Heather A. Crosby

Nomenclature of the finer branches of the biliary tree: Canals, ductules, and ductular reactions in human livers

The work of liver stem cell biologists, largely carried out in rodent models, has now started to manifest in human investigations and applications. We can now recognize complex regenerative processes in tissue specimens that had only been suspected for decades, but we also struggle to describe what we see in human tissues in a way that takes into account the findings from the animal investigations, using a language derived from species not, in fact, so much like our own. This international group of liver pathologists and hepatologists, most of whom are actively engaged in both clinical work and scientific research, seeks to arrive at a consensus on nomenclature for normal human livers and human reactive lesions that can facilitate more rapid advancement of our field. (HEPATOLOGY 2004; 39:1739–1745.)

Characterization and Isolation of Ductular Cells Coexpressing Neural Cell Adhesion Molecule and Bcl-2 from Primary Cholangiopathies and Ductal Plate Malformations

It has recently been shown that reactive bile ductules display neuroendocrine features, including immunoreactivity for the neural cell adhesion molecule (NCAM). In this study we have compared the immunohistochemical expression of NCAM with that of HEA-125 (biliary specific) and LKM-1 (hepatocyte specific) and other markers relevant to morphogenesis (Bcl-2, EMA) and cell proliferation (Ki-67) in cryostat sections from different chronic liver diseases and from fetal livers at different gestational ages. In parallel, viable NCAM-positive ductular cells were purified from collagenase digests of cirrhotic livers by immunomagnetic separation and characterized by immunocytochemistry and transmission electron microscopy. We demonstrated that reactive ductules with atypical morphology coexpressed NCAM and Bcl-2 and were found mainly in congenital diseases associated with ductal plate malformation and in primary cholangiopathies. On the contrary, reactive ductules with typical morphology were negative for NCAM/Bcl-2 and positive for EMA. Reactive ductules coexpressing NCAM/Bcl-2 were negative for the proliferation marker Ki-67 and appeared to be directly connected with periportal hepatocytes. In fetal livers NCAM/Bcl-2 was transiently expressed during the early developmental stages of ductal plate (10-16 weeks) and started to disappear as the ductal plate began duplicating. NCAM-positive ductal plate cells were Ki-67 negative, becoming positive in duplicated segments. Thus the histogenesis of ductular reactive cells seems to recapitulate the early stages of biliary ontogenesis. In primary cholangiopathies and ductal plate malformations, these cells do not appear to maturate further, and thus abundant ductular structures coexist with vanishing mature ducts. These NCAM-positive ductular cells were immunopurified from patients with chronic cholestatic liver diseases and showed ultrastructural features consistent with a less differentiated phenotype than mature cholangiocytes. These isolated cells represent a useful model for in vitro studies.

CD40 activation-induced, Fas-dependent apoptosis and NF-κB/AP-1 signaling in human intrahepatic biliary epithelial cells

Fas‐mediated mechanisms of apoptosis are thought to be involved in the bile duct loss that characterizes diseases such as primary biliary cirrhosis (PBC). We have previously shown that activation of CD40 on hepatocytes can amplify Fas‐mediated apoptosis; in the present study, we investigated interactions between CD40 and Fas in biliary epithelial cells (BEC). We report that the bile ducts in PBC liver tissue frequently express increased levels of Fas, Fas ligand (FasL), and CD40 associated with apoptotic BEC. The portal mononuclear infiltrate contains CD40L+ve T cells and macrophages, thereby demonstrating a potential mechanism for CD40 engagement in vivo. Primary cultures of human BEC also expressed Fas, FasL, and CD40 but not CD40L protein or mRNA. Activation of CD40 on BEC using recombinant CD40L increased transcriptional expression of FasL and induced apoptosis, which was inhibited by neutralizing antibodies to either Fas or FasL. Thus, CD40‐induced apoptosis of BEC is mediated through Fas/FasL. We then investigated the intracellular signals and transcription factors activated in BEC and found that NF‐κB and AP‐1 were both activated after CD40 ligation. Increased functional NF‐κB was seen early after CD40 ligation, but returned to baseline levels after 4 h. In contrast, the rapid up‐regulation of AP‐1 was sustained over 24 h. This study provides further functional evidence of the ability of CD40 to induce Fas/FasL‐dependent apoptosis of liver epithelial cells supporting the importance of cross‐talk between tumor necrosis factor (TNF) receptor family members as an amplification step in apoptosis induction. Sustained activation of AP‐1 in the absence of NF‐κB signaling may be a critical factor in determining the outcome of CD40 engagement.—Afford, S. C., Ahmed‐Choudhury, J., Randhawa, S., Russell, C., Youster, J., Crosby, H. A., Eliopoulos, A., Hubscher, S. G., Young, L. S., Adams, D. H. CD40 activation‐induced, Fas‐dependent apoptosis and NFkB/AP‐1 signaling in human intrahepatic biliary epithelial cells. FASEB J. 15, 2345‐2354 (2001)

Altered Notch ligand expression in human liver disease: further evidence for a role of the Notch signaling pathway in hepatic neovascularization and biliary ductular defects.

