Hydrogen peroxide upregulates TNF-related apoptosis-inducing ligand (TRAIL) expression in human astroglial cells, and augments apoptosis of T cells

Abstract

The brain is particularly vulnerable to oxygen free radicals, and these radicals have been implicated in the pathology of several neurological disorders. In this study, the modulation of TNF-related apoptosis-inducing ligand (TRAIL) expression by oxidative stress was shown in LN215 cells, an astroglioma cell line. Hydrogen peroxide (H2O2) treatment increased TRAIL expression in LN215 cells and H2O2-induced TRAIL augmented apoptosis in Peer cells, a cell line sensitive to TRAIL- mediated cell death. Our findings suggest that the upregulation of TRAIL in astroglial cells may abrogate immune cell effector functions.

Fig. 1

Gene expression of TRAIL in human astroglial cells after treatment with hydrogen peroxide (H₂O₂) in a dose or time-dependent manner. (A) LN215 cells were treated with varying doses of hydrogen peroxide (0 to 800 µM) for 4 hrs. (B) LN215 cells were incubated with hydrogen peroxide 800 µM for various times (0 to 24 hrs). Total RNA was measured for TRAIL mRNA by RT-PCR (A) or RNase protection assay (B). Cells not treated with H₂O₂ were used as control (CNTL). The data are representative of three experiments.

Fig. 2

Expression of TRAIL in human astroglial cells after treatment with H₂O₂ in a dose or time-dependent manner. (A) LN215 cells were treated with varying doses of hydrogen peroxide (0 to 800 µM) for 18 hrs. (C) LN215 cells were incubated with hydrogen peroxide 800 µM for various times (0 to 24 hrs). For western blot, 20 µg of total protein was loaded in 12% gel and anti-TRAIL monoclonal Ab was used as a primary Ab. (B) FACS analysis of TRAIL expression on LN215 were treated with 800 µM of hydrogen peroxide for 18 hrs. The data are representative of three independent experiments.

Fig. 3

Inhibition of TRAIL expression by cyclosporin A (CsA) in human astroglial cells after treatment with H₂O₂. (A) LN215 cells were pre-incubated with CsA (0.01 to 10 µM) for 30 min, were treated with hydrogen peroxide (800 µM) for 4 hrs and then gene expression of TRAIL were measured by RT-PCR. Cells not treated with H₂O₂ were used as a control. β-actin was used as the internal control. The fold induction was calculated as follows: (TRAIL/β-actin intensity in the study group)/(TRAIL/β-actin intensity in the control group). The fold induction value represents mean ± SD of triplicate experiments. (B) LN215 cells were pre-incubated with CsA (1 µM) for 30 min, were treated with hydrogen peroxide (800 µM) for 18 hrs and then expression of TRAIL were measured by western blot. The data are representative of three independent experiments.

Fig. 4

Induction of apoptosis in Peer cells by human astroglial cells treated with H₂O₂. LN215 cells were incubated with 800 µM of H₂O₂ for 18 hrs in serum free medium. LN215 cells were washed to remove H₂O₂ and then co-cultured with Peer cells for 24 hrs. Effector cells (LN215 cells) and target cells (Peer cells) were co-cultured at a ratio of 4 : 1. Cell death was measured by staining with annexin V. Control LN215 cells were not treated with H₂O₂, but maintained in serum free condition for 18 hrs. LN215 were pre-incubated for 30 min with CsA (1 µg/mL), and incubated with H₂O₂. The fold induction value represents a mean ± SD of triplicate experiments.