Date Published: July 10, 2013
Publisher: Blackwell Publishing Ltd
Author(s): J G Brain, H Robertson, E Thompson, E H Humphreys, A Gardner, T A Booth, D E J Jones, S C Afford, T von Zglinicki, A D Burt, J A Kirby.
Biliary epithelial cells (BEC) are important targets in some liver diseases, including acute allograft rejection. Although some injured BEC die, many can survive in function compromised states of senescence or phenotypic de-differentiation. This study was performed to examine changes in the phenotype of BEC during acute liver allograft rejection and the mechanism driving these changes. Liver allograft sections showed a positive correlation (p < 0.0013) between increasing T cell mediated acute rejection and the number of BEC expressing the senescence marker p21WAF1/Cip or the mesenchymal marker S100A4. This was modeled in vitro by examination of primary or immortalized BEC after acute oxidative stress. During the first 48 h, the expression of p21WAF1/Cip was increased transiently before returning to baseline. After this time BEC showed increased expression of mesenchymal proteins with a decrease in epithelial markers. Analysis of TGF-β expression at mRNA and protein levels also showed a rapid increase in TGF-β2 (p < 0.006) following oxidative stress. The epithelial de-differentiation observed in vitro was abrogated by pharmacological blockade of the ALK-5 component of the TGF-β receptor. These data suggest that stress induced production of TGF-β2 by BEC can modify liver allograft function by enhancing the de-differentiation of local epithelial cells.
The rate of acute cellular rejection in liver allografts has fallen from 60–70% to 10–20% of graft recipients and classical ductopaenic rejection has all but disappeared from clinical practice 1. However, this reduction has been accompanied by an increase in factors limiting the quality and length of life of liver transplant recipients 1–3. There are significant sequelae associated with the administration of immunosuppressive agents, stimulating interest in regimes designed to induce tolerance to liver grafts and the ability to maintain grafts with minimal or no immunosuppression 4. The limited evidence available also points to increased fibrosis in patients classed as tolerant, with no evidence of either acute or chronic rejection 4. There is also interest in other phenomena associated with graft immune responses, such as nonclassical antibody-mediated rejection and late acute cellular rejection 5,6.
This study suggests that cellular senescence plays a key role in acute cellular rejection of liver allografts. A clear positive correlation was demonstrated between the number of p21WAF1/Cip1 positive biliary epithelial cells and increasing grade of acute rejection. This is consistent with data from other groups detailing severity in chronic cholestatic liver diseases 10,13,16. In addition to its role as a marker of senescence, p21WAF1/Cip1 can also be upregulated during the G1 phase of the cell cycle 24. Ki67 is a marker of proliferation present throughout all active phases of the cell cycle 25. In order to differentiate senescent and proliferating cells, Ki67 was used in a triple label in place of p21WAF1/Cip1. This demonstrated that Ki67 and p21WAF1/Cip1 positive BEC occupy different cellular niches. Whereas p21WAF1/Cip1 positive cells never co-stained for S100A4 and were never seen adjacent to S100A4 stained BEC, Ki67 stained cells were always either co-stained for S100A4 or adjacent to S100A4 positive BEC in moderate to severe rejection (though in mild rejection Ki67 positive cells were seen in the absence of S100A4). These results are compatible with a model in which the BEC that express p21WAF1/Cip1 are nonproliferating, senescent cells while other BEC, which may also express S100A4, are actively proliferating. The presence of the senescence marker γH2AX was also assessed, this being regarded as a gold standard for the assessment of senescence 20. In sections of ACR; Ki67 negative, γH2AX positive cells were identified with increasing frequency in line with increasing Banff grade. This would strongly suggest that senescent cells are indeed present in increasing number with increased Banff grade, in line with our observations of p21WAF1/Cip1. The dual staining of p21WAF1/Cip1 and Ki67 was also performed, showing the presence of Ki67 negative p21WAF1/Cip1 positive cells in ACR.