Protein expression of double-stranded RNA-activated protein kinase (PKR) in intrahepatic bile ducts in normal adult livers, fetal livers, primary biliary cirrhosis, hepatolithiasis and intrahepatic cholangiocarcinoma

BACKGROUND/AIM: The protein expression of double-stranded RNA-activated protein kinase (PKR) in intrahepatic bile ducts has not been investigated. METHODS: Immunohistochemistry and a semiquantitative scoring method in normal liver and biliary diseases were used for the investigation. RESULTS: In "normal" adult livers (n=10), intrahepatic bile ducts were negative for PKR. In normal fetal livers (n=25), primitive biliary epithelia were almost negative for PKR. In primary biliary cirrhosis (PBC) (n=30), damaged bile ducts were frequently positive for PKR, while uninvolved bile ducts were negative. In hepatolithiasis (n=27), proliferated bile ducts were positive for PKR, and the PKR score correlated with the degree of proliferation. In cholangiocarcinoma (CC) (n=44), PKR expression was frequently noted, and the PKR score correlated with good differentiation of CC, being highest in well-differentiated CC and lowest in poorly-differentiated CC. The PKR score decreased in the following order: CC (mean PKR score=3.96), hepatolithiasis (2.56), PBC (1.60), normal fetal liver (0.40), and normal adult livers (0.00). The PKR expression in hepatocytes was "baseline" in normal adult livers, while moderately increased in fetal livers, PBC, hepatolithiasis and CC. CONCLUSIONS: Although the significance of these data is unclear, they suggest (i) that PKR is absent in bile ducts in normal adult and fetal livers, (ii) that PKR in bile duct cells newly emerges or increases in PBC, hepatolithiasis, and CC, (iii) that PKR accumulates in damaged bile ducts in PBC, (iv) that PKR increases in parallel with biliary cell proliferation in hepatolithiasis, and (v) that PKR expression correlates with differentiation in CC. PKR expression in intrahepatic bile ducts seems to be associated with inflammation or cell proliferation of the bile duct cells.     

Plasma cells and the chronic nonsuppurative destructive cholangitis of primary biliary cirrhosis

There has been increased interest in the role of B cells in the pathogenesis of primary biliary cirrhosis (PBC). Although the vast majority of patients with this disease have anti-mitochondrial antibodies, there is no correlation of anti-mitochondrial antibody titer and/or presence with disease severity. Furthermore, in murine models of PBC, it has been suggested that depletion of B cells may exacerbate biliary pathology. To address this issue, we focused on a detailed phenotypic characterization of mononuclear cell infiltrates surrounding the intrahepatic bile ducts of patients with PBC, primary sclerosing cholangitis, autoimmune hepatitis, chronic hepatitis C, and graft-versus-host disease, including CD3, CD4, CD8, CD20, CD38, and immunoglobulin classes, as well as double immunohistochemical staining for CD38 and IgM. Interestingly, CD20 B lymphocytes, which are a precursor of plasma cells, were found in scattered locations or occasionally forming follicle-like aggregations but were not noted at the proximal location of chronic nonsuppurative destructive cholangitis. In contrast, there was a unique and distinct coronal arrangement of CD38 cells around the intrahepatic ducts in PBC but not controls; the majority of such cells were considered plasma cells based on their expression of intracellular immunoglobulins, including IgM and IgG, but not IgA. Patients with PBC who manifest this unique coronal arrangement were those with significantly higher titers of anti-mitochondrial antibodies. CONCLUSION: These data collectively suggest a role for plasma cells in the specific destruction of intrahepatic bile ducts in PBC and confirm the increasing interest in plasma cells and autoimmunity.     

