Targeting of aminopeptidase N to bile canaliculi correlates with secretory activities of the developing canalicular domain.

We have used human hepatoma cell lines as an in vitro model to study the development of hepatic bile canaliculi (BC). Well-differentiated hepatoma cells cultured for 72 hours could develop characteristic spheroid structures at sites of cell-cell contact that contained tight junctions and various membrane protein markers, resembling BC found in vivo. Intact cytoskeleton was essential for this differentiation process. In the coculture experiments in which cells of different origins were populated together, BC only formed between hepatic cells and preferentially among well-differentiated cells. Poorly differentiated hepatoma cells never formed BC among themselves, but could be induced to undergo canalicular differentiation by interacting with well-differentiated cells. During BC morphogenesis, integral canalicular membrane proteins were gradually delivered and accumulated at the developing BC. Among them, targeting of aminopeptidase N (APN) seemed to correlate with activation of certain secretory functions. Specifically, only APN-positive BC supported excretion of fluorescein diacetate (FDA) and 70-kd dextran, but had no relationship with secretion of horseradish peroxidase (HRP). Targeting of another BC protein, dipeptidyl peptidase IV (DPPIV), on the other hand, bore no association with any secretory activity examined. In addition, inhibition of enzymatic activity of APN could perturb canalicular differentiation without affecting cell proliferation. Our results suggest that targeting of APN proteins may reflect or even play an important role in the development and functional maturation of the canalicular structures.

Endothelial cell binding by human polyclonal anti-DNA antibodies: relationship to disease activity and endothelial functional alterations.

Polyclonal anti-dsDNA and anti-ssDNA antibodies (PoAb) that showed significant binding to human umbilical vein endothelial cells (HUVEC) were isolated from eight patients with systemic lupus erythematosus (SLE). Anti-dsDNA PoAbs from five patients and anti-ssDNA PoAbs from seven patients demonstrated enhanced binding to HUVEC during active disease, compared with PoAbs obtained from corresponding patients during remission. Reduction of the DNA content in the PoAb preparations by DNase treatment was associated with enhanced binding to HUVEC in 20 of 32 PoAbs tested, which included 75% 'active disease' PoAbs, and with reduced binding to HUVEC in three of 32 PoAbs tested, all obtained during remission. Such altered endothelial cell binding was reversed with DNA reconstitution. Binding of the remaining nine PoAbs to HUVEC was not altered by variations in their DNA content. Induced plasma membrane expression of E-selectin, but reduced expression of vascular cell adhesion molecule-1 (VCAM-1) by HUVEC, was observed following incubation of HUVEC with 'active disease' PoAbs from three and two of the eight patients, respectively. PoAbs and serum samples from two of the eight patients during active disease induced von Willebrand factor release from HUVEC, which was not observed during remission. We conclude that anti-DNA antibodies from selected patients with SLE can bind to endothelial cells. Correlation between cellular binding and disease activity suggests that such binding of anti-DNA antibodies to endothelial cells could be of pathogenic significance. Preliminary data also suggest that the expression of adhesion molecules and haemostatic factor(s) by endothelial cells may be modified following their binding by anti-DNA antibodies.