IL-2-mediated apoptosis of kidney tubular epithelial cells is regulated by the caspase-8 inhibitor c-FLIP

Background. Tubular epithelial cells (TECs) are essential in the maintenance of kidney function. Apoptosis of TECs occur during acute and chronic renal allograft rejection as well as other forms of renal injury, including autoimmune nephritis. The regulation of TEC apoptosis by proinflammatory cytokines associated with renal inflammation [e.g., interleukin (IL)-2 and interferon-gamma (IFN-gamma)] has not been extensively investigated. Methods. Apoptosis in murine TECs was determined by FACS with annexin-V or ligation-mediated-polymerase chain reaction (LM-PCR) and mRNA levels by reverse transcription (RT)-PCR or Northern blot. Protein expression was observed using Western blot. Results. IL-2R (CD25) was expressed by murine TECs and up-regulated by IL-2. Both IL-2 and IFN-gamma induced TEC apoptosis and activated caspase-8. Apoptosis with IL-2 was concentration-dependent and blocked by z-IETD-fmk, a specific caspase-8 inhibitor. Apoptosis with IFN-gamma was associated with increased surface expression of Fas, while IL-2 had no effect on Fas. IL-2 did not induce apoptosis in Fas-deficient TECs (M3.1-lpr) suggesting IL-2 regulation of caspase-8 activity requires Fas. Consistent with this, IL-2 but not IFN-gamma was found to decrease mRNA and protein expression of c-FLIP, an endogenous caspase-8 inhibitor in murine TECs. Overexpression of c-FLIP in TECs (CS3.7-FLIP) blocked apoptosis and caspase-8 activation with both IFN-gamma and IL-2. c-FLIP expression was found in kidney cortex, primary and cloned TECs, suggesting c-FLIP is likely a key regulator of caspase-8-mediated apoptosis in vivo. Conclusion. This is the first report of c-FLIP regulation by IL-2 in renal TECs. Augmentation of c-FLIP in TECs may enhance an endogenous mechanism by which TECs normally resist injury to caspase-8-mediated apoptosis and thus may be a useful and novel strategy to prevent tubular injury in transplant rejection and autoimmune nephritis.