Electron microscopic observation of destruction of biliary epithelium in primary biliary cirrhosis

ABSTRACT— Electron microscopic studies of the intrahepatic biliary tree in 16 patients with primary biliary cirrhosis (PBC) disclosed four types of biliary epithelial injury suggesting cell death in the ducts: 1) coagulative and 2) lytic necrosis without detachment of affected cells from the biliary epithelial layer, and 3) apoptosis and 4) detachment of several adjoining biliary cells from the basement membrane and neighboring biliary cells. Lesions 1), 2) and 3) were also found in livers with extrahepatic cholestasis without bile duct loss, and 1) and 2) were found in PBC livers irrespective of the degree of bile duct loss. 3) was rare and mostly confined to bile ductules, when present. By contrast, 4) was only observed in PBC, especially in livers with a moderate degree of bile duct loss in which extensive bile duct destruction appeared to be progressing. Detached biliary cells in lesion 4) were occasionally in contact with and/or surrounded by migrating lymphocytes with pseudopod formation, suggesting lymphocyte-target cell interactions. It therefore seems possible that epithelial detachment is an important ultrastructural lesion associated with extensive bile duct destruction in PBC livers.     

Expression of intercellular adhesion molecule-1 and lymphocyte function-associated antigen-1 protein and messenger RNA in primary biliary cirrhosis

OBJECTIVE: This study examined the role of intercellular adhesion molecule-1 (ICAM-1) and lymphocyte function-associated antigen-1 (LFA-1) in the autoimmune process of bile duct destruction in the early stages of primary biliary cirrhosis (PBC). MATERIALS AND METHODS: Ten PBC liver samples and five control samples were studied. Immunohistochemical studies of ICAM-1 and LFA-1, and Western blot of ICAM-1 were performed. Immunoelectron microscopy was conducted using immunoglobulin-gold and silver staining. Human ICAM-land LFA-1 peptide nucleic acid probes were used for in situ hybridization. RESULTS: In PBC liver samples, immunohistochemistry showed aberrant ICAM-1 expression on bile duct epithelial plasma membrane and also luminal sites of endothelial plasma membrane of terminal portal venules. Western blot confirmed ICAM-1 protein expression. LFA-1-positive lymphocytes were associated with epithelial cells of septal and interlobular bile ducts. Immunoelectron microscopy localized ICAM-1 on the luminal and basal surfaces as well as on lymphocytes around damaged bile duct epithelial cells, and LFA-1 on lymphocytes around damaged bile ducts. Messenger RNA expression of ICAM-1 was demonstrated in bile ducts, and LFA-1 in lymphocytes around bile ducts. CONCLUSION: De novo expression of ICAM-1 and LFA-1 at protein and mRNA levels in PBC may imply an inductive role of ICAM-1 through binding with its ligand LFA-1 in the extravasation of activated lymphocytes and lymphocyte-mediated bile duct destruction.     

Protein expression of double‐stranded RNA‐activated protein kinase (PKR) in intrahepatic bile ducts in normal adult livers,fetal livers,primary biliary cirrhosis,hepatolithiasis and intrahepatic cholangiocarcinoma

Abstract: Background/Aim: The protein expression of double‐stranded RNA‐activated protein kinase (PKR) in intrahepatic bile ducts has not been investigated. Methods: Immunohistochemistry and a semiquantitative scoring method in normal liver and biliary diseases were used for the investigation. Results: In “normal” adult livers (n=10), intrahepatic bile ducts were negative for PKR. In normal fetal livers (n=25), primitive biliary epithelia were almost negative for PKR. In primary biliary cirrhosis (PBC) (n=30), damaged bile ducts were frequently positive for PKR, while uninvolved bile ducts were negative. In hepatolithiasis (n=27), proliferated bile ducts were positive for PKR, and the PKR score correlated with the degree of proliferation. In cholangiocarcinoma (CC) (n=44), PKR expression was frequently noted, and the PKR score correlated with good differentiation of CC, being highest in well‐differentiated CC and lowest in poorly‐differentiated CC. The PKR score decreased in the following order: CC (mean PKR score=3.96), hepatolithiasis (2.56), PBC (1.60), normal fetal liver (0.40), and normal adult livers (0.00). The PKR expression in hepatocytes was “baseline” in normal adult livers, while moderately increased in fetal livers, PBC, hepatolithiasis and CC. Conclusions: Although the significance of these data is unclear, they suggest (i) that PKR is absent in bile ducts in normal adult and fetal livers, (ii) that PKR in bile duct cells newly emerges or increases in PBC, hepatolithiasis, and CC, (iii) that PKR accumulates in damaged bile ducts in PBC, (iv) that PKR increases in parallel with biliary cell proliferation in hepatolithiasis, and (v) that PKR expression correlates with differentiation in CC. PKR expression in intrahepatic bile ducts seems to be associated with inflammation or cell proliferation of the bile duct cells.     

