Rituximab mode of action.

<p>Rituximab is a monoclonal antibody that recognises the transmembrane receptor CD20 on B cells. Patient treatment with rituximab results in rapid depletion of CD20+ B cells from the circulation, where B cells are deleted predominantly via antibody-mediated and complement-dependent cytotoxicity (ADCC and CDC respectively). NK cells are thought to be the major ADCC-mediating effector cells.</p

An in vitro model to study the effect(s) of rituximab on B cell-associated HCV.

<p>L3055 B cells were exposed to HCVcc JFH-1 for 2 hours, washed to remove non-associated virus and treated with rituximab or control antibody. Resting or IL-2 stimulated PBMC from healthy individuals were co-cultured with the target cells for 4 hours and CD107a content measured in NK cells by flow cytometry. The plots indicate gating on the CD3-CD56+ NK cells, which were further analysed for CD56 expression levels and CD107a marker for degranulation (A). CD56^low NK cells are the subpopulation responsible for degranulation following rituximab treatment. The level of infectious virus released by L3055 B cells treated with rituximab (white squares) or control antibody (black squares) was measured by harvesting supernatants from the ADCC assay and assessing infectivity using the permissive Huh-7 hepatoma cell line. Data are presented as focus forming units/ml (FFU/ml) (B). HCVcc JFH-1 infectivity was comparable following incubation in media for 5 hours at 37°C (virus only), or in the presence of B cell ADCC assays in the presence of rituximab or control antibody (C). Error bars represent means from at least 4 replicates ± SD, * p<0.05 compared to control treatment.</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

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

The effects of PCM and PCM CD154-depleted upon human hepatocyte ROS accumulation and cell death.

<p><a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0030867#pone-0030867-g005" target="_blank">Figures 5a</a> demonstrates representative flow cytometry plots to illustrate the effect of PCM (hatched red) and PCM CD154-depleted (solid grey) upon human hepatocyte ROS accumulation during H-R. The gate used to analyse primary human hepatocytes is the same as that shown in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0030867#pone-0030867-g001" target="_blank">Figures 1</a> & <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0030867#pone-0030867-g002" target="_blank">2</a>. The area of interest within the flow cytometry plots is marked by the vertical ellipse. The area on the left of each ellipse again represents cell debris. <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0030867#pone-0030867-g005" target="_blank">Figure 5b</a>, human hepatocytes were treated with PCM or PCM CD154-depleted during H-R and the percentage of cells staining with both the ROS probe DCF and apoptotic marker, Annexin-V, were assessed by flow cytometry.. The percentage of human hepatocytes that stain for both DCF and Annexin-V are shown in parentheses. Data is representative 3 separate experiments (*p<0.05 relative to basal, **p<0.05 relative to PCM).</p

CD40 Expression on Primary Human Hepatocytes.

<p><a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0030867#pone-0030867-g001" target="_blank">Figure 1a</a> demonstrates a representative flow cytometry plot of CD40 expression on primary human hepatocytes during normoxia, hypoxia and H-R. The plot on the left hand side represents a typical forward scatter (FS) versus side scatter (SS) plot of primary human hepatocytes. The FS versus SS plots shown is from the H-R sample of a liver preparation but similar plots were obtained during normoxia and hypoxia (data not shown). <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0030867#pone-0030867-g001" target="_blank">Figure 1b</a> shows a bar chart with the pooled data of three separate experiments illustrating the level of CD40 expression on primary human hepatocytes. Data are expressed as MFI and readings are based upon values taken from cells within the gated region in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0030867#pone-0030867-g001" target="_blank">Figure 1a</a>.</p

Proposed mechanism of CD40-CD154 mediated apoptosis and necrosis in human hepatocytes during normoxia, hypoxia and H-R.

<p>During normoxia (blue arrows) activation of CD40 expressed upon human hepatocyte by CD154 results in the translocation of the TRAF adaptor molecules to the cell membrane. The TRAF molecules are then responsible for the recruitment of the flavoenzyme NADPH Oxidase to the CD40∶TRAF complex. Here NADPH Oxidase can induce the production of ROS, primarily in the form of hydrogen peroxide. This process can be inhibited by the NADPH Oxidase inhibitor DPI. The resultant accumulation of ROS can result directly in necrotic cell death or it can activate the MAPK members, JNK and p38, to induce human hepatocyte apoptosis. However, during hypoxia (red arrows) CD40 activation on human hepatocytes does not result in ROS accumulation but can induce FasL expression via mechanisms that NADPH Oxidase-dependent and ROS-independent (dotted black line). This increased FasL expression can result in autocrine (green arrow) and/or paracrine (orange arrows) Fas-mediated apoptosis. Due to the increases in intracellular antioxidants levels during hypoxia CD154 does not increase apoptosis. However, mitochondrial ROS does contribute to apoptosis during hypoxia. During H-R, CD40 activation can lead to NADPH Oxidase-ROS-JNK/p38 dependent apoptosis, NADPH Oxidase-ROS-dependent necrosis and NADPH Oxidase∶ROS∶FasL expression with resultant Fas-mediated apoptosis. <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0030867#pone-0030867-g006" target="_blank">Figure 6</a> highlights the importance of local microenvironment in shaping the effects of CD40∶CD154.</p

CD40∶CD154 regulates ROS accumulation, apoptosis and necrosis within human hepatocytes in a NADPH Oxidase dependent manner during normoxia and H-R.

