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Enhancement of Programmed Death Ligand 2 on Hepatitis C Virus Infected Hepatocytes by Calcineurin Inhibitors.

Koike K, Takaki A, Yagi T, Iwasaki Y, Yasunaka T, Sadamori H, Shinoura S, Umeda Y, Yoshida R, Sato D, Nobuoka D, Utsumi M, Miyake Y, Ikeda F, Shiraha H, Fujiwara T, Yamamoto K - Transplantation (2015)

Bottom Line: Treatment of hepatoma cell lines with CNIs resulted in increased PD-L2 expression, especially in combination with HCV core or NS3 protein.The PD-L2 is highly expressed on CH-C-OLT hepatocytes, whereas HCV proteins, in combination with CNIs, induce high expression of PD-L2 resulting in elevated expression of ICAM-1.These findings demonstrate the effect of CNIs on inducing PD-L2 and subsequent ICAM-1 expression, effects that may produce inflammatory cell infiltration in post-OLT hepatitis C.

View Article: PubMed Central - PubMed

Affiliation: 1 Department of Gastroenterology and Hepatology, Okayama University Graduate School of Medicine, Dentistry and Pharmaceutical Sciences, Okayama City, Japan. 2 Department of Gastroenterological Surgery Transplant and Surgical Oncology, Okayama University Graduate School of Medicine, Dentistry and Pharmaceutical Sciences, Okayama City, Japan. 3 Health Service Center, Okayama University, Okayama, Japan.

ABSTRACT

Background: Post orthotopic liver transplantation (OLT) viral hepatitis is an immunological condition where immune cells induce hepatitis during conditions of immune-suppression. The immune-regulatory programmed death-1 (PD-1)/PD-ligand 1 system is acknowledged to play important roles in immune-mediated diseases. However, the PD-1/PD-L2 interaction is not well characterized, with PD-L2 also exhibiting an immunostimulatory function. We hypothesized that this atypical molecule could affect the recurrence of post-OLT hepatitis. To test this hypothesis, we conducted immunohistochemical staining analysis and in vitro analysis of PD-L2.

Methods: The expression of PD-L2 was evaluated in liver biopsy specimens from patients with chronic hepatitis B (n = 15), post-OLT hepatitis B (n = 8), chronic hepatitis C (n = 48), and post-OLT hepatitis C (CH-C-OLT) (n = 14). The effect of calcineurin inhibitors (CNIs) and hepatitis C virus (HCV) on PD-L2 expression was investigated in hepatoma cell lines.

Results: The PD-L2 was highly expressed on CH-C-OLT hepatocytes. Treatment of hepatoma cell lines with CNIs resulted in increased PD-L2 expression, especially in combination with HCV core or NS3 protein. Transfection of cell lines with PD-L2 containing plasmid resulted in high intercellular adhesion molecule-1 (ICAM-1) expression, which might enhance hepatitis activity.

Conclusions: The PD-L2 is highly expressed on CH-C-OLT hepatocytes, whereas HCV proteins, in combination with CNIs, induce high expression of PD-L2 resulting in elevated expression of ICAM-1. These findings demonstrate the effect of CNIs on inducing PD-L2 and subsequent ICAM-1 expression, effects that may produce inflammatory cell infiltration in post-OLT hepatitis C.

No MeSH data available.


Related in: MedlinePlus

Effect of CNI treatment on PD-L2 expression in hepatoma cell lines. A, Expression levels of PD-L2 mRNA determined using RT-PCR (upper section). The PLC/PRF/5 and Huh7 cell lines were treated with IFN-γ2 (1000 IU/mL) and/or FK (1 μM) or CyA (10 μM). Expression levels of PD-L2 protein were determined using Western blotting (lower section). B, Effect of CNI treatment, in combination with HCV proteins, on PD-L2 expression in hepatoma cell lines. Relative PD-L2 mRNA expression levels were determined using real-time PCR in hepatoma cell lines transfected with HCV plasmid and/or treated with CNIs. C, Expression levels of PD-L2 determined using flow cytometric analysis in hepatoma cell lines treated with CNIs in combination with HCV protein expression. The MFI is presented as fold increase relative to control cells. The data represent mean ± SD of triplicate measurements. D, Effect of CNI treatment, in combination with HBV proteins, on PD-L2 expression in hepatoma cell lines. Relative PD-L2 mRNA expression levels were determined using real-time PCR in hepatoma cell lines transfected with HBV plasmid and/or treated with CNIs. MFI, mean fluorescence intensity.
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Figure 2: Effect of CNI treatment on PD-L2 expression in hepatoma cell lines. A, Expression levels of PD-L2 mRNA determined using RT-PCR (upper section). The PLC/PRF/5 and Huh7 cell lines were treated with IFN-γ2 (1000 IU/mL) and/or FK (1 μM) or CyA (10 μM). Expression levels of PD-L2 protein were determined using Western blotting (lower section). B, Effect of CNI treatment, in combination with HCV proteins, on PD-L2 expression in hepatoma cell lines. Relative PD-L2 mRNA expression levels were determined using real-time PCR in hepatoma cell lines transfected with HCV plasmid and/or treated with CNIs. C, Expression levels of PD-L2 determined using flow cytometric analysis in hepatoma cell lines treated with CNIs in combination with HCV protein expression. The MFI is presented as fold increase relative to control cells. The data represent mean ± SD of triplicate measurements. D, Effect of CNI treatment, in combination with HBV proteins, on PD-L2 expression in hepatoma cell lines. Relative PD-L2 mRNA expression levels were determined using real-time PCR in hepatoma cell lines transfected with HBV plasmid and/or treated with CNIs. MFI, mean fluorescence intensity.

