Interferon-γ-mediated apoptosis in intestinal epithelial cells: Implications for potentially beneficial cytokine balancing

Aims: Interferon (IFN)-γ represents one of the key mediators in the pathogenesis of inflammatory bowel diseases. In our hands, IFN-γ shifts the interleukin (IL)-18/IL-18 binding protein (bp) ratio in intestinal epithelial cells (IEC) towards the latter; however, even in the absence of other pro-apoptotic cytokines, cell death occurs.

Aim: To determine the mechanisms involved in IFN-γ mediated cell death of IEC.

Methods: IEC lines (HT-29/Caco-2/T84) were stimulated with different concentrations of IFN-γ. Staining of the cells was performed with Annexin, Sytox Green and Draq 5. DNA was taken from cell lysates to perform DNA laddering, and LDH activity was measured in supernatants of cell cultures. Total cell lysates from IFN-γ stimulated cells and controls were used to detect phosphorylated STAT-1, cleavage of Parp-1, and activation of Caspase 3, 8 and 9 by Western blot analysis. PCR was performed to assess involvement of pro-apoptotic B-cell leukemia/lymphoma (Bcl) family members BAD/BAX as well as of anti-apoptotic members Bcl-2/Bcl-XL in IFN-γ mediated apoptosis. Levels of IL-18/IL-18bp were determined in supernatants and cell lysates by ELISA.

Results: IFN-γ up-regulated IL-18bp in IEC, while IL-18 remained unchanged. Morphological alterations occurred in cells treated with INF-γ (1–100ng/ml) for up to 72h. Depending on the time point cell adhesion was impaired. Apoptosis was confirmed by positive staining of phosphatidylserin by Annexin, while there was no indication for necrosis. DNA cleavage occurred and LDH was released. IFN-γ treatment of the cells caused early phosphorylation of STAT 1. Activation of Caspase 3 and PARP-cleavage was detectable in stimulated cells, while cleavage of Caspase 8 and 9 was not present. Bcl-XL was downregulated on mRNA as well as protein level, whereas BAD mRNA was induced. mRNA of Bcl-2 and BAX was neither found in stimulated nor in unstimulated cells.

Conclusions: Differently to previous reports, IFN-γ alone induces apoptosis in intestinal epithelial cells. Selective inhibition of IFN-γ-mediated pro-apoptotic effects, while preserving the beneficial consequences on the IL-18/IL-18bp ratio, could contribute to the integrity of the mucosal barrier in intestinal inflammation.