Maintained IL-18bp secretion by intestinal epithelial cells upon selective inhibition of IFN-γ-dependent apoptosis

Introduction: Interferon (IFN)-γ is capable of inducing apoptosis in intestinal epithelial cells (IEC), even in absence of other pro-apoptotic stimuli. At the same it alters the interleukin (IL)-18/IL-18 binding protein (bp) ratio in vitro as well as in vivo.

Aim: To study presence of IL-18bp in vivo and to inhibit IFN-γ mediated apoptosis, while preserving IFN-γ dependent up-regulation of IL-18bp in vitro.

Methods: Cultivated IEC lines HT-29, Caco-2 and T84 were stimulated with different concentrations of IFN-γ. Staining of phosphatidylserine, DNA laddering, cleavage of poly-ADP-ribose-polymerase (Parp) and activation of caspase3 was detected in IFN-γ stimulated cells and controls to determine apoptosis. To inhibit IFN-γ mediated apoptosis caspase3 inhibitor Z-DEVD-FMK and p38 inhibitor SB203580 were used. Biopsies of 11 patients with Crohn's disease (CD), 7 patients with ulcerative colitis (UC) and 6 controls were cultivated for 48h in Rosweli Park Memorial Institute (RPMI) medium. IL-18bp was measured by ELISA.

Results: Apoptosis in IEC treated with IFN-γ was confirmed by positive staining of phosphatidylserin and DNA cleavage. Activation of caspase3 and Parp cleavage was detectable only in IFN-γ treated cells, but not in untreated. IL-18bp expression was >100fold higher (p<0.05) in supernatants and >20fold (p<0.05) higher in lysates of IFN-γ (100ng/ml) treated IEC as compared to controls. Presence of Z-DEVD-FMK and SB203580 preserved the IL-18bp expression, while at the same time IFN-γ mediated apoptosis was inhibited. IL-18bp expression in supernatants of patients with UC and CD was about 2.5times higher than in healthy controls. IL-18bp concentrations in inflamed areas of patients with CD and CU were 20% and 50% higher, respectively, than in controls. IL-18bp expression in inflamed areas of patients with CD was significantly higher than in patients with CU. Stimulation of biopsies of healthy controls with IFN-γ (100ng/ml) resulted in up-regulation of IL-18bp.

Conclusions: IFN-γ induces expression of IL-18bp in vivo and in vitro. Selective inhibition of IFN-γ mediated apoptosis, while preserving beneficial consequences on the ratio of IL-18/IL-18bp, could contribute to the integrity of the mucosal barrier in intestinal inflammation.