Human brain astrocytes mediate TRAIL-mediated apoptosis after treatment with IFN-gamma

Abstract

TNF-related apoptosis inducing ligand (TRAIL) expressions were studied in primary human brain astrocytes in response to pro-inflammatory cytokines. When astrocytes were treated with IL-1beta, TNF-alpha or IFN-gamma, TRAIL was induced in cultured fetal astrocytes. In particular, IFN-gamma induced the highest levels of TRAIL in cultured astrocytes. When astrocytes were pre-treated with IFN-gamma, they induced apoptosis in TRAIL-sensitive Peer cells. Our results suggest that IFN-gamma modulates the expression of TRAIL in astrocytes, which may enhance cytotoxic sensitivity of infiltrating immune cells or brain cells other than astrocytes during inflammation of brain.

Fig. 1

Effect of IFN-γ on TRAIL expression in astrocytes. 2 × 10⁵ astrocytes were pre-treated with IFN-γ (250 U/mL) for 8 hr. (A) RPA was performed using 10 µg total RNA. IL-1 (100 U/mL) or TNF-α (750 U/mL) was treated for 8 h. (B) TRAIL expression was determined by semi-quantitative RT-PCR, and PCR product obtained was subjected to southern blotting. β-actin was used as an internal control. RT-PCR was performed in quadruplicate and representative results are shown. (C) For western blotting 40 µg of each cell homogenate were loaded on a 12% PAGE. Proteins were blotted onto a nitrocellulose membrane and incubated with 5 µg/mL of polyclonal mouse anti-human TRAIL antibody.

Fig. 2

Induction of apoptosis of Peer cells by astrocytes treated with IFN-γ. 2 × 10⁵ fetal brain astrocytes were pre-treated with 250 U/mL of IFN-γ for 24 hr. Effector cells (astrocytes) and target cells (Peer cells) were cocultured for 24 hr at an E:T ratio of 1:1, 2:1 or 4:1. After collecting floating Peer cells, cell death was measured by staining with annexin V-FITC and propidium iodide. Total 1 × 10⁵ cells were analyzed by a flow cytometry. Astrocytes not treated with IFN-γ were used as control effector cells. The assay was performed in quadruplicate and representative results are shown.