Gliovascular and cytokine interactions modulate brain endothelial barrier in vitro

Abstract

The glio-vascular unit (G-unit) plays a prominent role in maintaining homeostasis of the blood-brain barrier (BBB) and disturbances in cells forming this unit may seriously dysregulate BBB. The direct and indirect effects of cytokines on cellular components of the BBB are not yet unclear. The present study compares the effects of cytokines and cytokine-treated astrocytes on brain endothelial barrier. 3-dimensional transwell co-cultures of brain endothelium and related-barrier forming cells with astrocytes were used to investigate gliovascular barrier responses to cytokines during pathological stresses. Gliovascular barrier was measured using trans-endothelial electrical resistance (TEER), a sensitive index of in vitro barrier integrity. We found that neither TNF-α, IL-1β or IFN-γ directly reduced barrier in human or mouse brain endothelial cells or ECV-304 barrier (independent of cell viability/metabolism), but found that astrocyte exposure to cytokines in co-culture significantly reduced endothelial (and ECV-304) barrier. These results indicate that the barrier established by human and mouse brain endothelial cells (and other cells) may respond positively to cytokines alone, but that during pathological conditions, cytokines dysregulate the barrier forming cells indirectly through astrocyte activation involving reorganization of junctions, matrix, focal adhesion or release of barrier modulating factors (e.g. oxidants, MMPs).

Figure 1

Effect of mouse cytokines on bend-3 mono-culture barrier and bEnd-3/HFA co-culture barrier. a) Cumulative effect of mouse cytokines (TNF-α (20 ng/ml), IL-1β (20 ng/ml) and IFN-γ (1000 U/ml)) applied to apical + basal sides of mouse brain endothelial mono-cultures. Resistance was recorded daily (7 d). Significant increase in the resistance of mouse brain endothelium was observed in a rank order of IFN-γ > TNF-α > IL-1β compared with control. 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 mouse cytokines (TNF-α (20 ng/ml), IL-1β (20 ng/ml) and IFN-γ (1000 U/ml)) of mouse brain endothelial solute permeability. Solute permeability was measured at 30', 1 h, 2 h, 3 h, 4 h and 6 h after 3 d of treatment. TNF-α and IFN-γ treated cultures showed lesser permeability than control or IL-1b treated cultures. The solute permeability of mouse brain endothelium in this experiment was in a rank order of IFN-γ ≈ TNF-α > IL-1β ≈ Con. 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. c) Effect of mouse cytokines (TNF-α (20 ng/ml), IL-1β (20 ng/ml) and IFN-γ (1000 U/ml)) on contact dependent bEnd-3/HFA co-culture system. Resistance was recorded daily. Significant increase in mouse brain endothelial barrier was observed with IFN-γ > IL-1β ≥ TNF-α compared to controls. Inset shows the mode of contact dependent system used and cytokine addition. Bars indicate standard error. Repeated measures ANOVA with Dunnett's post-hoc test. d) Effect of mouse cytokines (TNF-α (20 ng/ml), IL-1β (20 ng/ml) and IFN-γ (1000 U/ml)) on contact independent bEnd-3/HFA co-culture system. Resistance was recorded daily. Significant increase in the resistance of brain endothelium was observed with IFN-γ > IL-1β ≥ TNF-α compared with control. Inset shows the mode of contact dependent system used and cytokine addition. Bars indicate standard error. Repeated measures 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.

Figure 2

Effect of human cytokines on human astrocytes in contact-independent mouse brain endothelial co-culture barrier. Astrocytes were treated with human cytokines (TNF-α (20 ng/ml), IL-1β (20 ng/ml) and IFN-γ (1000 U/ml)) in a contact independent bEnd-3/HFA co-culture system. Resistance was recorded daily. hIFN-γ treated co-cultures from 5 d- 7 d showed decreased barrier compared to other treatment and control conditions. Inset shows the mode of co-culture system and cytokine addition. Bars indicate standard error. Repeated measures 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.

Figure 3

Effect of mouse cytokines (TNF-α (20 ng/ml), IL-1β (20 ng/ml) and IFN-γ (1000 U/ml)) on mouse brain endothelial metabolism. TNF-α (20 ng/ml) and IFN-γ (1000 U/ml)) significantly decreased mouse brain endothelial cell metabolism by 4 d but not IL-1β (20 ng/ml).

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).

Figure 5

Effect of human cytokines on HBMEC-3 mono-culture barrier and HBMEC-3/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 (HBMEC-3) mono-cultures. Resistance was recorded daily. No significant difference in the resistance of cytokine treated HBMEC-3 barrier to that of untreated HBMEC-3 barrier was noted in this experiment. 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 HBMEC-3/HFA co-culture barrier. Human cytokines were added to both apical and basal sides of the co-culture system and TEER recorded daily. Co-cultures treated with cytokines showed slightly lesser barrier integrity than untreated controls. The rank order of this experiment is Con> IL-1β ≈TNF-α> IFN-γ *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 (1000 U/ml)) on HCMEC-D3 metabolism. TNF-α (20 ng/ml), IL-1β (20 ng/ml) and IFN-γ (1000 U/ml)) significantly decreased mouse brain endothelial cell metabolism by 3 d.

Figure 6

Effect of human cytokines in ECV-304 mono-culture barrier and ECV-304/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 ECV-304 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 ≈ TNF-α ≈> IL-1β > Con 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 contact independent ECV-304/HFA co-culture system. Resistance was recorded daily. After 5 d barrier was pronouncedly lost in all conditions. TEER readings obtained until 5 d were plotted to observe the effect of human cytokines on species matched co-culture barrier. A rank order of Con>TNF-α>IL-1β ≈ IFN-γ was observed. Inset shows the mode of contact dependent system used and cytokine addition. Bars indicate standard error. Repeated measures 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. c) Effect of human cytokines (TNF-α (20 ng/ml), IL-1β (20 ng/ml) and IFN-γ (1000 U/ml)) on in vitro cell metabolism. TNF-α (20 ng/ml), IL-1β (20 ng/ml) and IFN-γ (1000 U/ml)) significantly decreased ECV-304 metabolism by 3 d. Bars indicate standard error. One way ANOVA with Dunnett's post-test. *p < 0.05 was considered to be statistically significant, **p < 0.01 very significant, and ***p < 0.001 highly significant.