Fig. 7
Astrocyte-derived CXCL1 and endothelial CXCR2 are essential for cerebral endothelial activation. A i.c.v. LPS injection (4 h) induced significant CXCR2 mRNA expression in WT mice. B Levels of CXCR2 protein after i.c.v. LPS injection from 4 to 24 h gradually increased compared with the control group (4 h after i.c.v. saline injection). C The expression of CXCR2 mRNA in primary brain microvascular endothelial cells, microglia, and astrocytes stimulated with either vehicle or TNF-α (100 ng/ml) was measured via real-time PCR. The results are represented as the means ± SEM of three independent experiments; *P < 0.05. D Primary endothelial cells, astrocytes, and microglia were seeded at 2 × 106 cells/well in six-well plates and were incubated overnight. The following day, the cells were stimulated with 100 ng/ml LPS or 100 ng/ml TNF-α for 12 h. Cell lysates were collected and analyzed for CXCR2 expression via Western blotting. E Primary astrocytes and microglia from wild-type mice were seeded at 2 × 106 cells/well in six-well plates and were incubated overnight. The following day, the cells were stimulated with 100 ng/ml LPS or 100 ng/ml TNF-α for 12 h. Then, the conditioned supernatants and cell lysates were collected and analyzed for CXCL1 expression via ELISA. The results are represented as the means ± SEM of three independent experiments; **P < 0.01. F Astrocyte culture conditioned medium was added into primary cerebral endothelial cells from wild-type or CXCR2−/− mice, and the levels of VCAM-1 and ICAM-1 were measured via Western blotting