Mentions: Because the CH-C-OLT group showed elevated PD-L2 expression on hepatocytes, we surmised that the combination of HCV and CNIs might induce PD-L2 expression. First, we examined the expression levels of PD-L2 mRNA and protein by RT-PCR and Western blotting, respectively, to explore the role of PD-L2 in hepatoma cells treated with CNIs. Treatment of hepatoma cell lines with CNIs resulted in elevated PD-L2 gene and protein expression (Figure 2A). We also examined PD-L2 mRNA expression using real-time PCR in a hepatoma cell line transfected with HCV plasmids and treated with CNIs. PD-L2 mRNA expression was not altered by HCV core protein expression; however, it was enhanced in combination with CNIs (especially with CyA) (Figure 2B). Similarly, transfection with HCV NS3 protein revealed that NS3 protein itself was capable of enhancing PD-L2 mRNA expression and CNI (especially CyA) treatment further elevated this effect. The surface expression of PD-L2 on the hepatoma cell line was assessed using flow cytometric analysis. Surface expression appeared to be increased in the presence of CyA and in combination with HCV proteins, especially with HCV-NS3 (Figure 2C). However, no significant differences were observed following treatment with HCV and/or CNIs (Figure 2C). Next, we investigated whether HBV and CNI treatment altered PD-L2 levels. PD-L2 mRNA expression was not altered with HBV protein (Figure 2D).


Enhancement of Programmed Death Ligand 2 on Hepatitis C Virus Infected Hepatocytes by Calcineurin Inhibitors.

Koike K, Takaki A, Yagi T, Iwasaki Y, Yasunaka T, Sadamori H, Shinoura S, Umeda Y, Yoshida R, Sato D, Nobuoka D, Utsumi M, Miyake Y, Ikeda F, Shiraha H, Fujiwara T, Yamamoto K - Transplantation (2015)

Effect of CNI treatment on PD-L2 expression in hepatoma cell lines. A, Expression levels of PD-L2 mRNA determined using RT-PCR (upper section). The PLC/PRF/5 and Huh7 cell lines were treated with IFN-γ2 (1000 IU/mL) and/or FK (1 μM) or CyA (10 μM). Expression levels of PD-L2 protein were determined using Western blotting (lower section). B, Effect of CNI treatment, in combination with HCV proteins, on PD-L2 expression in hepatoma cell lines. Relative PD-L2 mRNA expression levels were determined using real-time PCR in hepatoma cell lines transfected with HCV plasmid and/or treated with CNIs. C, Expression levels of PD-L2 determined using flow cytometric analysis in hepatoma cell lines treated with CNIs in combination with HCV protein expression. The MFI is presented as fold increase relative to control cells. The data represent mean ± SD of triplicate measurements. D, Effect of CNI treatment, in combination with HBV proteins, on PD-L2 expression in hepatoma cell lines. Relative PD-L2 mRNA expression levels were determined using real-time PCR in hepatoma cell lines transfected with HBV plasmid and/or treated with CNIs. MFI, mean fluorescence intensity.
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Figure 2: Effect of CNI treatment on PD-L2 expression in hepatoma cell lines. A, Expression levels of PD-L2 mRNA determined using RT-PCR (upper section). The PLC/PRF/5 and Huh7 cell lines were treated with IFN-γ2 (1000 IU/mL) and/or FK (1 μM) or CyA (10 μM). Expression levels of PD-L2 protein were determined using Western blotting (lower section). B, Effect of CNI treatment, in combination with HCV proteins, on PD-L2 expression in hepatoma cell lines. Relative PD-L2 mRNA expression levels were determined using real-time PCR in hepatoma cell lines transfected with HCV plasmid and/or treated with CNIs. C, Expression levels of PD-L2 determined using flow cytometric analysis in hepatoma cell lines treated with CNIs in combination with HCV protein expression. The MFI is presented as fold increase relative to control cells. The data represent mean ± SD of triplicate measurements. D, Effect of CNI treatment, in combination with HBV proteins, on PD-L2 expression in hepatoma cell lines. Relative PD-L2 mRNA expression levels were determined using real-time PCR in hepatoma cell lines transfected with HBV plasmid and/or treated with CNIs. MFI, mean fluorescence intensity.
Mentions: Because the CH-C-OLT group showed elevated PD-L2 expression on hepatocytes, we surmised that the combination of HCV and CNIs might induce PD-L2 expression. First, we examined the expression levels of PD-L2 mRNA and protein by RT-PCR and Western blotting, respectively, to explore the role of PD-L2 in hepatoma cells treated with CNIs. Treatment of hepatoma cell lines with CNIs resulted in elevated PD-L2 gene and protein expression (Figure 2A). We also examined PD-L2 mRNA expression using real-time PCR in a hepatoma cell line transfected with HCV plasmids and treated with CNIs. PD-L2 mRNA expression was not altered by HCV core protein expression; however, it was enhanced in combination with CNIs (especially with CyA) (Figure 2B). Similarly, transfection with HCV NS3 protein revealed that NS3 protein itself was capable of enhancing PD-L2 mRNA expression and CNI (especially CyA) treatment further elevated this effect. The surface expression of PD-L2 on the hepatoma cell line was assessed using flow cytometric analysis. Surface expression appeared to be increased in the presence of CyA and in combination with HCV proteins, especially with HCV-NS3 (Figure 2C). However, no significant differences were observed following treatment with HCV and/or CNIs (Figure 2C). Next, we investigated whether HBV and CNI treatment altered PD-L2 levels. PD-L2 mRNA expression was not altered with HBV protein (Figure 2D).

