Lack of hepatocellular CD10 along bile canaliculi is physiologic in early childhood and persistent in Alagille syndrome

Abstract

Many tissues, including hepatobiliary cells, express neutral endopeptidase (CD10), encoded by MME. Serum neutral endopeptidase activity (NEA) has been recommended as a marker of cholestasis in adults but not in children with Alagille syndrome (AGS). We investigated ontogenic and disease-related differences in the expression of CD10. CD10 was found on canalicular surfaces of hepatocytes throughout the lobule in 16 adults and in 31 children aged Z24 months, with and without cholestasis, but not in 39 children aged o24 months, with and without cholestasis. Ten AGS children aged 2 months to 6 years lacked any canalicular CD10 expression. Cholangiocyte apices and/or intrasinusoidal granulocytes marked for CD10 in all subjects. Liver membrane fractions from a child with cholestasis aged o24 months and from 2 AGS patients aged 424 months contained reduced levels of CD10. In contrast, AGS children and all controls expressed CD10 similarly on granulocytes. MME mRNA was found in the liver of children aged o24 months and of adults, all with cholestasis, and of AGS patients. Granulocyte MME mRNA levels were similar among all study subjects; however, liver MME mRNA levels were 6-to 140-fold less than in normal adults in all cholestatic subjects, including AGS children. Methylation of the MME promoter was not detected in the liver of AGS children. In conclusion, hepatocytes in early childhood physiologically lack immunohistochemically detectable CD10. Reduced MME mRNA in AGS is not due to MME promoter methylation. Liver CD10 in childhood appears to undergo reduced synthesis or rapid degradation, which persists in AGS. Absence of CD10 expression thus may limit NEA as a marker of cholestasis in young patients and in AGS.

Diagnostic Utility of CD10 in Benign and Malignant Extrahepatic Bile Duct Lesions

“…6,22 In the gastrointestinal tract, CD10 expression is limited to microvilli of the small intestinal and biliary epithelium, where it regulates the concentration of bradykinin, cholecystokinin, and other bioactive substances in the postsecretory modification of bile. 4,5,24 The immunostaining pattern of CD10 in the intestinal villi, gallbladder, and bile ducts is consistently strong with continuous apical expression on the brush border, 14,21,30 whereas in hepatocytes CD10 expression is discontinuous, with patchy apical labeling with a canalicular distribution. 4,6,13,21 A few reports also showed that CD10 expression is mostly preserved in benign lesions such as hepatic bile duct adenoma, 36 pyloric gland adenoma of the gallbladder, 24 and benign intrahepatic bile ducts.…”

“…4,5,24 The immunostaining pattern of CD10 in the intestinal villi, gallbladder, and bile ducts is consistently strong with continuous apical expression on the brush border, 14,21,30 whereas in hepatocytes CD10 expression is discontinuous, with patchy apical labeling with a canalicular distribution. 4,6,13,21 A few reports also showed that CD10 expression is mostly preserved in benign lesions such as hepatic bile duct adenoma, 36 pyloric gland adenoma of the gallbladder, 24 and benign intrahepatic bile ducts. 4,32 In contrast, intrahepatic cholangiocarcinomas were negative to CD10.…”

Missense mutations and single nucleotide polymorphisms in ABCB11 impair bile salt export pump processing and function or disrupt pre-messenger RNA splicing

“…Membrane fractions were prepared essentially as previously described. 26,27 The blots shown are representative of two separate transfections for each mutant. For each set of transfections and subsequent membrane preparations, there was always a control experiment with wild-type BSEP, and so the mutants were always standardized to the 100% expression level of wild-type processed at the same time.…”

“…One gel was stained with Coomassie blue to confirm uniformity of protein loading among samples. The contents of the other gel were western blotted as previously described, 26 except using a 1:300 dilution of an anti-hemagglutinin (Y-11) polyclonal primary antibody (Santa Cruz Biotechnology, Santa Cruz, CA) and a 1:1,000 dilution of horseradish peroxidase- conjugated goat anti-rabbit secondary antibody (DAKO, Ely, UK).…”

“…Real-time PCR was performed as previously described, 26 except that data were normalized to HPRT1 expression [delta (d) Ct] and plotted as fold difference of the mean Ct values (dd Ct) compared with the wild-type exon.…”