure 1D)
Summary
Alzheimer’s disease is a neurodegenerative disorder classically associated with amyloid β peptide (Aβ) plaques and phosphorylated τ tangles. The presence of monocytes and lymphocytes, as well as cytokines production, have been described in the brain of AD patients and in AD animal models (Sugimoto et al, 2003; Zand et al, 2005; Ferrara et al, 2007). This is not the case in physiological conditions, in which the access of immune cells to the CNS is strictly regulated, and only few cells go through the BBB to undertake immune surveillance (Argaw et al, 2012). We investigated whether astrocytes may influence endothelial-leukocytes interaction in the presence of amyloid-β (Aβ)
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