JOURNAL OF PATHOLOGY, VOL.
165: 153-1 61 (1991)
NEW MONOCLONAL ANTIBODIES REACTING WITH BILE DUCTS: FURTHER INSIGHTS INTO THE PATHOGENESIS OF BILE DUCTULAR PROLIFERATION IN BILIARY DISEASES REUBEN c. s. AYRES*, STEFAN G. HUBSCHER?, JEAN SHAW*, CHRIS GARNER*, RUTH JOPLIN*, ANNE WILLIAMS? AND JAMES M. NEUBERGER*
*The Liver Unit, Queen Elizabeth Hospital, Birmingham B15 2TH, U . K . ; ?The Department of Pathology, The Medical School, University of Birmingham, Birmingham B15 2TT, U.K. Received 20 December 1990 Accepted 29 April 1991
SUMMARY We have produced a range of monoclonal antibodies which stain human intrahepatic bile ducts of different sizes. Amongst 26 monoclonal antibodies produced, five clones reacted specifically with bile ducts of different sizes, of which three have been maintained in culture and their viability following freezing and thawing confirmed. Staining patterns varied between normal adult liver tissue, normal fetal liver tissue and a variety of hepatobiliary diseases. The antibodies provide further evidence of the immunological heterogeneity of the human intrahepatic biliary tree and support the hypothesis that proliferating bile ductules are derived from periseptal hepatocytes. The preparation of the antibodies, their staining reactions in normal adult, normal fetal and a variety of liver diseases are described. KEY
WORDS-Monoclonal antibodies, biliary epithelial cells.
INTRODUCTION Previous studies of surface antigen expression have demonstrated the heterogeneity of the human intrahe atic biliary tree.' In primary biliary cirrhosis2*'and other liver disorders: surface antigen expression on intrahepatic biliary epithelial cells is altered. Primary biliary cirrhosis is an idiopathic chronic disorder associated with destruction of medium sized intrahepatic bile ducts5y6 and the presence of proliferating bile ductules, the origin of which is Despite evidence that primary biliary cirrhosis is an immune-mediated disease," no surface biliary epithelial marker has been convincingly identified against which immune attack might be targetted. Our original intention was to find a monoclonal antibody specific for biliary epithelial cells in primary biliary cirrhosis. We have prepared a series of monoclonal antibodies usin a variation of the method of Kohler and Milstein I?, I * Addressee for correspondence: Dr R. C. S. Ayres, Liver Research Laboratories, Queen Elizabeth Hospital, Edgbaston, Birmingham B15 ZTH, U.K.
0022-341 7/9 1/ I 0015 3 4 9 $05.00 0 1991 by John Wiley & Sons, Ltd.
following immunization of a Balb C mouse with human intrahepatic biliary epithelial cells derived from primary biliary cirrhosis livers. The monoclonal antibodies produced selectively label human intrahepatic bile ducts of different sizes. Staining patterns vary between normal adult liver, normal fetal liver and liver tissue from a variety of adult liver diseases. METHODS Isolation and characterization of human biliary epithelial cells
Human biliary epithelial cells were isolated from explanted livers of patients undergoing orthotopic liver transplantation for primary biliary cirrhosis by differential density separation and immunopurification as described in detail e1~ewhere.l~ Characterization was by immunocytostaining with y-glutamyl transferase, anti-human epithelial antigen- 125 and anti-cytokeratin 19 and by light and electron microscopy. Purity and viability were both above 95 per cent.
154 Table I-Staining
R. C. S. AYRES E T A L .
reactions of Clone 5 with frozen sections of liver tissue Structure stained
Tissue Normal adult Median Interquartile range n Normal fetal Median Interquartile range n NANB Median Interquartile range n NBC Median Interquartile range n PBC Median Interquartile range n PSC Median Interquartile range n
Septa1 ducts
1.25 1G 2 . 0 6
1.75 I G2.5 2 NS
ILD
BD
Hepat. diffuse
Hepat. perisept.
