Use of Flow Cytometric Analysis to Examine the Uptake of Apoptotic Bodies by Healthy Hepatocytes

Excerpt

The spectrum of liver disease after alcohol abuse is similar to many other types of liver diseases in that damage to the hepatocyte, or parenchymal cell, is the cardinal event in the pathogenesis of injury. Damage to the liver, however, involves not only the hepatocyte but also the nonparenchymal cells: the Kupffer, endothelial, and stellate cells which release inflammatory and fibrogenic factors (such as cytokines and chemokines) that lead to altered liver pathology. We have studied extensively the effects of ethanol administration on protein trafficking in the liver, focusing most of our work on the hepatocyte and the process of endocytosis by these cells, using the endocytic pathway of the asialoglycoprotein receptor (ASGP-R) as a model [1‐8]. Currently in our laboratory we are examining the effect of ethanol administration on the process of apoptosis (programmed cell death) to determine a role for altered endocytosis in this process. Apoptosis is recognized not only as one of the initiating events in toxic liver injury, but is also increasingly recognized as a key mechanism in tissue inflammation and fibrosis [9, 10]. Of interest to us is work showing that healthy hepatocytes can bind and internalize apoptotic bodies [11, 12], and that the hepatocellular ASGP-R is involved in this clearance. We have utilized an in vitro system to characterize uptake of apoptotic bodies in isolated hepatocytes and define a role for the ASGP-R in this uptake. Our goal is to determine if there is an impaired ability to take up bodies in hepatocytes from ethanol-fed animals compared with control hepatocytes. An ethanol-induced impairment in the ability to remove apoptotic cells in the liver parenchyma could lead to an accumulation of bodies thereby disturbing the hepatic architecture and contributing to the initiation of fibrosis, specifically by activation of the non-parenchymal cell population. The work described in this present study was performed to characterize the in vitro system using flow cytometry to examine uptake of apoptotic bodies by healthy hepatocytes. Our long-term goal is to determine whether inadequate removal of apoptotic cells, presumably via altered receptor-mediated endocytosis (RME), plays a role in the course of pathogenesis of alcoholic liver injury. …