Tumor Necrosis Factor-α Renders Human Peritoneal Mesothelial Cells Sensitive to Anti-Fas Antibody-Induced Apoptosis

To investigate the role of Fas in the apoptosis of human peritoneal mesothelial cells (HPMC),HPMC were exposed to cytokines (tumor necrosis factor-α (TNF-α)and/or interferon-γ(IFN-γ)) for 48 hours and then stimulated with anti-Fas antibody (Clone CH11) for 24 hours. RT-PCR and flow cytometry were used to assess the expression of Fas. Western blot was used to assess the expression of Bcl-2 protein. Propidium iodide(PI)staining, TUNEL method and M30 Cytodeath antibody were used to assess apoptosis. Inhibitor of caspase 3 and 8 were used to prevent the apoptotic process. Our results showed that resting HPMC could express Fas and, after 48 hours treatment, TNF-α (5 ng/ml) increased the expression by 48% (P＜0.001). INF-γ(5 ng/ml) did not increase the expression of Fas. While resting HPMC were resistant to anti-Fas treatment, TNF-α rendered HPMC sensitive to anti-Fas antibody-induced apoptosis. PI staining showed nuclear condensation of HPMC and a hypodiploid peark on DNA histogram. There were also DNA fragmentation and cytokeratin cleavage. TNF-α treatment was also accompanied by down-regulation of Bcl-2 protien. Western blot showed there was activation of caspase 3 and 8. The Fas-induced apoptosis could be prevented by pre-incubating HPMC in caspase 8 and 3 inhibitors. In conclusion, TNF-α upergulates Fas expression in HPMC and renders HPMC sensitive to anti-Fasantibody induced apoptosis. The apoptotic process is executed through caspase 8 and 3. This might be an important mechanism of HPMC cell death during peritonitis.

並列關鍵字

apoptosis ； fas ； mesothelial cells ； peritoneal dialysis

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