The Jagged and Delta family of transmembrane proteins are ligands for Notch receptors, which control the proliferation and/or differentiation of many cell lineages. Expression and localization of these ligands in the adult human liver has not been fully elucidated, nor whether dysregulation of these proteins contributes to liver disease processes. We have examined expression of the five known Notch ligands in human liver. Expression of Jagged-1 and Delta-4 mRNA was seen in normal and diseased liver tissue, whereas Jagged-2, Delta-1, and Delta-3 mRNA was undetectable. In primary liver cell isolates, Jagged-1 expression was found in all cell types, whereas Delta-4 was present in biliary epithelial and liver endothelial cells, but absent in hepatocytes. Interestingly, Jagged-1 mRNA expression was significantly up-regulated in diseased liver tissue. By immunohistochemistry, Jagged-1 expression was present on most structures in normal tissue. However in disease, strikingly strong Jagged-1 immunoreactivity was observed on many small neovessels and bile ductules. The expression of downstream modulators and effectors of Notch signaling was also detectable in purified cell isolates. This, together with aberrant Jagged-1 expression suggests that the Notch signaling pathway may play a role in the neovascularization and biliary defects observed in the liver during the development of cirrhosis.

The role of Notch receptor expression in bile duct development and disease.

Mutations in the Jagged1 gene, a ligand for the Notch signalling pathway, have been implicated in the pathogenesis of Alagille syndrome (AGS), resulting in bile duct paucity. Recently, a mouse model for AGS suggested that abnormalities of the Notch2 receptor, as well as of Jagged1, may be present. Expression patterns of Notch receptors have not been described in the developing human liver or in paediatric liver. The expression of Notch receptors and ligands was examined in fetal, paediatric normal, and diseased human liver by RT‐PCR and immunohistochemistry. RT‐PCR showed Notch1–4 mRNA expression to be present. In fetal liver, Notch3 protein was expressed on mesenchymal cells, closely adjacent to ductal plate cells that expressed Jagged1. In paediatric normal liver, Notch1 and Notch2 were present on mature bile duct cells. Notch expression was altered in disease, with distinct differences in AGS from extrahepatic biliary atresia (EHBA) and α1‐anti‐trypsin deficiency (α1AT). In AGS, where extensive ductular reaction was present, Jagged1 was expressed on ductular reactive cells (DRCs), along with marked Notch2 and Notch3 staining. Where there was ductular paucity, Notch2 and Notch3 were not expressed on remaining biliary epithelial cells. In EHBA and α1AT, Notch receptor expression was not seen on DRCs. Instead, Notch2 and Notch3 were expressed by stromal cells. In all diseases, Notch3 was expressed on neovessels in portal tracts and cirrhotic fibrous septa. In conclusion, Notch3 is expressed in close proximity to Jagged1 at the time of ductal plate formation, suggesting that Notch3 is important for bile duct development. The expression of both Notch2 and Notch3 in AGS on DRCs confirms that these receptors may be important in the pathogenesis of this disease. Further studies are required to investigate the presence of Notch2 and Notch3 at other periods in liver development and to clarify the role of Notch signalling in paediatric cholestases. Copyright

Progenitor cells of the biliary epithelial cell lineage

Stem-like cells have been identified in liver that are able to differentiate in vivo and in culture to biliary epithelial cells (BEC), hepatocytes and oval cells. The growth factors/cytokines and signal pathways required for the differentiation processes are beginning to be evaluated. There is increasing evidence to suggest that these stem-like cells may originate from both the bone marrow population or from a precursor remnant from liver embryogenesis, as they share many of the same markers (CD34, c-kit, CD45). Most recently, it has been shown that a population of progenitor cells can copurify with mesenchymal bone marrow cells and differentiate under specific culture conditions to form both hepatic epithelial and also endothelial cells. The interaction of haemopoietic and mesenchymal stem cells needs further evaluation. The close association of ductular reactive cells and neovessels in end-stage cholestatic liver diseases and the relation to Jagged/Notch signalling pathway may be important in the regulation of stem cells to form both biliary epithelial and endothelial cells.