Myeloperoxidase-positive inflammatory cells participate in bile duct damage in primary biliary cirrhosis through nitric oxide-mediated reactions

Previous studies have suggested that increased nitric oxide (NO)-mediated products are found in the livers of subjects with primary biliary cirrhosis (PBC), but the mechanisms involved remain enigmatic. We took advantage of immunohistochemistry and several unique monoclonal antibodies to study inflammatory cells responsible for the generation of NO, the enzymes responsible for NO production, the expression of 3-nitrotyrosine, and the presence of CD68(+) and/or myeloperoxidase (MPO)(+) cells. We examined a total of 113 liver specimens, including 64 with PBC, 19 with primary sclerosing cholangitis (PSC), 6 with non-A, non-B hepatitis, 6 with alcoholic liver disease, 4 with cryptogenic cirrhosis, 4 with biliary atresia, and 10 normal subjects. Twenty-two percent of PBC had elevated expression of 3-nitrotyrosine in their bile duct epithelial cells (BECs) (P =.0316). Furthermore, the BECs in PBC also demonstrated apoptotic changes. MPO-positive inflammatory cells were also noted adjacent to the basement membrane. In contrast, the liver of normal subjects showed few apoptotic changes in the bile ducts, with no evidence of MPO staining in the portal area. Furthermore, sections from livers of subjects with stage I or stage II PBC demonstrated significantly increased inflammatory cell infiltration (P =.0064) and elevated 3-nitrotyrosine expression in BECs (P =.0246) compared with stage III and IV. The presence of 3-nitrotyrosine was closely associated with infiltrating CD68- and/or MPO-positive cells. There was also a stage-associated difference in the presence of bile duct infiltrating cells and 3-nitrotyrosine in PBC with an increase dominant in early stage disease. In conclusion, NO and reactive oxygen species, collectively determined as 3-nitrotyrosine, are associated with bile duct destruction in PBC and are particularly prevalent in early stage disease.     

B7-2 positive cells around interlobular bile ducts in primary biliary cirrhosis and chronic hepatitis C

Bile duct damage in patients with chronic hepatitis C (hepatitis-associated bile duct lesion) as well as that in patients with primary biliary cirrhosis (PBC; chronic non-suppurative destructive cholangitis), may be causally related to immunological assaults. Efficient antigen presentation is known to require the provision of a costimulatory signal which is dependent on the CD28 on T cell surfaces, and that at least two molecules, B7-1 and B7-2, work as costimulatory ligands for CD28. In this study, we examined immunohistochemically, the expression of B7-2 in portal tracts of liver biopsy specimens obtained from 75 patients with chronic hepatitis C who had hepatitis-associated bile duct lesions, and from 63 PBC patients with chronic non-suppurative destructive cholangitis. B7-2 positive cells were recognizable as large mononuclear cells scattered in portal tracts. Some of these cells showed a dendritic cell-like appearance. B7-2 positive cells were observed more frequently (41%) in PBC liver specimens than in chronic hepatitis C specimens (17%, P< 0.05). In PBC livers, such cells were preferentially observed around the damaged bile duct with a few located in the biliary epithelial layer. There was no such finding in chronic hepatitis C livers. The frequency and density of B7-2 positive cells in the liver specimens tended to decrease according to the stage of PBC (45% in stages 1 and 2, and 33% in stages 3 and 4; P=0.10), whereas with chronic hepatitis C, no such tendency was observed. These findings suggest that B7-2 positive cells may play a role in the bile duct lesions that appear in the early histological stages of PBC and that the immunological mechanisms of bile duct damage, particularly of antigen presentation and B7-2 expression, differ between PBC and chronic hepatitis C.     

