Figure 4

Effect of human cytokines on HCMEC-D3 mono-culture barrier and HCMEC-D3/HFA co-culture barrier. a) Effect of human cytokines (TNF-α (20 ng/ml), IL-1β (20 ng/ml) and IFN-γ (1000 U/ml)) applied to apical + basal sides of human brain endothelial (HCMEC-D3) mono-cultures. Resistance was recorded daily. Significant increase in the resistance of human brain endothelium treated with cytokines in a rank order of IFN-γ ≈ Con ≈ IL-1β > TNF-α was observed. Inset shows the mode of culture and cytokine treatment. Bars indicate standard error. Repeated measured ANOVA with Dunnett's post-hoc test. *p < 0.05 was considered to be statistically significant, **p < 0.01 very significant, and ***p < 0.001 highly significant. b) Effect of human cytokines (TNF-α (20 ng/ml), IL-1β (20 ng/ml) and IFN-γ (1000 U/ml)) on HCMEC-D3/HFA contact dependent co-culture barrier. Human cytokines were added to both apical and basal sides of the contact dependent co-culture system and TEER recorded daily. Co-cultures treated with TNF-α showed a higher loss in barrier integrity than other conditions. The rank order of this experiment is Con≈IL-1β>IFN-γ> TNF-α *p < 0.05 was considered to be statistically significant, **p < 0.01 very significant, and ***p < 0.001 highly significant. c) Effect of human cytokines (TNF-α (20 ng/ml), IL-1β (20 ng/ml) and IFN-γ (1000U/ml)) on HCMEC-D3/HFA contact independent co-culture barrier. Human cytokines were added to both apical and basal chamber of the co-culture system and TEER recorded daily. Co-cultures treated with cytokines showed lesser barrier integrity than untreated controls. The rank order of this experiment is Con> IL-1β ≈IFN-γ > TNF-α *p < 0.05 was considered to be statistically significant, **p < 0.01 very significant, and ***p < 0.001 highly significant. d) Effect of human cytokines (TNF-α (20ng/ml), IL-1β (20ng/ml) and IFN-γ (1000U/ml)) on HCMEC-D3 metabolism. TNF-α (20ng/ml) and IFN-γ (1000U/ml)) significantly decreased mouse brain endothelial cell metabolism by 3 d but not IL-1β (20ng/ml).