Figure 4

WEAK increased Lucifer yellow permeability in an in vitro model of the BBB. (A) hCMEC/D3 cells were differentiated on coated Transwell® filters and stimulated (TWEAK, TNF) or not (CTRL) during 24 hours. At time t = 0, Lucifer yellow (LY) was applied in the apical compartment. After 60 min, LY fluorescence was assessed in the lower compartment and the permeability coefficient (Pe) was calculated taking into account the relation between the permeability of the monolayer and the permeability of empty filters (pre-coated, without cells). Each condition was run in triplicate in three independent experiments. TWEAK induces increased passage of LY across the hCMEC/D3 cell monolayer. (B) The passage of BSA-FITC was used to assess transport through primary HCMEC seeded and differentiated on coated filters. Primary HCMEC were stimulated with Fc-TWEAK or TNF for 24 h or not (CTRL). At time t = 0, BSA-FITC was applied in the apical compartment. After 60 min, the fluorescence levels were assessed in the basal compartment. Each condition was run in triplicate. (C) The assay setup of HCMEC for TEER was the same as for the BBB permeability assay described in (B). HCMEC TEER was measured by the Electrical Resistance System Millicell-ERS-2. Coated inserts without cells were used as a blank (minimum resistance). The electrical resistance of each insert following treatment with TWEAK, P1.17, or TNF was calculated by subtracting the blank from each reading. Each condition was run in duplicate, and the resistance measured twice for each well. In (A), (B) and (C) * p < 0.05 according to Student’s t test.