The cytoskeleton protein β-actin may mediate T cell apoptosis during acute rejection reaction after liver transplantation in a rat model

Abstract

Cytoskeletal proteins and associated regulatory proteins are essential for maintaining cell structure and growth. β-actin is a major component of the cytoskeleton, and β-actin remodeling is involved in lymphocyte migration, infiltration and apoptosis. However, little is known about whether changes in β-actin expression affect lymphocyte cell fate, particularly during acute rejection after liver transplantation in a rat model. In our studies, grafts were harvested on days 5, 7 or 9 after xenogeneic rat liver transplantation. The acute rejection grade was histopathologically evaluated. Recipient-derived CD8⁺ T lymphocytes gradually infiltrated into liver allografts in cases of severe acute rejection. The apoptotic rate of CD8⁺ T lymphocytes peaked on day 7 and then decreased. Moreover, changes in β-actin expression were consistent with the apoptotic rate of CD8⁺ T lymphocytes in both allografts and peripheral blood based on western blotting and immunohistochemistry results. Additionally, jasplakinolide (an actin-stabilizing drug) evoked CD8⁺ T lymphocyte apoptosis. In conclusion, our study is the first to describe the fluctuating expression levels and dynamics of the cytoskeletal protein β-actin and its potential roles in the pathogenesis of acute rejection following rat liver transplantion. Our results enhance the understanding of the roles of CD8⁺ T lymphocytes during acute rejection and suggest that β-actin regulation leads to apoptosis.

Keywords: Acute rejection; CD8+ T lymphocyte; liver transplantation; β-actin.

Figure 1

Time course of pathologic characteristics of liver grafts on days 5, 7, 9 after LT. A: Analysis of pathologic characteristics of allogenic and homogenic groups by H&E staining (original magnification, 200×). B: Bar graph showing the rejection activity index (RAI) results in two groups. C, D: Analysis of serum levels of ALT, AST, TBIL and cytokine (IFN-γ, IL-2 and TNF-α). E: Relative RNA expression for cytokine genesin liver grafts of allogenic and homogenic groups detected by qPCR, GAPDH was used as loading controls. All data representative of three independent experiments and calculated data are shown as mean ± SD. All statistical analyses were performed by student t test, *p<0.05, **p<0.01, ***p<0.001 compared to homogenic group.

Figure 2

The ratio of CD8⁺ T cells in liver grafts and peripheral blood on days 5, 7, 9 after LT. A: Immunofluorescent staining for CD8 in liver grafts of allogenic and homogenic groups (original magnification 200×). CD8 antibody was labeled with cyanidin-3 (red) and cell nucleus was labeled DAPI (blue). B, C: Flow cytometry analysis of ratio of peripheral CD8⁺ T cells in allogenic and homogenic groups. All data representative of three independent experiments and calculated data are shown as mean ± SD. All statistical analyses were performed by student t test, *p<0.05, ***p<0.001 compared to homogenic group.

Figure 3

Alterations of the expression of β-actin in CD8⁺ T cells in liver grafts on days 5, 7, 9 after LT. A: Triple double fluorescent staining for CD8 (red), β-actin (green) and DAPI (blue) in allogenic group and homogenic group (original magnification 200×). B: Histogram showing relative fluorescence intensity of β-actin expression in CD8⁺ T cells. C: qPCR was performed to analyze the expression of β-actin in liver tissues, GAPDH was used as loading controls. All data representative of three independent experiments and calculated data are shown as mean ± SD. All statistical analyses were performed by student t test, *p<0.05, **p<0.01 compared to homogenic group.

Figure 4

Correlation between the expression of β-actin in CD8⁺ T cells and the counts of apoptosis CD8⁺ T cells in liver grafts and peripheral blood. A: Immunohistochemical images showing the expression of β-actin in CD8⁺ T cells and the counts of apoptosis CD8⁺ T cells in the grafts slices of allogenic and homogenic groups (original magnification 200×). B: Histogram showing the alternated counts of apoptosis CD8⁺ T cells with time course in the liver slices. C: Western blot was performed to analyze the levels of cofilin, β-actin, cleaved-caspase 3 in the liver grafts at different time point. D: The apoptosis rate of CD8⁺ T cells isolated by using CD8 magnetic beads from peripheral blood was measured by Annexin V/PI assay. E: Histogram showing that apoptosis rate of CD8⁺ T cells from allogenic liver allografts was significant greater than homogenic liver grafts at each time point postoperatively. F: The expression of β-actin in CD8⁺ T cells isolated from peripheral blood were analyze by western blot. All data representative of three independent experiments and calculated data are shown as mean ± SD. All statistical analyses were performed by student t test, *p<0.05, **p<0.01, ***p<0.001 compared to homogenic group.

Figure 5

Correlation analysis in allografts during acute rejection episodes. A-C: Correlation between β-actin and the inflammatory cytokines (IFN-γ, IL-2 and TNF-α) mRNA levels; D: Correlation between β-actin mRNA levels and the severity of allograft rejection which was represented as RAI. Statistical analyses were performed by Spearman’s test to assess any correlation. A probability level of P<0.05 was considered statistically significant.

Figure 6

The number of CD8⁺ T cells undergone apoptosis was significant increased by using actin stabilization drug jasplakinolide. A: CD8⁺ T cells were treated with DMSO (0.05%) or jasplakinolide (1 μg/mL). Cells were harvested after 48 h incubation to analyze apoptosis rate using Annexin V/PI assay by flow cytometry. B: Histogram showing apoptosis rate of CD8⁺ T cells in JASP group was significant higher than the control group. C: Expression levels of β-actin protein in control group and JASP group was detected by western blot. All data representative of three independent experiments and calculated data are shown as mean ± SD. All statistical analyses were performed by student t test, ***p<0.001 compared to homogenic group.