A Novel Cell‐Surface Marker Found on Human Embryonic Hepatoblasts and a Subpopulation of Hepatic Biliary Epithelial Cells

The nature of the cells that contribute to the repopulation of the liver after hepatic necrosis or cirrhosis remains uncertain, in part because we lack specific markers to facilitate identification and prospective isolation of progenitor cells. The monoclonal antibody GCTM‐5 reacts with a minority subpopulation of cells in spontaneously differentiating cultures of pluripotent human embryonal carcinoma or embryonic stem cells. The epitope recognized by GCTM‐5 is found on a 50‐kDa protein present on the surface of these cells. In tissue sections of first‐trimester human embryos, GCTM‐5 specifically stained hepatoblasts and no other cell type examined. In normal pediatric or adult liver, GCTM‐5 reacted with a minority population of luminal bile duct cells. In diseased livers, the numbers of GCTM‐5–positive cells were increased compared with normal liver; antibody staining was restricted to a subpopulation of ductular reactive cells, and among this subpopulation we observed GCTM‐5–positive cells that did not express cytokeratin 19 or N‐CAM, classical makers of ductular reactive cells. Live GCTM‐5–positive cells could be isolated from diseased livers by immunomagnetic sorting. These results suggest that GCTM‐5 will be a useful reagent for defining cell lineage relationships between putative progenitor populations in embryonic liver and in the biliary epithelium during tissue repair.

Adhesion of human haematopoietic (CD34(+)) stem cells to. human liver compartments is integrin and CD44 dependent and modulated by CXCR3 and CXCR4

BACKGROUND/AIMS Haematopoietic stem cells (HSC) have previously been shown in some studies to migrate to damaged and diseased liver where a small proportion will engraft. Such cells can promote liver repair in rodent models of liver injury and lead to improved liver function in uncontrolled clinical studies. In order to maximize the engraftment of cells for clinical applications it is necessary to understand the molecular mechanisms that regulate stem cell recruitment and retention. Our aim therefore was to determine which factors where involved in adhesion of circulating HSC to liver endothelium and sequestration around epithelial cells within the liver. METHODS We examined the ability of CD34+ populations from peripheral and mobilized blood and the CD34-expressing cell line KG1a to bind to human hepatic sinusoidal endothelial (HSEC) and biliary epithelial cells (BEC) in vitro. RESULTS We report that all CD34(+) populations express alpha4beta1, beta2 integrins and CD44. Liver tissue sections and primary liver cells expressed the corresponding ligands VCAM-1/fibronectin, ICAM-1 and CD44. Pertussis toxin was shown to decrease binding of CD34(+) cells and the cells migrated to CXCR3 and CXCR4 ligands. CONCLUSIONS CD34(+) populations use alpha4beta1, beta2 integrins and CD44 receptors to bind to the ligands VCAM-1/fibronectin, ICAM-1, and hyaluronic acid expressed on sinusoidal vessels in tissue sections and to primary human HSEC. Binding to BEC was mediated by the interaction of beta1 and beta2 integrins with VCAM-1 and ICAM-1 respectively. A role for chemokines is supported by our finding that pertussis toxin inhibits CD34(+) cell adhesion to BEC and HSEC and by the ability of CD34(+) cells to migrate to CXCR3 and CXCR4 ligands.

Haematopoietic stem cell recruitment to injured murine liver sinusoids depends on α4β1 integrin/VCAM-1 interactions

Objective: Evidence suggests haematopoietic stem cells (HSCs) can migrate to injured liver and influence tissue repair. However, mechanisms governing HSC recruitment to injured hepatic microcirculation are poorly understood. These were investigated in vivo following hepatic ischaemia–reperfusion (IR) injury and in vitro using flow-based adhesion assays. Design: Partial IR was induced in anaesthetised WT or PECAM-1−/− mice for 90 min. Recruitment of systemically administered HSCs was monitored and effects of function blocking antibodies against α4β1 integrin, CD18, CD44, PECAM-1 or VCAM-1 investigated. The kinetics and molecular events governing adhesion to murine cardiac endothelial cells in vitro were also determined. Effects of conditioned media from IR injured liver on HSC adhesion molecule expression was determined by FACS. Results: Administered HSCs homed predominantly to lungs rather than liver, highlighting a potential therapeutic hurdle. Hepatic HSC recruitment following IR injury was inhibited by anti-α4β1 and anti-VCAM-1 antibodies. A role for α4β1 was also confirmed using flow-based adhesion assays. Incubating HSCs with conditioned media from IR injured liver increased α4β1 expression. CD18, CD44 and PECAM-1 were not involved in recruitment. Conclusions: This novel study demonstrates that the α4β1/VCAM-1 pathway mediates HSC recruitment to injured liver. Manipulating this pathway may enhance delivery of HSCs to the liver.