Leukocyte infiltration into the brain contributes to the development of ischemic brain damage and is mediated by endothelial/
leukocyte adhesion molecules,
cytokines, and
chemokines released by ischemic brain cells. In this study, we provide evidence that human astrocytes (FHAs) subjected to in vitro
hypoxia produce proinflammatory mediator(s) capable of up-regulating inflammatory genes, including
intercellular adhesion molecule-1,
interleukin (IL)-1beta,
tumor necrosis factor-alpha,
IL-8, and
monocyte chemotactic protein-1 (MCP-1) in human cerebromicrovascular endothelial cells (HCECs). FHAS were exposed to
hypoxia in an anaerobic chamber for 4 hours, followed by reoxygenation for 24 hours. Astrocyte-
conditioned media (ACM) collected from normoxic FHAS or FHAS subjected to
hypoxia/reoxygenation were applied to
HCEC cultures for 4 to 24 hours. Semiquantitative reverse transcription-polymerase chain reaction, immunocytochemistry, and
enzyme-linked
immunosorbent assay demonstrated up-regulation of
intercellular adhesion molecule-1 in HCECs exposed to hypoxic ACM. A pronounced elevation in
cytokine IL-1beta and
tumor necrosis factor-alpha, and
chemokine IL-8 and MCP-1
mRNA, accompanied by increased release of immunoreactive
cytokines and
chemokines into cell media was observed in HCECs exposed to hypoxic ACM.
Hypoxia/reoxygenation induced a transient (4 to 18 hours of reoxygenation) up-regulation of IL-1beta
mRNA in FHAS and a two- to threefold increase in IL-1beta levels secreted into ACM. Pretreatment of FHAS with 10 micromol/L
dexamethasone inhibited both
hypoxia-induced expression/secretion of IL-1beta and the ability of hypoxic ACM to induce inflammatory phenotype in HCECs. The ability of hypoxic ACM to up-regulate inflammatory genes in HCECs was inhibited in the presence of
IL-1 receptor antagonist (IL-1Ra) and by pretreating ACM with the blocking anti-IL-1beta antibody. These findings strongly implicate IL-1beta secreted by hypoxic astrocytes in triggering inflammatory activation of HCECs and thereby influencing inflammatory responses at the site of the blood-brain barrier.