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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

The effect of increased PD-L2 expression on immunological parameters. A, RT-PCR and Western blotting were performed to confirm the efficiency of PD-L2 containing plasmid transfection. The ability of the PD-L2 containing plasmid to induce PD-L2 mRNA expression was confirmed in two cell lines, with the more efficiently transfected Huh7 cells used for confirmation of protein expression. B, Representative flow cytometric analysis of Huh7 cells transfected with PD-L2 containing plasmid (green) and control plasmid (purple). Expressions of Fas, CD1d, HLA-class 1 and intercellular adhesion molecule-1 (ICAM-1) are shown. C, The mean fluorescence intensity (MFI) is presented as fold increase relative to control cells. The data represent mean ± SD of triplicate measurements. D, Immunohistochemical staining pattern of ICAM-1. Left panel is the representative staining pattern of ICAM-1 in liver tissue. Expression intensity was scored as follows: 0, negative; 1, weak expression; 2, moderate expression; 3, strong expression. Right: The concordance coefficients (κ statistics) were used to evaluate agreement between elevated PD-L2 expression and elevated ICAM-1 expression.
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Figure 3: The effect of increased PD-L2 expression on immunological parameters. A, RT-PCR and Western blotting were performed to confirm the efficiency of PD-L2 containing plasmid transfection. The ability of the PD-L2 containing plasmid to induce PD-L2 mRNA expression was confirmed in two cell lines, with the more efficiently transfected Huh7 cells used for confirmation of protein expression. B, Representative flow cytometric analysis of Huh7 cells transfected with PD-L2 containing plasmid (green) and control plasmid (purple). Expressions of Fas, CD1d, HLA-class 1 and intercellular adhesion molecule-1 (ICAM-1) are shown. C, The mean fluorescence intensity (MFI) is presented as fold increase relative to control cells. The data represent mean ± SD of triplicate measurements. D, Immunohistochemical staining pattern of ICAM-1. Left panel is the representative staining pattern of ICAM-1 in liver tissue. Expression intensity was scored as follows: 0, negative; 1, weak expression; 2, moderate expression; 3, strong expression. Right: The concordance coefficients (κ statistics) were used to evaluate agreement between elevated PD-L2 expression and elevated ICAM-1 expression.

Mentions: To determine whether elevated expression of PD-L2 affects other host immune responses, we analyzed the expression of cell surface markers on hepatoma cell lines (Hep3B and Huh7) transfected with PD-L2. Induced PD-L2 mRNA expression was confirmed in Hep3B and Huh7 cells, with the more efficiently transfected Huh7 cells used for protein expression confirmation (Figure 3A). Cell surface expression of ICAM-1, Fas, CD1d, and major histocompatibility complex-class 1 were determined in the transfected cells. Intercellular adhesion molecule-1 was upregulated on PD-L2 transfected cells (Figure 3B and C). As a result, we subsequently evaluated the immunostaining patterns of PD-L2 and ICAM-1 (Figure 3D). Tissues that exhibited strong ICAM-1 staining and strong hepatocyte PD-L2 staining (≥2) were strongly correlated in CH-C and CH-C-OLT patients (κ = 0.30, P = 0.015).


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)

The effect of increased PD-L2 expression on immunological parameters. A, RT-PCR and Western blotting were performed to confirm the efficiency of PD-L2 containing plasmid transfection. The ability of the PD-L2 containing plasmid to induce PD-L2 mRNA expression was confirmed in two cell lines, with the more efficiently transfected Huh7 cells used for confirmation of protein expression. B, Representative flow cytometric analysis of Huh7 cells transfected with PD-L2 containing plasmid (green) and control plasmid (purple). Expressions of Fas, CD1d, HLA-class 1 and intercellular adhesion molecule-1 (ICAM-1) are shown. C, The mean fluorescence intensity (MFI) is presented as fold increase relative to control cells. The data represent mean ± SD of triplicate measurements. D, Immunohistochemical staining pattern of ICAM-1. Left panel is the representative staining pattern of ICAM-1 in liver tissue. Expression intensity was scored as follows: 0, negative; 1, weak expression; 2, moderate expression; 3, strong expression. Right: The concordance coefficients (κ statistics) were used to evaluate agreement between elevated PD-L2 expression and elevated ICAM-1 expression.
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Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC4539199&req=5

Figure 3: The effect of increased PD-L2 expression on immunological parameters. A, RT-PCR and Western blotting were performed to confirm the efficiency of PD-L2 containing plasmid transfection. The ability of the PD-L2 containing plasmid to induce PD-L2 mRNA expression was confirmed in two cell lines, with the more efficiently transfected Huh7 cells used for confirmation of protein expression. B, Representative flow cytometric analysis of Huh7 cells transfected with PD-L2 containing plasmid (green) and control plasmid (purple). Expressions of Fas, CD1d, HLA-class 1 and intercellular adhesion molecule-1 (ICAM-1) are shown. C, The mean fluorescence intensity (MFI) is presented as fold increase relative to control cells. The data represent mean ± SD of triplicate measurements. D, Immunohistochemical staining pattern of ICAM-1. Left panel is the representative staining pattern of ICAM-1 in liver tissue. Expression intensity was scored as follows: 0, negative; 1, weak expression; 2, moderate expression; 3, strong expression. Right: The concordance coefficients (κ statistics) were used to evaluate agreement between elevated PD-L2 expression and elevated ICAM-1 expression.
Mentions: To determine whether elevated expression of PD-L2 affects other host immune responses, we analyzed the expression of cell surface markers on hepatoma cell lines (Hep3B and Huh7) transfected with PD-L2. Induced PD-L2 mRNA expression was confirmed in Hep3B and Huh7 cells, with the more efficiently transfected Huh7 cells used for protein expression confirmation (Figure 3A). Cell surface expression of ICAM-1, Fas, CD1d, and major histocompatibility complex-class 1 were determined in the transfected cells. Intercellular adhesion molecule-1 was upregulated on PD-L2 transfected cells (Figure 3B and C). As a result, we subsequently evaluated the immunostaining patterns of PD-L2 and ICAM-1 (Figure 3D). Tissues that exhibited strong ICAM-1 staining and strong hepatocyte PD-L2 staining (≥2) were strongly correlated in CH-C and CH-C-OLT patients (κ = 0.30, P = 0.015).

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