Letters to the Editor

EXPRESSION OF HLA-DR ANTIGENS ON BILEDUCT CELLS OF REJECTED LIVER TRANSPLANT

Background/Aims: Intra-hepatic bile ducts are the primary site of damage in several immunologically mediated liver diseaes. However, immunological processes underlying biliary epithelial cell recognition by T lymphocytes are poorly understood. Therefore, a convenient in vitro model that could mimic these immunologic disorders would be of great interest.

Methods: A human cell line (HuGB) was established from a metastasis of gallbladder adenocarcinoma in the liver. Intermediate filament expression was analysed by immunostaining, and gamma-glutamyl transpeptidase and albumin secretion were measured. VLA integrin expression pattern, expression of HLA class I and II antigens and ICAM-1 protein were analysed by flow cytometry and their modulation by interferon-γ was quantitated using a QIFIKIT commercial kit.

Results: Histological analysis showed high similarity between the initial gallbladder adenocarcinoma and the established cell line. Cytokeratins 8 and 19 and vimentin showed strong positive staining in the established cell line. Gamma-glutamyl transpeptidase was secreted by these cells while albumin expression was negative. HuGB cells also expressed VLA-α2, VLA-α3, VLA-α6, VLA-β1, but not VLA-α1, VLA-α4 and NCAM, a pattern of adhesion molecule expression compatible with the biliary epithelium. Also, similar to the biliary epithelium found in normal liver, HuGB cells expressed abundant HLA class I but few HLA class II antigens. We found that the expression of HLA antigens and ICAM-1 protein were increased during interferon-γ treatment of HuGB cell line.

Conclusions: Both phenotypic and morphological characteristics of HuGB cells suggested their biliary origin. Sensitivity of HuGB cells to interferon-γ suggests that this new cell line could represent a suitable model to investigate the up-regulation of membrane antigens occurring in immune diseases involving biliary epithelial cells.

From histopathological analyses after liver transplantation it is evident that the biliary epithilium is an important target for leucocytes of the graft recipient. Besides clinical and histopathological investigations undertaken by several authors it was also endeavoured to determine the immunological impact of the biliary epithelial cells (BEC) in vitro. As for the intrahepatic BEC, in vitro studies proved to be restricted owing to difficult isolation procedures and the limited number of cells yielded from transplanted organs. Therefore, studies on cultured extrahepatic BEC served as a model for the immunological features of the biliary epithelium in transplantation. Herein, in vivo and in vitro studies dealing with BEC and immunologically mediated hepatic disorders are reviewed in order to understand better the pathogenesis after liver transplantation. Furthermore, possible underlying mechanisms of BEC-directed immunity with regard to BEC-leucoycte interactions are discussed.

The biliary epithelium is a major target for allograft-directed immune responses during rejection crises after liver transplantation. This paper deals with in vitro studies on the immunogenetic potential of cultured biliary epithelial cells (BECs) to elicit an allogeneic cellular immune response. Therefore, BECs were cocultured with syngeneic and allogeneic lymphocytes in order to study lymphocyte activation. The respective lymphocytes [³H]thymidine incorporation was a measure for the proliferative activity. While syngeneic peripheral blood lymphocytes (PBL) never exhibited BEC-induced proliferation allogeneic PBL were significantly (P < 0.05) activated in all experiments (n = 6). In experiments with purified subpopulations CD8⁺ cells but not CD4⁺ cells proved to be activated by BECs. In time kinetics (n = 5) the maximum of the BEC-induced proliferation was on day 9 while the endothelial cell-induced proliferation was found to be 2 days earlier on day 7 after onset of the experiments (P < 0.05). BEC-induced proliferation was accompanied by induced IL-2 secretion (> 300 pg/ml) by activated lymphocytes as determined by ELISA. Stimulation of BECs with 500 U/ml interferon-γ, 1000 U/ml interferon-α or blocking expression of HLA molecules on the surface membrane of BECs by monoclonal antibodies did not alter BEC-induced allogeneic lymphocyte proliferation. Monoclonal antibodies against CD8⁺ but not CD4⁺ suppressed proliferative activity of PBL and CD8⁺ cells by 40 and 45%, respectively. Overall, these results provide evidence that BECs may induce CD8⁺ lymphocyte activation in vivo and therefore might play a crucial role in triggering immune responses related to liver transplant rejection episodes.