Primary and malignant cholangiocytes undergo CD40 mediated Fas dependent apoptosis, but are insensitive to direct activation with exogenous Fas ligand

Abstract

Introduction: Cholangiocarcinoma is a rare malignancy of the biliary tract, the incidence of which is rising, but the pathogenesis of which remains uncertain. No common genetic defects have been described but it is accepted that chronic inflammation is an important contributing factor. We have shown that primary human cholangiocyte and hepatocyte survival is tightly regulated via co-operative interactions between two tumour necrosis family (TNF) receptor family members; CD40 and Fas (CD95). Functional deficiency of CD154, the ligand for CD40, leads to a failure of clearance of biliary tract infections and a predisposition to cholangiocarcinoma implying a direct link between TNF receptor-mediated apoptosis and the development of cholangiocarcinoma.

Aims: To determine whether malignant cholangiocytes display defects in CD40 mediated apoptosis. By comparing CD40 and Fas-mediated apoptosis and intracellular signalling in primary human cholangiocytes and three cholangiocyte cell lines.

Results: Primary cholangiocytes and cholangiocyte cell lines were relatively insensitive to direct Fas-mediated killing with exogenous FasL when compared with Jurkat cells, which readily underwent Fas-mediated apoptosis, but were extremely sensitive to CD154 stimulation. The sensitivity of cells to CD40 activation was similar in magnitude in both primary and malignant cells and was STAT-3 and AP-1 dependent in both.

Conclusions: 1) Both primary and malignant cholangiocytes are relatively resistant to Fas-mediated killing but show exquisite sensitivity to CD154, suggesting that the CD40 pathway is intact and fully functional in both primary and malignant cholangiocytes 2) The relative insensitivity of cholangiocytes to Fas activation demonstrates the importance of CD40 augmentation of Fas dependent death in these cells. Agonistic therapies which target CD40 and associated intracellular signalling pathways may be effective in promoting apoptosis of malignant cholangiocytes.

Figure 1. Expression of CD40, Fas and FasL protein on the cell surface assessed by flow cytometry.

Cells were grown in media alone or stimulated with IFN-γ at 50 ng/ml for 48 hours. (a) flow cytometry histograms of matched isotype control (red outline) and CD40, Fas and FasL expression (green solid colour) (b) shows the percentage positive cells for CD40, Fas and FasL protein. n = 3+/− standard deviation. *p<0.05, **p<0.01 as compared to untreated controls.

Figure 2. Fas ligand induction to promote apoptosis assessed by RT-PCR at 4 hours.

Cells were grown in culture media alone, stimulated with srh CD154 at 1 µg/ml or srh FasL at 50 ng/ml for 4 hours. (a) densitometry scanning data for FasL PCR gels at 4 hours. n = 4+/− standard error of the mean. (b i) a representative FasL PCR gel and (b ii) the corresponding β-actin gel. *p<0.05, **p<0.01 as compared to untreated controls.

Figure 3. Transcription factor activation in cells undergoing apoptosis after stimulation with soluble TNF ligands.

Cells were grown in media alone, with srh CD154 at 1 µg/ml, srh FasL at 50 ng/ml or srh TNFα at 10 ng/ml for 24 hours. Graphs show densitometry scanning data, n = 3+/− standard deviation and representative autoradiographs of primary cholangiocyte samples showing (a) pSTAT-3 (60 kDa), (b) c-Fos (60 kDa) and (c) c-Jun (35 kDa) all with their corresponding β-actin blots (45 kDa). *p<0.05, **p<0.01, ***p<0.001 as compared to untreated controls.

Figure 4. Apoptosis induction in cholangiocytes after stimulation with soluble TNF ligands.

All cells were cytocentrifuged and stained by the standard ISEL technique to detect apoptosis after culture in media alone, media with srh CD154 at 1 µg/ml or media with srh FasL at 50 ng/ml for 24 hours. All control slides without DNA polymerase klenow fragment were negative (a i). (a ii) primary cholangiocytes in media alone, (a iii) shows primary cholangiocytes stimulated with soluble CD154 and (a iv) primary cholangiocytes challenged with soluble FasL. (b) shows the percentage positive ISEL stained cells in all cell types when grown in media alone, with CD154 or with FasL. n = 3+/− standard deviation. *p<0.05, **p0.01, ***p<0.001 as compared to untreated controls.