0.0
0.0 0.045 8
1.75
2.0
1.5-2.0
1.0-23
o.(M.o
8
8
8
1.5 1.0-1.5
1 .o 1.0-1.0 5
0.5-1 .O
1 .O 0.5-1.5
NS
6 P
165: 153-1 61 (1991)
NEW MONOCLONAL ANTIBODIES REACTING WITH BILE DUCTS: FURTHER INSIGHTS INTO THE PATHOGENESIS OF BILE DUCTULAR PROLIFERATION IN BILIARY DISEASES REUBEN c. s. AYRES*, STEFAN G. HUBSCHER?, JEAN SHAW*, CHRIS GARNER*, RUTH JOPLIN*, ANNE WILLIAMS? AND JAMES M. NEUBERGER*
*The Liver Unit, Queen Elizabeth Hospital, Birmingham B15 2TH, U . K . ; ?The Department of Pathology, The Medical School, University of Birmingham, Birmingham B15 2TT, U.K. Received 20 December 1990 Accepted 29 April 1991
SUMMARY We have produced a range of monoclonal antibodies which stain human intrahepatic bile ducts of different sizes. Amongst 26 monoclonal antibodies produced, five clones reacted specifically with bile ducts of different sizes, of which three have been maintained in culture and their viability following freezing and thawing confirmed. Staining patterns varied between normal adult liver tissue, normal fetal liver tissue and a variety of hepatobiliary diseases. The antibodies provide further evidence of the immunological heterogeneity of the human intrahepatic biliary tree and support the hypothesis that proliferating bile ductules are derived from periseptal hepatocytes. The preparation of the antibodies, their staining reactions in normal adult, normal fetal and a variety of liver diseases are described. KEY
WORDS-Monoclonal antibodies, biliary epithelial cells.
INTRODUCTION Previous studies of surface antigen expression have demonstrated the heterogeneity of the human intrahe atic biliary tree.' In primary biliary cirrhosis2*'and other liver disorders: surface antigen expression on intrahepatic biliary epithelial cells is altered. Primary biliary cirrhosis is an idiopathic chronic disorder associated with destruction of medium sized intrahepatic bile ducts5y6 and the presence of proliferating bile ductules, the origin of which is Despite evidence that primary biliary cirrhosis is an immune-mediated disease," no surface biliary epithelial marker has been convincingly identified against which immune attack might be targetted. Our original intention was to find a monoclonal antibody specific for biliary epithelial cells in primary biliary cirrhosis. We have prepared a series of monoclonal antibodies usin a variation of the method of Kohler and Milstein I?, I * Addressee for correspondence: Dr R. C. S. Ayres, Liver Research Laboratories, Queen Elizabeth Hospital, Edgbaston, Birmingham B15 ZTH, U.K.
0022-341 7/9 1/ I 0015 3 4 9 $05.00 0 1991 by John Wiley & Sons, Ltd.
following immunization of a Balb C mouse with human intrahepatic biliary epithelial cells derived from primary biliary cirrhosis livers. The monoclonal antibodies produced selectively label human intrahepatic bile ducts of different sizes. Staining patterns vary between normal adult liver, normal fetal liver and liver tissue from a variety of adult liver diseases. METHODS Isolation and characterization of human biliary epithelial cells
Human biliary epithelial cells were isolated from explanted livers of patients undergoing orthotopic liver transplantation for primary biliary cirrhosis by differential density separation and immunopurification as described in detail e1~ewhere.l~ Characterization was by immunocytostaining with y-glutamyl transferase, anti-human epithelial antigen- 125 and anti-cytokeratin 19 and by light and electron microscopy. Purity and viability were both above 95 per cent.
154 Table I-Staining
R. C. S. AYRES E T A L .
reactions of Clone 5 with frozen sections of liver tissue Structure stained
Tissue Normal adult Median Interquartile range n Normal fetal Median Interquartile range n NANB Median Interquartile range n NBC Median Interquartile range n PBC Median Interquartile range n PSC Median Interquartile range n
Septa1 ducts
1.25 1G 2 . 0 6
1.75 I G2.5 2 NS
ILD
BD
Hepat. diffuse
Hepat. perisept.
0.0
0.0 0.045 8
1.75
2.0
1.5-2.0
1.0-23
o.(M.o
8
8
8
1.5 1.0-1.5
1 .o 1.0-1.0 5
0.5-1 .O
1 .O 0.5-1.5
NS
6 P