<p><a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0030867#pone-0030867-g002" target="_blank">Figure 2a</a> demonstrates representative flow cytometry plots to illustrate the effect of CD154 (hatched red) and CD154 in presence of DPI (solid grey) upon human hepatocyte ROS accumulation during normoxia, hypoxia and H-R. Typical FS versus SS plots of primary human hepatocytes during normoxia, hypoxia and H-R are shown to the left of each flow cytometric plot. The FS versus SS plots shown is from the H-R alone sample of a liver preparation but similar plots were obtained during normoxia and hypoxia (data not shown). The areas of interest on the flow cytometric plots are marked by the vertical ellipses. The area on the left of each ellipse represents cell debris. Cell debris is included within the plot as human hepatocytes vary considerably in size and therefore to include all viable human hepatocytes in the analysis a large gate is required on the flow cytometer, this by necessity includes the cell debris. <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0030867#pone-0030867-g002" target="_blank">Figure 2b</a>. shows a bar chart with the pooled data of three separate experiments illustrating the effects of CD154 and CD154+DPI upon human hepatocytes ROS accumulation. Data is expressed as MFI and readings are based upon values taken from cells within the gated region shown in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0030867#pone-0030867-g002" target="_blank">Figure 2a</a>. Data are expressed as mean ± S.E. (*p<0.01 relative to basal, **p<0.05 relative to basal, †p<0.001 relative to CD154). <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0030867#pone-0030867-g002" target="_blank">Figure 2c</a> demonstrates representative flow cytometry plots to illustrate the effect of CD154 (hatched red) and CD154 in presence of DPI (solid grey) upon human hepatocyte apoptosis and necrosis during normoxia, hypoxia and H-R. Again, the area of interest within the flow cytometric plots is marked by the vertical ellipse. The same gate has been applied to primary human hepatocytes for these plots as those shown in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0030867#pone-0030867-g002" target="_blank">Figure 2a</a>. <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0030867#pone-0030867-g002" target="_blank">Figure 2d</a>. shows a bar chart with the pooled data of three separate experiments illustrating the effects of CD154 and CD154+DPI upon human hepatocytes apoptosis and necrosis during normoxia, hypoxia and H-R. Data is expressed as increase/decrease relative to basal, where basal refers to the level of apoptosis or necrosis during normoxia alone. Data are expressed as mean ± S.E. (**p<0.01 relative to basal, *p<0.05 relative to basal, †p<0.05 relative to CD154, §p<0.05 relative to basal). <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0030867#pone-0030867-g002" target="_blank">Figure 2e</a>. Human hepatocytes were treated with CD154 or CD154 following pre-treatment with rotenone (Rot) during normoxia, hypoxia and H-R. The percentage of cells staining with both the ROS probe DCF and apoptotic marker, Annexin-V, were assessed by flow cytometry. The percentage of human hepatocytes that stain for both DCF and Annexin-V are shown in parentheses. Data are representative of 3 separate experiments (†p<0.05 relative to basal, *p<0.05 relative to CD154).</p

FasL Expression on Primary Human Hepatocytes.

<p><a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0030867#pone-0030867-g004" target="_blank">Figure 4a</a> demonstrates a representative flow cytometry plot of FasL expression on primary human hepatocytes during normoxia, hypoxia and H-R. The gate used to analyse primary human hepatocytes is the same as that shown in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0030867#pone-0030867-g001" target="_blank">Figures 1</a> & <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0030867#pone-0030867-g002" target="_blank">2</a>. <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0030867#pone-0030867-g004" target="_blank">Figure 4b</a>. demonstrates representative flow cytometry plots to illustrate the effect of CD154 (hatched red) and CD154 in presence of DPI (solid grey) upon human hepatocyte FasL expression during normoxia, hypoxia and H-R. <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0030867#pone-0030867-g004" target="_blank">Figure 4c</a>. shows a bar chart with the pooled data of three separate experiments illustrating the effects of CD154 upon human hepatocyte FasL expression during normoxia, hypoxia and H-R. Data are expressed as MFI and readings are based upon values taken from cells within the gated region in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0030867#pone-0030867-g001" target="_blank">Figure 1a</a>. Data are expressed as mean ± S.E. (*p<0.05 relative to basal, **p<0.05 relative to CD154).</p