Morphological and immunohistochemical assessment of intrahepatic bile duct development in the rat

The hypotheses that intrahepatic bile ducts are derived either by a transformation of periportal liver cells or by dichotomous branching of the extrahepatic bile ducts were investigated in fetal and postnatal rat livers by histological and immunohistochemical methods using an antiserum to prekeratin which, in the liver, binds to biliary epithelial cells (BEC). In conventionally stained sections, bile duct development was observed to begin in the 19 day fetus around the larger branches of the portal vein, with the formation of lumina surrounded by cuboidal or elongated hepatoblast-like cells on the portal aspect and readily distinguished hepatoblasts on the lobular aspect. At 21 days, these structures had developed into canals of Hering lined jointly by recognizable liver cells and BEC. The number of canals of Hering per portal tract peaked at 22 days' gestation and diminished in number at birth and over the ensuing 56 h, with a concomitant increase in fully formed ducts. Bile ducts lined completely by BEC were first found at 20 days. Immunohistochemically, prekeratin antigens were first detected at 20 days in duct-like structures not only in phenotypic BEC but also in adjacent cells with an hepatoblast phenotype. Such intermediate cells were present until birth. These findings support the view that intrahepatic bile ducts develop by a reorganization and modulation of the periportal hepatoblasts to BEC.     

原发性胆汁性肝硬化免疫分子机制研究进展

原发性胆汁性肝硬化(primary biliary cirrhosis,PBC)是一种免疫介导损伤胆管上皮细胞及肝内小胆管的胆汁淤积性自身免疫性肝病,具有发展为肝硬化的倾向.多种免疫相关基因通过影响疾病易感性、免疫调节等途径,促使PBC的发生、发展.本文就PBC的免疫分子机制研究动态进行综述.     

A quantitative analysis of T lymphocyte populations in human liver allografts undergoing rejection: the use of monoclonal antibodies and double immunolabeling

The aim of this study was to quantitate T-cell populations infiltrating portal tracts, bile ducts and hepatic lobules in 82 biopsy specimens from 25 patients after orthotopic liver transplantation. Biopsy specimens taken immediately after revascularization of the graft were used as controls. Patients studied include 18 with initial rejection episodes, 11 with unresolved rejection, five with vanishing bile duct syndrome and eight patients with other forms of liver injury. Quantitation was done in a blinded fashion for the first 20 biopsy specimens. A double immunolabeling technique was used to simultaneously immunolabel bile duct structures (with anti-major histocompatibility complex class II or antikeratins) and lymphoid populations (with anti-CD2, anti-CD4 or anti-CD8). This facilitated the accurate quantitation of intraepithelial lymphocytes within bile ducts. This technique also enabled simultaneous detection of CD4 and CD8 antigens on lymphocytes in portal tracts. The predominant lymphocyte subtype within biliary epithelium during acute and chronic rejection was of the CD2+/CD8+ phenotype. CD8+/CD4+ ratio in bile ducts was approximately 5:1 in acute, unresolved and chronic rejection. In vanishing bile duct syndrome, double immunolabeling enabled the detection of destroyed interlobular bile duct remnants that were not apparent on routine hematoxylin and eosin staining. Attached to some of these structures were CD8+ lymphocytes. Lobular CD8+ cells were not prominent in acute rejection but increased significantly in biopsy specimens from patients with unresolved and chronic rejection. In chronic rejection, a selective increase was seen in these CD8+ cells in centrizonal regions.(ABSTRACT TRUNCATED AT 250 WORDS)     

Apoptosis of biliary epithelial cells in primary biliary cirrhosis and primary sclerosing cholangitis

BACKGROUND/AIMS: Primary biliary cirrhosis (PBC) is an autoimmune disease characterized by inflammatory destruction of small bile ducts. Primary sclerosing cholangitis (PSC) is a different, presumed autoimmune cholestatic liver disease where the bile ducts are also destroyed. In this study, apoptosis and portal triad inflammation in liver tissue from patients with PBC is examined and compared to that from patients with PSC and patients with normal liver. METHODS: Explanted liver tissue from patients with PBC and PSC and normal liver from patients with metastases to liver were examined. The liver samples were stained for apoptosis using the terminal deoxynucleotidyl triphosphate (TdT)-mediated deoxyuridine triphosphate nick end labelling (TUNEL) assay. The biliary epithelial cells (BEC) were then scored on the basis of their TUNEL stain and the degree of periductal inflammation. RESULTS: In PBC, apoptosis of BEC, as detected by the TUNEL assay, was significantly increased in the presence of inflammation. Regardless of the presence or absence of inflammation, the small bile ducts in PBC liver tissue exhibited greater evidence of apoptosis than did similar ducts from PSC or control livers. CONCLUSION: These findings suggest that in PBC, unlike PSC, the apoptosis of BEC in PBC is secondary to the invasion of inflammatory cells.     

