Cytotoxicity of cytokines in cerebral microvascular endothelial cell.
OBJECTIVE
Several studies reported that the levels of proinflammatory cytokines such as TNF-alpha, IL-1beta, IL-6, and IL-8 are elevated in the cerebrospinal fluid (CSF) of patients after subarachnoid hemorrhage (SAH). Cytokines in CSF may contribute to the development of vasospasm and cerebral ischemia. In the present study, we investigated the possible cytotoxic effects of these cytokines on cultured cerebral microvascular endothelial cells.


METHODS
The effects of TNF-alpha, IL-1beta, IL-6, and IL-8 were tested using cell viability assay, DNA fragmentation analysis (DNA laddering), Western blot analysis (Anti-poly-(ADP-ribose) polymerase [PARP] antibody), and caspase-3 activity.


RESULTS
TNF-alpha and IL-1beta, but not IL-6 or IL-8, caused cell detachment in a dose-dependent manner (p<0.05). TNF-alpha (200 pg/ml) and IL-1beta (150 pg/ml) produced DNA ladders at 24-72 h. TNF-alpha but not IL-1beta cleaved the PARP from 116- to 85-kDa fragments and enhanced caspase-3 activity at 24-72 h after incubation with endothelial cells. Caspase-3 inhibitor at 10 micromol/l significantly prevented TNF-alpha-induced cell detachment (p<0.05).


CONCLUSIONS
TNF-alpha induces apoptosis in cultured cerebral endothelial cells through the cleavage of caspase-3. IL-1beta decreases the adherent cells, produces DNA ladders, but fails to cleave PARP or increase caspase-3 activity. IL-1beta may induce apoptosis in cerebral endothelial cells through different pathway from that of TNF-alpha.
Department of Neurosurgery, University of Mississippi Medical Center, Jackson, MS 39216, USA.
Hitoshi
H
Kimura
Ilker
I
Gules
Toshinari
T
Meguro
John H
JH
Zhang
D000818:Animals; D015153:Blotting, Western; D002196:Capillaries / Q000166:cytology / Q000187:drug effects; D053148:Caspase 3; D061945:Caspase Inhibitors; D020169:Caspases / Q000378:metabolism; D002417:Cattle; D002452:Cell Count; D002460:Cell Line; D002470:Cell Survival / Q000187:drug effects; D002560:Cerebrovascular Circulation / Q000187:drug effects*; D016207:Cytokines / Q000633:toxicity*; D053938:DNA Fragmentation / Q000187:drug effects; D042783:Endothelial Cells / Q000187:drug effects*; D004791:Enzyme Inhibitors / Q000494:pharmacology; D007375:Interleukin-1 / Q000633:toxicity; D015850:Interleukin-6 / Q000633:toxicity; D016209:Interleukin-8 / Q000633:toxicity; D011065:Poly(ADP-ribose) Polymerases / Q000378:metabolism; D013345:Subarachnoid Hemorrhage / Q000473:pathology; D014409:Tumor Necrosis Factor-alpha / Q000633:toxicity
D061945:Caspase Inhibitors; D016207:Cytokines; D004791:Enzyme Inhibitors; D007375:Interleukin-1; D015850:Interleukin-6; D016209:Interleukin-8; D014409:Tumor Necrosis Factor-alpha; D011065:Poly(ADP-ribose) Polymerases; D053148:Caspase 3; D020169:Caspases