Human liver sinusoidal endothelial cells promote intracellular crawling of lymphocytes during recruitment - a new step in migration

The recruitment of lymphocytes via the hepatic sinusoidal channels and positioning within liver tissue is a critical event in the development and persistence of chronic inflammatory liver diseases. The hepatic sinusoid is a unique vascular bed lined by hepatic sinusoidal endothelial cells (HSEC), a functionally and phenotypically distinct sub-population of endothelial cells. Using flow based adhesion assays to study the migration of lymphocytes across primary human HSEC, we found that lymphocytes enter into HSEC, confirmed by electron microscopy demonstrating clear intracellular localization of lymphocytes in vitro and by studies in human liver tissues. Stimulation by interferon gamma increased intracellular localization of lymphocytes within HSECs. Furthermore using confocal imaging and time-lapse recordings we demonstrated ‘intracellular crawling’ of lymphocytes entering into one endothelial cell from another. This required the expression of ICAM-1 and stabilin-1 and was facilitated by the junctional complexes between HSEC. Conclusion: We demonstrate a new step in lymphocyte migration which is facilitated by the unique structure of HSEC. We believe ‘intracellular crawling’ contributes to optimal lymphocyte positioning in liver tissue during chronic hepatitis.

Chronic inflammation is a major cause of global morbidity and mortality, often leading to tissue fibrosis and organ failure and is also a recognized risk factor for carcinogenesis(1-4). It is characterized by the recruitment of immune cells into organs via their interaction with endothelial cells followed by their positioning in strategic locations within the tissue(5). This is seen in nearly all adult liver diseases which are driven by chronic inflammation where leukocytes are recruited via specialized channels termed sinusoids which are lined by hepatic sinusoidal endothelial cells (HSEC)(6). This influx of immune cells often leads to lymphoid aggregates/follicles around the portal tract in a range of liver diseases(7).

Despite this common pathway of chronic inflammation, there are several features which contribute to the liver being a unique site for leukocyte recruitment. The extravasation occurs within the hepatic sinusoidal channels in contrast to the post-capillary venules as seen in most other organs(8, 9). These channels are characterized by a low flow environment and the sinusoidal endothelium, HSEC, has a unique morphology and performs specialized functions including scavenging and filtration(10). Conventional adhesion molecules, such as selectins which mediate leukocyte rolling, are absent from this vascular bed and recruitment is mediated by atypical adhesion molecules such as vascular adhesion protein-1 (VAP-1) and the common lymphatic endothelial and vascular endothelial receptor-1 (CLEVER-1) also known as stabilin-1 (11, 12).

The aim of this study was to perform a detailed analysis of the transendothelial pathway used by lymphocytes to cross human liver sinusoidal endothelium in order to identify organ specific targets of chronic inflammation within the liver. We developed real-time cell imaging by laser scanning confocal microscopy under conditions of physiologically relevant shear stress to visualize the migration of lymphocytes across HSEC. We visualized lymphocyte migration into the cytoplasm of HSECs from where the cells crossed junctional membranes to allow them to crawl from within one HSEC into another. We noted this process more frequently in HSEC compared to conventional vascular endothelium and found it was enhanced by interferon gamma treatment of the endothelium. Whilst crawling of leukocytes on the luminal surface has been described previously (13) we believe this is the first description of ‘intracellular crawling’ which may play an important role in leukocyte recruitment and positioning within the liver.