Significance of periductal Langerhans cells and biliary epithelial cell‐derived macrophage inflammatory protein‐3α in the pathogenesis of primary biliary cirrhosis

Background/aims: To clarify the primary biliary cirrhosis (PBC)‐specific antigen‐presenting mechanism, we examined the distribution and phenotypic characteristics of infiltrating dendritic cells (DCs) with respect to bile ducts and the mechanism of migration in terms of the periductal cytokine milieu and biliary innate immunity. Methods and results: Immunohistochemistry using liver sections from patients with PBC and controls revealed that blood dendritic cell antigen (BDCA)‐2⁺ plasmacytoid DCs were found mainly in the portal tracts in PBC and the controls, but their distribution was not related to bile ducts. BDCA‐1⁺ and CD19^? myeloid DCs were also found in portal tracts in PBC and the controls and, in particular, Langerin+Langerhans cells (LCs) were dominantly scattered around or within biliary epithelial layers of the damaged bile ducts in PBC. Moreover, experiments with cultured human biliary epithelial cells (BECs) showed that an LC‐attracting chemokine, macrophage inflammatory protein‐3α, was produced by BECs in the response to cytokines [interleukin (IL)‐1β, tumour necrosis factor‐α and IL‐17] and pathogen‐associated molecular patterns. Conclusions: LCs existing around or within biliary epithelial layers are important as periductal antigen‐presenting cells in PBC and the migration of LCs into bile ducts is closely associated with the periductal cytokine milieu and biliary innate immunity in PBC.     

Site-characteristic expression and induction of trefoil factor family 1, 2 and 3 and malignant brain tumor-1 in normal and diseased intrahepatic bile ducts relates to biliary pathophysiology.

BACKGROUND/AIM: Trefoil factor family (TFF)1,2,3 are involved in a homeostasis/repair process of mucosal epithelia. In this study, the significance of TFF family and deleted in the malignant brain tumor-1 (DMBT1), a putative receptor of TFF2, in the intrahepatic biliary tree was investigated in normal and diseased livers. MATERIALS AND METHODS: Expression of TFF1,2,3 and DMBT1 were examined immunohistochemically in primary biliary cirrhosis (PBC), primary sclerosing cholangitis (PSC), chronic viral hepatitis (CVH), extrahepatic biliary obstruction (EBO), and normal livers. RESULTS: In normal livers, TFF1,3 and DMBT1 were infrequently detectable in large and rarely in small bile ducts, respectively. TFF2 was not detectable in large bile ducts. In large bile duct diseases (PSC and EBO), expression of TFF3 and DMBT1 were increased. In small bile duct diseases (PBC and CVH), expression of TFF2/DMBT1 was induced in moderately to severely damaged ducts irrespective of etiology. CONCLUSION: The intrahepatic biliary tree shows a site-characteristic expression and induction of TFF1,2,3 and DMBT1. In large bile ducts, TFF1,3 were constitutively expressed and increased in pathologic bile ducts. In small bile ducts, TFF2/DMBT1 is induced in damaged ducts irrespective of etiologies. However, the cytoprotective/repair property of TFF2/DMBT1 may not be enough to prevent the following bile duct loss in PBC.     

