Hydrogen peroxide induces mRNA for tumour necrosis factor α in human endothelial cells

Abstract

Reactive oxygen intermediates are important mediators of inflammation. We investigated if hydrogen peroxide (H₂O₂) induces tumour necrosis factor α (TNFα) expression in cultured human cells from umbilical vein endothelium (HUVEC), aortic smooth muscle cells (SMC), peripheral blood mononuclear cells (PBMC), or the cell line Mono Mac 6. Cultures were stimulated with 200 μmol/L H₂O₂ for 15 min. After 4h cells were harvested, mRNA extracted, and amplified by semi-quantitative reverse transcription polymerase chain reaction (RT-PCR) with histone (H3) as reference gene. In HUVECs, mRNA for TNFα increased with a factor of 4 after stimulation (p < 0.001), in PBMC with a factor of 2 (p < 0.05), while mRNA from SMC and Mono Mac 6 did not increase significantly. Cellular TNFα protein in HUVECs was measured with flow cytometry (FACS) before and 6, 12, and 24h after stimulation. TNFα protein was detectable in small, but reproducible amounts 12h after stimulation, and increased further after 24h. However, no secretion of TNFα was detected by ELISA. FACS analysis of the passaged HUVEC cultures did not reveal any contamination with non-endothelial cells. In conclusion, H₂O₂ induces TNFα mRNA in HUVECs and PBMC. In HUVECs an increase of intracellular TNFα protein was also detected, indicating that endothelial cells can produce small amounts of TNFα.

Keywords:

• Endothelial cells
• hydrogen peroxide
• tumour necrosis factor α
• mononuclear blood cells
• Mono Mac 6
• reactive oxygen intermediates
• smooth muscle cells