Bottom Line: Treatment of hepatoma cell lines with CNIs resulted in increased PD-L2 expression, especially in combination with HCV core or NS3 protein.The PD-L2 is highly expressed on CH-C-OLT hepatocytes, whereas HCV proteins, in combination with CNIs, induce high expression of PD-L2 resulting in elevated expression of ICAM-1.These findings demonstrate the effect of CNIs on inducing PD-L2 and subsequent ICAM-1 expression, effects that may produce inflammatory cell infiltration in post-OLT hepatitis C.

View Article: PubMed Central - PubMed

Affiliation: 1 Department of Gastroenterology and Hepatology, Okayama University Graduate School of Medicine, Dentistry and Pharmaceutical Sciences, Okayama City, Japan. 2 Department of Gastroenterological Surgery Transplant and Surgical Oncology, Okayama University Graduate School of Medicine, Dentistry and Pharmaceutical Sciences, Okayama City, Japan. 3 Health Service Center, Okayama University, Okayama, Japan.

ABSTRACT

Background: Post orthotopic liver transplantation (OLT) viral hepatitis is an immunological condition where immune cells induce hepatitis during conditions of immune-suppression. The immune-regulatory programmed death-1 (PD-1)/PD-ligand 1 system is acknowledged to play important roles in immune-mediated diseases. However, the PD-1/PD-L2 interaction is not well characterized, with PD-L2 also exhibiting an immunostimulatory function. We hypothesized that this atypical molecule could affect the recurrence of post-OLT hepatitis. To test this hypothesis, we conducted immunohistochemical staining analysis and in vitro analysis of PD-L2.

Methods: The expression of PD-L2 was evaluated in liver biopsy specimens from patients with chronic hepatitis B (n = 15), post-OLT hepatitis B (n = 8), chronic hepatitis C (n = 48), and post-OLT hepatitis C (CH-C-OLT) (n = 14). The effect of calcineurin inhibitors (CNIs) and hepatitis C virus (HCV) on PD-L2 expression was investigated in hepatoma cell lines.

Results: The PD-L2 was highly expressed on CH-C-OLT hepatocytes. Treatment of hepatoma cell lines with CNIs resulted in increased PD-L2 expression, especially in combination with HCV core or NS3 protein. Transfection of cell lines with PD-L2 containing plasmid resulted in high intercellular adhesion molecule-1 (ICAM-1) expression, which might enhance hepatitis activity.

Conclusions: The PD-L2 is highly expressed on CH-C-OLT hepatocytes, whereas HCV proteins, in combination with CNIs, induce high expression of PD-L2 resulting in elevated expression of ICAM-1. These findings demonstrate the effect of CNIs on inducing PD-L2 and subsequent ICAM-1 expression, effects that may produce inflammatory cell infiltration in post-OLT hepatitis C.

No MeSH data available.


Related in: MedlinePlus