Apoptosis induction in cholangiocytes after stimulation with soluble TNF ligands.

<p>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.</p

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

<p>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.</p

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

<p>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.</p

Figure 5

<p>Representative Western blots showing NFkB, c-Fos and c-Jun and pSTAT 3 levels in response to CD154/C4BP stimulation. Aliquots of nuclear or cytoplasmic extracts as appropriate (40 ug protein) from cultured and stimulated primary human cholangiocytes were probed for NFkB (panel I); c-Fos and c-Jun(panel II) or pSTAT3 content. Blots were stripped and re-probed for beta-actin which allowed for normalisation of data for variations in protein loading.</p

Figure 6

<p>Densitometry Histograms showing changes in NFkB, c-Fos and c-Jun and pSTAT3 levels in response to sCD154 and C4BP stimulation determined using Western blotting (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0000159#pone-0000159-g005" target="_blank">figure 5</a>). Changes in levels were assessed by densitometric analysis. These data show that the CD154/C4BP complex had no significant influence upon levels of NFkB or c-Jun, but did decrease levels of c-Fos at 24 hours compared with either unstimulated 24 hour control or the stimulated level observed at 4 hours. *p<0.05 a p<0.05These data show that stimulation of cells with CD154/C4BP complex resulted in a lower level of pSTAT3 which was sustained at 24 hours. *p<0.05 c.f. time point matched untreated control a p<0.05 c.f. treatment matched 4 hour time point.</p

Figure 8

<p>Co-localisation of C4BP and CD40 in liver tumour tissue using dual immunofluorescence. Panels a–c shows a representative section of tumour tissue from a patient with cholangiocarcinoma stained for C4BP (green - FITC) and CD40 (red - PE ). In panel a, the arrows identify an epithelial ductular structure (DS) surrounded by tumour cells (TC) and stromal tissue. Positive C4BP staining is seen within the epithelia, tumour cells, and in mononuclear infiltrate in the surrounding stromal tissue. Panel b shows the same tissue section stained for CD40 with the arrow identifying the inflammatory cells within the surrounding stroma. Panels d and e shows a sequential section from the same specimen where the primary antibodies have been substituted for non immune serum (control). Panel c shows the merged image for panels a and b. The bright yellow areas indicate regions of C4BP and CD40 co-localisation within the epithelial cells of the ductular structure, many surrounding tumour cells, and the inflammatory cells within the surrounding stromal tissue.</p

Figure 7

<p>Immunolocalisation of C4BP in human liver tissue. This figure shows three representative sections of liver tissue stained for C4BP. Panel a shows normal liver tissue which is predominantly negative. Panel b shows PSC liver tissue showing the presence of strongly staining inflammatory cells surrounding the portal tract and within the sinusoids. Panel c shows hepatic tumour tissue taken from a hepatic resection for secondary liver cancer (colorectal hepatic metastasis) where very strong staining was observed in the tumour tissue and the inflammatory infiltrate at the tumour margin.</p

Figure 1

<p>Panel a) This histogram shows inhibition of sCD154 mediated apoptosis by C4BP but not apoptosis induced by 0.2 mM TDC. * sCD154 or **TDC induced similar levels of cholangiocyte apoptosis when experimental error was taken into account (59.7%+/−7.5 and 83.4%+/−7.7 and respectively) relative to control (p<.005)***. C4BP + sCD154 reduced apoptosis to control levels (p<0.005) whereas C4BP had no effect on TDC induced apoptosis (81.5%+/−6.7). C4BP/sCD154 also had no effect on TDC mediated apoptosis (data not shown) Panel b and c show representative cytospins stained for fragmented DNA using ISEL. Panel d) Histogram summary of the effects of sCD154 and C4BP on Cholangiocyte proliferation. Primary human cholangiocytes were cultured in 24-well culture plates and simulated with either sCD154, C4BP or a mixture of both. Following incubation for 24 hours, the cells were fixed and proliferation assessed by immunohistochemical staining for Ki-67 antigen. No significant difference was seen between the un-stimulated controls and treated samples, implying that sCD154, C4BP or the mixture had any effect on cholangiocyte proliferation. These data represent the mean of three different counted areas per well repeated for three different liver preparations.</p

Figure 4

<p>Fractionation of C4BP/sCD154 complex by gel filtration. C4BP and sCD154 were incubated together at 37°C for 1 hour. After this time the solution was eluted on a Sepacryl-300 with 100 ul fractions collected up to a final eluted volume of 20 ml, which encompassed void volume through to the lower limit of the fractionation range ( Dextran blue to cytochrome C). Fractions were assayed for the presence of sCD154 using a commercially available ELISA kit.</p

Effect of Percoll upon Cell Viability and Absolute Cell Count.

<p>The effects of Percoll upon human hepatocyte isolation procedures. We used Percoll after eight human hepatocyte isolation procedures (normal resected liver tissue 1, biliary cirrhosis 5, donor liver tissue 1 and normal benign liver tissue 1). All values are represented as medians and the values in parentheses represent the range.</p