Tumor necrosis factor and reactive oxygen species cooperative cytotoxicity is mediated via inhibition of NF-kappaB.
BACKGROUND
Tumor necrosis factor alpha (TNFalpha) plays a key role in pathogenesis of brain injury. However, TNFalpha exhibits no cytotoxicity in primary cultures of brain cells. This discrepancy suggests that other pathogenic stimuli that exist in the setting of brain injury precipitate TNFalpha cytotoxicity. The hypothesis was tested that reactive oxygen species (ROS), that are released early after brain injury, act synergistically with TNFalpha in causing cell death.


METHODS
Cultured human and rat brain capillary endothelial cells (RBEC), and cortical astrocytes were treated with TNFalpha alone or together with different doses of H2O2, and apoptotic cell death and DNA fragmentation were measured by means of 3'-OH-terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling (TUNEL) and Hoechst fluorescence assay, respectively. The effect of H2O2 on TNFalpha-induced activation of nuclear factor kappa B (NF-kappaB) was measured by Western blots of cytoplasmic and nuclear extracts of RBEC using anti-inhibitor of NF-kappaB (IkappaB) and anti-p65 subunit of NF-kappaB antibodies. Nuclear translocation of NF-kappaB was investigated by immunofluorescent staining of RBEC with anti-p65 antibodies.


RESULTS
TNFalpha alone had no cytotoxic effect in brain endothelial cells and astrocytes at concentrations up to 100 ng/ml. Co-treatment with 5-10 microM of H2O2 caused a two-fold increase in the number of apoptotic cells 24 hr later. Similar doses (1-3 microM) of H2O2 initiated early DNA fragmentation. H2O2 inhibited TNFalpha-induced accumulation of p65 in the nucleus, although it had no effect on degradation of the IkappaB in cytoplasm. Immunostaining confirmed that H2O2 inhibited p65 transport to the nucleus.


CONCLUSIONS
Reactive oxygen species could act synergistically with TNFalpha in causing cytotoxicity via inhibition of a cytoprotective branch of TNFalpha signaling pathways, which starts with NF-kappaB activation.
Ginis
I
I
Stroke Branch, National Institute of Neurological Disorders and Stroke, NIH, Bethesda, MD 20892-4128, USA. ginisi@ninds.nih.gov
Hallenbeck
J M
JM
Liu
J
J
Spatz
M
M
Jaiswal
R
R
Shohami
E
E
D000818:Animals; D017209:Apoptosis; D001253:Astrocytes / Q000166:cytology / Q000378:metabolism; D015153:Blotting, Western; D001921:Brain / Q000378:metabolism*; D002196:Capillaries / Q000378:metabolism; D002467:Cell Nucleus / Q000378:metabolism; D002478:Cells, Cultured; D003600:Cytosol / Q000378:metabolism; D053938:DNA Fragmentation; D004305:Dose-Response Relationship, Drug; D004730:Endothelium, Vascular / Q000166:cytology / Q000378:metabolism; D004789:Enzyme Activation; D006801:Humans; D006861:Hydrogen Peroxide / Q000494:pharmacology; D020287:In Situ Nick-End Labeling; D008856:Microscopy, Fluorescence; D016328:NF-kappa B / Q000378:metabolism* / Q000502:physiology*; D051381:Rats; D017382:Reactive Oxygen Species / Q000378:metabolism*; D013997:Time Factors; D051996:Transcription Factor RelA; D014409:Tumor Necrosis Factor-alpha / Q000378:metabolism*
D016328:NF-kappa B; D017382:Reactive Oxygen Species; D051996:Transcription Factor RelA; D014409:Tumor Necrosis Factor-alpha; D006861:Hydrogen Peroxide