A flow adhesion assay to study leucocyte recruitment to human hepatic sinusoidal endothelium under conditions of shear stress

J Vis Exp. 2014 Mar 21:(85):51330. doi: 10.3791/51330.

Abstract

Leucocyte infiltration into human liver tissue is a common process in all adult inflammatory liver diseases. Chronic infiltration can drive the development of fibrosis and progression to cirrhosis. Understanding the molecular mechanisms that mediate leucocyte recruitment to the liver could identify important therapeutic targets for liver disease. The key interaction during leucocyte recruitment is that of inflammatory cells with endothelium under conditions of shear stress. Recruitment to the liver occurs within the low shear channels of the hepatic sinusoids which are lined by hepatic sinusoidal endothelial cells (HSEC). The conditions within the hepatic sinusoids can be recapitulated by perfusing leucocytes through channels lined by human HSEC monolayers at specific flow rates. In these conditions leucocytes undergo a brief tethering step followed by activation and firm adhesion, followed by a crawling step and subsequent transmigration across the endothelial layer. Using phase contrast microscopy, each step of this 'adhesion cascade' can be visualized and recorded followed by offline analysis. Endothelial cells or leucocytes can be pretreated with inhibitors to determine the role of specific molecules during this process.

Publication types

  • Research Support, Non-U.S. Gov't
  • Video-Audio Media

MeSH terms

  • Cell Adhesion / immunology
  • Cytokines / pharmacology
  • Endothelial Cells / cytology*
  • Endothelial Cells / immunology
  • Humans
  • Liver / cytology*
  • Liver / immunology
  • Lymphocytes / cytology*
  • Lymphocytes / immunology
  • Microscopy / methods

Substances

  • Cytokines