Natural killer T cells exacerbate liver injury in a transforming growth factor beta receptor II dominant-negative mouse model of primary biliary cirrhosis

Primary biliary cirrhosis (PBC) is an organ-specific autoimmune liver disease characterized by the presence of antimitochondrial antibodies and the destruction of small intrahepatic bile ducts with portal inflammation. In previous studies, we reported that both CD1d expression and the frequency of CD1d-restricted natural killer T (NKT) cells were increased in the livers of patients with PBC. To define a specific role of CD1d-restricted NKT cells in the pathogenesis of PBC, particularly early events, we investigated the function of hepatic CD1d-restricted NKT cells in our transforming growth factor beta (TGF-beta) receptor II dominant-negative (dnTGFbetaRII) mouse model of PBC. We generated CD1d(-/-) and CD1d(+/-) dnTGFbetaRII mice and performed a comparative study of liver immunopathology. We report herein that these dnTGFbetaRII mice demonstrate a massive increase of hyperactive CD1d-restricted NKT cells within the hepatic tissues. CD1d(-/-)dnTGFbetaRII mice, which lack CD1d-restricted CD1d-restricted NKT cells, exhibit significantly decreased hepatic lymphoid cell infiltrates and milder cholangitis compared with CD1d(+/-)dnTGFbetaRII mice. Interestingly, there was a significant increase in the production of interferon-gamma in hepatic CD1d-restricted NKT cells activated by alpha-galactosylceramide in young but not older dnTGFbetaRII mice, suggesting an age-dependent role of CD1d-restricted NKT cells. CONCLUSION: These data demonstrate that CD1d-restricted NKT cells in dnTGFbetaRII mice are a critical factor in liver injury.     

原发性胆汗性肝硬化中B细胞免疫的研究进展

原发性胆汁性肝硬化（PBC）以肝内小胆管的损伤为特征，但其损伤机制尤其是B细胞免疫在其中发挥的作用尚不明确。从B细胞和浆细胞在PBC肝组织中的分布、B细胞清除、PBC血清中异常增高的IgM产生机制及其组织器官来源、线粒体抗体的产生机制以及在胆管损伤中的可能作用、PBC中特征性的自身抗体等方面，回顾了B细胞免疫在PBC发病机制中的可能作用。通过以上分析，认为B细胞免疫的确在PBC的发病机制中发挥了重要作用，但仍有很多关键的问题尚未得到阐明。     

In situ nucleic acid hybridization of pyruvate dehydrogenase complex-E2 in primary biliary cirrhosis: Pyruvate dehydrogenase complex-E2 messenger RNA is expressed in hepatocytes but not in biliary epithelium

Pyruvate dehydrogenase-E2, or a cross-reactive molecule, has been shown by a variety of immunohistochemical methods to be present in increased amounts in biliary epithelial cells (BEC) in primary biliary cirrhosis (PBC). In this study, to further understand the nature of the immunoreactive molecule in BEC, we examined the expression of pyruvate dehydrogenase complex-E2 (PDC-E2) messenger RNA (mRNA) and PDC-E2 protein in sections of livers from patients and controls to help identify the molecule found in BEC. We performed in situ hybridization using an antisense probe against the major epitope of PDC-E2. The data were very striking and suggested that there was no increased production of PDC-E2 in BEC. For example, in livers from patients with PBC, PDC-E2 mRNA was found in periportal hepatocytes in 16 of 17 cases (94%). In contrast, interlobular bile ducts and septal bile ducts had detectable levels of PDC-E2 mRNA in only 1 of 17 (6%) and 3 of 8 (38%) cases, respectively. Interestingly, proliferating bile ductules contained detectable levels of mRNA in 12 of 15 cases (80%). In control liver, periportal hepatocytes were positive in 15 of 17 cases (88%). Interlobular bile ducts, septal bile ducts, and proliferating bile ductules expressed mRNA signals in 4 of 17 (24%), 2 of 10 (20%), and 14 of 16 (88%), respectively. When formalin-fixed, paraffin-embedded sections were examined by immunohistochemical staining with anti-PDC-E2 monoclonal antibody (mAb) C355.1, the interlobular bile ducts showed typical aberrant apical staining in all 10 PBC cases, but 0 of 9 liver controls. Periportal hepatocytes, proliferating bile ductules and infiltrating mononuclear cells stained with C355.1 but in a characteristic mitochondrial staining pattern. The presence of a PDC-E2-like molecule recognized by C355.1 is not reflected by the expression levels of PDC-E2 mRNA in the BEC of patients with PBC.(Hepatology 1997 Jan;25(1):27-32)     

