IFN-α reduces hepatocyte sensitivity to CTL cytotoxicity.

<p>Hepatocytes have been described as expressing low to no MHC Class I. As expected, IFN-α treatment increased the levels of MHC Class I on HepG2 (A) and HHL (B); filled curve represents an isotype control, the solid line represents the MCH Class I expression. C) HepG2 cells were stimulated for 16 hours with a serial dilution of IFN-α at 0 IU/ml (closed circles), 10 IU/ml (open diamonds), 100 IU/ml (open squares), and 1000 IU/ml (open inverted triangles), prior to cytotoxicity assay with the CTL line 2. Treatment with IFN-α reduced the HepG2 cells sensitivity to CTL cytotoxicity in a dose dependent manner. D) This phenomenon was also found with the novel human hepatocyte cell lines (HHL). HHL-17 cells were either left untreated (closed circles) or stimulated for 16 hours with 1000 IU/ml (open inverted triangles) prior to co-incubation with the CTL line 1.</p

Analysis of alternative triggers for PI-9 expression.

<p>A) The up-regulation of PI-9 was also found to be triggered by other inflammatory cytokines. HepG2 cells were treated for 16 hours with either IFN-γ at 100 IU/ml, or IL-1β at 50 ng/ml. GAPDH expression was used as a positive control. B) Stimulation of the HepG2 cell line with either IFN-γ (open squares) or IL-1β (open triangles) also inhibited CTL killing compared to the un-stimulated HepG2 cells (closed circle). C) HepG2 cells were left un-infected (Nil), or infected with either a baculovirus expressing a sub-genomic replicon (NS-replicon), or with a control baculovirus expressing LacZ (+Control). PI-9 expression was analysed by RT-PCR. PI-9 was strongly up-regulated only in the cells expressing the sub-genomic replicon. GAPDH expression was used as a positive control.</p

IFN- treatment did not protect a B-cell line.

<p>Treatment of a BCL with 1000 IU/ml IFN-α (open inverted triangles) prior to the cytotoxicity assay, did not reduce CTLs ability to kill the treated BCL compared to the untreated BCL (closed circles).</p

Clinical data of HCV patients at time of biopsy.

<p>Clinical data of HCV patients at time of biopsy.</p

Expression of the granzyme B inhibitor, serine proteinase inhibitor 9 (PI-9).

<p>A) RT-PCR revealed that IFN-α stimulated cells, HepG2 and HHL-9 cells, were able to upregulate PI-9. GAPDH expression was analysed as a positive control. Bi and ii) This was confirmed by FACS analysis as described in the <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0000791#s4" target="_blank">methods and materials</a>. Upregulation of PI-9 was shown to be dose dependent.</p

Expression of PI-9 in liver tissue from patients with chronic hepatitis C.

<p>Liver specimens obtained from diagnostic biopsy were stained for PI-9 expression as described in the <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0000791#s4" target="_blank">methods</a>. A) Representative negative isotype control. B–D) PI-9 was detected in the majority of hepatocytes. Stronger PI-9 expression, as expected, was seen within the mononuclear infiltrate (highlighted by red arrows), while all the hepatocytes stained positively, albeit at a lower level (the strongest are highlighted by blue arrows).</p

IFN-α treated hepatocytes remain susceptible to FASL-induced apoptosis.

<p>Aii) Induction of apoptosis by FASL was assessed across a concentration range. HepG2 cells, either stimulated with IFN-α (1000 IU/ml) (closed inverted triangles) or left un-stimulated (open diamonds), were incubated with cross-linked rFASL at concentrations ranging from 0.1 pg/ml to 2 µg/ml. Apoptosis was assessed by Annexin V binding and propidium iodine (P.I) incorporation. Ai) Raw FACS data is shown. Bii) HepG2 cells, either un-stimulated (open and closed circles), stimulated with 100 IU/ml (open and closed squares) IFN-α or 1000 IU/ml (open and closed inverted triangles) IFN-α, were incubated with 1 µg/ml (left, open symbols) or 2 µg/ml (right, closed symbols) cross-linked rFASL over a time course of up to 6 hours. Apoptosis was assessed by the cytosolic presence of the activated form of caspase 3. Bi) Raw FACS data.</p