ATP Induces Disruption of Tight Junction Proteins via IL-1 Beta-Dependent MMP-9 Activation of Human Blood-Brain Barrier In Vitro

Abstract

Disruption of blood-brain barrier (BBB) follows brain trauma or central nervous system (CNS) stress. However, the mechanisms leading to this process or the underlying neural plasticity are not clearly known. We hypothesized that ATP/P2X7R signaling regulates the integrity of BBB. Activation of P2X7 receptor (P2X7R) by ATP induces the release of interleukin-1β (IL-1β), which in turn enhances the activity of matrix metalloproteinase-9 (MMP-9). Degradation of tight junction proteins (TJPs) such as ZO-1 and occludin occurs, which finally contributes to disruption of BBB. A contact coculture system using human astrocytes and hCMEC/D3, an immortalized human brain endothelial cell line, was used to mimic BBB in vitro. Permeability was used to evaluate changes in the integrity of TJPs. ELISA, Western blot, and immunofluorescent staining procedures were used. Our data demonstrated that exposure to the photoreactive ATP analog, 3'-O-(4-benzoyl)benzoyl adenosine 5'-triphosphate (BzATP), induced a significant decrease in ZO-1 and occludin expression. Meanwhile, the decrease of ZO-1 and occludin was significantly attenuated by P2X7R inhibitors, as well as IL-1R and MMP antagonists. Further, the induction of IL-1β and MMP-9 was closely linked to ATP/P2X7R-associated BBB leakage. In conclusion, our study explored the mechanism of ATP/P2X7R signaling in the disruption of BBB following brain trauma/stress injury, especially focusing on the relationship with IL-1β and MMP-9.

Figure 1

A438079 attenuates BzATP-induced disruption of blood-brain barrier in vitro. (a) The IL-1β level was significantly higher in BzATP groups than in control groups (^∗ P < 0.05). A438079 treatment significantly reduced the levels of IL-1β compared with the vehicle group (^∗∗ P < 0.05). (b) The level of MMP-9 was significantly higher in BzATP groups than in control groups (^∗ P < 0.05). A438079 treatment significantly reduced the level of MMP-9 compared with the vehicle group (^∗∗ P < 0.05). (c) BzATP induced hyperpermeability compared with the control groups (^∗ P < 0.05), which was decreased by the P2X7 inhibitor A438079 in the coculture system (^∗∗ P < 0.05). (d) Western blot analysis showed that the expression of ZO-1 and occludin in the BzATP groups was decreased compared with the controls (^∗ P < 0.05), while A438079 treatment significantly attenuated the disruptions of ZO-1 and occludin (^∗∗ P < 0.05).

Figure 2

Confocal microscopy images of immunofluorescence staining of ZO-1 and occludin in hCMEC/D3 cells demonstrating the protective effect of A438079 against BzATP-induced tight junction disruption.

Figure 3

BzATP-induced tight junction disruption and hyperpermeability of endothelial coculture system were attenuated by IL-1β inhibition. (a) The levels of MMP-9 were significantly higher in the BzATP groups than in the control groups (^∗ P < 0.05). IL-1RA treatment significantly reduced the level of MMP-9 versus vehicle group (^∗∗ P < 0.05). (b) BzATP induces hyperpermeability versus control groups (^∗ P < 0.05), which was decreased by IL-1RA in the coculture system (^∗∗ P < 0.05). (c) Confocal microscopy images of immunofluorescence staining of ZO-1 and occludin demonstrating the protective effect of IL-1RA against BzATP-induced tight junction disruption.

Figure 4

MMP-9-induced tight junction disruption and endothelial coculture system hyperpermeability were not attenuated by IL-1β inhibition. (a) The levels of IL-1β were significantly higher in BzATP groups than in control groups (^∗ P < 0.05). However, batimastat + BzATP treatment did not alter the level of IL-1β versus BzATP group (^∗∗ P > 0.05). (b) Exposure to BzATP significantly increased the permeability to FITC-dextran compared with the control groups (^∗ P < 0.05). Batimastat attenuated BzATP-induced paracellular permeability versus BzATP group (^∗∗ P < 0.05). (c) Confocal microscopy images of immunofluorescence staining of ZO-1 and occludin demonstrating the protective effect of batimastat against BzATP-induced tight junction disruption.

Figure 5

IL-1β-induced tight junction disruption and endothelial coculture system hyperpermeability were attenuated by MMP-9 inhibition. (a) The levels of MMP-9 were significantly higher in IL-1β groups than in control groups (^∗ P < 0.05). Batimastat treatment significantly reduced the level of MMP-9 versus vehicle group (^∗∗ P < 0.05). (b) Exposure to IL-1β significantly increased the permeability to FITC-dextran compared with the control groups (^∗ P < 0.05), which was decreased by batimastat in coculture system (^∗∗ P < 0.05). (c) Confocal microscopy images of immunofluorescence staining of ZO-1 and occludin demonstrating the protective effect of batimastat against IL-1β-induced tight junction disruption.

Figure 6

BzATP-induced tight junction disruption and endothelial coculture system hyperpermeability were attenuated by MMP inhibition. (a) The levels of IL-1β were not significantly altered in MMP-9-treated groups versus control groups (^∗ P > 0.05). No statistically significant differences were found between IL-1RA + MMP-9 groups and MMP-9 groups in the levels of IL-1β (^∗∗ P > 0.05). (b) Exposure to MMP-9 significantly increased the permeability to FITC-dextran compared with the control group (^∗ P < 0.05). However, IL-1RA failed to attenuate MMP-9-induced paracellular hyperpermeability (^∗∗ P > 0.05). (c) Immunofluorescence staining showed that the expression of ZO-1 and occludin in MMP-9 groups was significantly decreased compared with controls, while IL-1RA + MMP-9 groups showed no improvement in the levels of the two tight junction proteins.