Immunohistochemical differences in the portal tract and acinar infiltrates between primary biliary cirrhosis and autoimmune cholangitis

BACKGROUND: Antimitochondrial antibody-negative primary biliary cirrhosis, or autoimmune cholangitis, may be indistinguishable clinically and histologically from antimitochondrial antibody-positive primary biliary cirrhosis. AIMS: We aimed to compare the phenotypic markers of the portal and acinar infiltrates in autoimmune cholangitis and antimitochondrial antibody-positive primary biliary cirrhosis. PATIENTS AND METHODS: Formalin-fixed, paraffin-embedded liver sections were identified from 32 patients with a clinical and histological diagnosis of primary biliary cirrhosis. Thirteen were antimitochondrial antibody-negative (autoimmune cholangitis group) and 19 were antimitochondrial antibody-positive. The groups were well matched for age, histological stage, liver biochemistry and drug treatment. Immunohistochemical staining was performed using monoclonal antibodies against CD3 (pan T cell), CD8 (cytotoxic), CD45RO (memory), CD45RA (naive), CD68 (macrophages) and against the secreted form of eosinophilic cationic protein (EG2). RESULTS: In autoimmune cholangitis, both portal and acinar CD3 cell counts were significantly higher than in antimitochondrial antibody-positive primary biliary cirrhosis (median portal count 421 vs 257 cells/graticule, P< 0.03; median acinar count 18 vs 9 cells/graticule, P< 0.02). There were no differences between the groups in portal or lobular CD8, CD45RO, CD45RA, CD68 or EG2. Of the total group (antimitochondrial antibody positives and negatives), there were significantly more CD45RA cells in early (stage 1) compared with cirrhotic (stage 4) disease (median 19.3 vs 14 cells/graticule, P< 0.03). EG2 staining was found in eight of the 32 sections overall, but not in the patients with stage 1 disease (P< 0.04). CONCLUSION: CD3 counts are higher in autoimmune cholangitis than in antimitochondrial antibody-positive primary biliary cirrhosis in both portal and acinar areas. However, there are no significant differences in memory/na?ve T-cell subsets between both conditions and, in both, loss of naive T lymphocytes and secretion of eosinophilic cationic protein occur with disease progression. This implies that the effector pathways of bile duct destruction are similar in autoimmune cholangitis and primary biliary cirrhosis.     

Differential expressions of aquaporin proteins in human cholestatic liver diseases.

Aquaporins (AQPs) are the channel forming membranous proteins involved in the biliary physiological homeostasis. Recently, we have reported the heterogeneous expression of AQPs in intrahepatic biliary epithelial cells or cholangiocytes in mice. However, the involvements of AQPs in hepatobiliary disorder are still unclear. Thus, we hypothesized that the AQP protein expressions are altered in human cholestatic disorders. METHODS: The AQP expressions of the immortalized human cholangiocytes cell line (H69) were assessed by immunoblotting. The expression of AQPs in liver biopsy specimens from various human cholestatic diseases as well as viral hepatitis were evaluated immunohistochemically. The degrees of staining were classified into four grades by comparison with staining intensity from controls. RESULTS: AQP1 expression, predominantly membranous, was confirmed by immunoblotting analysis. However, the other subtypes of AQP expression were not detected. In human pathological tissues, AQP1 expression by interlobular bile ducts was similar to normal and viral hepatitis, although this expression was attenuated according to bile duct injuries in PBC. On the contrary, the AQP1 expression by proliferating bile ductile (equivalent for small cholangiocytes) was enhanced. In intrahepatic cholestasis, AQP1 expressions were diminished, which was further associated with the aberrant expressions by periportal hepatocytes. CONCLUSIONS: AQP1 was expressed intensely in smaller proliferating bile ducts in human cholestatic liver disease. Also, the AQP1 expression was decreased in injured duct cells undergoing degeneration in PBC. The AQP1 expression was decreased in intrahepatic cholestasis probably due to negative feed back of the decreased bile flow. The role of AQP expression profiles may help the understanding of the pathogenesis of human cholestatic liver diseases.