Inflammatory activation of human brain endothelial cells by hypoxic astrocytes in vitro is mediated by IL-1 beta

Leukocyte infiltration into the brain contributes to the development of isc hemic brain damage and is mediated by endothelial/leukocyte adhesion molecu les, cytokines, and chemokines released by ischemic brain cells. In this st udy, we provide evidence that human astrocytes (FWAs) subjected to in vitro hypoxia produce proinflammatory mediator(s) capable of up-regulating infla mmatory genes, including intercellular adhesion molecule-1, interleukin (IL )-1 beta, tumor necrosis factor-alpha, IL-8, and monocyte chemotactic prote in-1 (MCP-1) in human cerebromicrovascular endothelial cells (HCECs). FHAS were exposed to hypoxia in an anaerobic chamber fur 4 hours, followed by re oxygenation for 24 hours. Astrocyte-conditioned media (ACM) collected from normoxic FHAS or FHAS subjected to hypoxia/reoxygenation were applied to HC EC cultures for 4 to 24 hours. Semiquantitative reverse transcription-polym erase chain reaction, immunocytochemistry, and enzyme-linked immunosorbent assay demonstrated up-regulation of intercellular adhesion molecule-1 in HC ECs exposed to hypoxic ACM. A pronounced elevation in cytokine IL-1 beta an d tumor necrosis faotor-alpha, and chemokine IL-8 and MCP-1 mRNA, accompani ed by increased release of immunoreactive cytokines and chemokines into cel l media was observed in HCECs exposed to hypoxic ACM. Hypoxia/reoxygenation induced a transient (4 to 18 hours of reoxygenation) upregulation of IL-1 beta mRNA in FHAS and a two- to threefold increase in IL-1 beta levels secr eted into ACM. Pretreatment of FHAS with 10 mu mol/L. dexamethasone inhibit ed both hypoxia-induced expression/secretion of IL-1 beta and the ability o f hypoxic ACM to induce inflammatory phenotype in HCECs. The ability of hyp oxic ACM to up-regulate inflammatory genes in HCECs was inhibited in the pr esence of IL-1 receptor antagonist (IL-1Ra) and by pretreating ACM with the blocking anti-IL-1 beta antibody. These findings strongly implicate IL-1 b eta secreted by hypoxic astrocytes in triggering inflammatory activation of HCECs and thereby influencing inflammatory responses at the site of the bl ood-brain barrier.