In Vivo F-Actin Filament Organization during Lymphocyte Transendothelial and Interstitial Migration Revealed by Intravital Microscopy

Serena L.S Yan,Il-Young Hwang,Olena Kamenyeva,John H Kehrl

doi:10.1016/j.isci.2019.05.040

Serena L.S Yan, Il-Young Hwang + Show 2 more

Open Access

https://doi.org/10.1016/j.isci.2019.05.040

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Journal: iScience	Publication Date: May 30, 2019
Citations: 16	License type: cc-by

Affiliation: National Institutes of Health

Abstract

SummaryActin is essential for many cellular processes including cell motility. Yet the organization of F-actin filaments during lymphocyte transendothelial migration (TEM) and interstitial migration have not been visualized. Here we report a high-resolution confocal intravital imaging technique with LifeAct-GFP bone marrow reconstituted mice, which allowed visualization of lymphocyte F-actin in vivo. We find that naive lymphocytes preferentially cross high endothelial venules (HEVs) using paracellular rather than the transcellular route. During both modes of transmigration F-actin levels rise at the lymphocyte leading edge as the cell engages the TEM site. Once the lymphocytes breach the endothelium, they briefly reside in HEV pockets before crossing into the parenchyma. During interstitial migration dynamic actin-based protrusions rapidly form and collapse to help drive motility. Using a panel of inhibitors, we established roles for actin regulators and myosin II in lymphocyte TEM. This study provides further insights into lymphocyte TEM and interstitial migration in vivo.

Highlights

The trafficking of immune cells through lymph nodes (LNs) plays a critical role in immunity
transendothelial migration (TEM) occurs through micro-wide gaps in high endothelial venules (HEVs) generated by transmigrating lymphocytes where lymphocytes provide the mechanical force needed to overcome the endothelial cell (EC) barriers allowing their cell body to squeeze through EC gaps and pores (Carman and Springer, 2008; Muller, 2003)
Both blood B and T cells had detectable levels of LifeAct-GFP expression as did B and T cells prepared from spleen and LNs (Figure 1A)

Summary

Introduction

The trafficking of immune cells through lymph nodes (LNs) plays a critical role in immunity. During immune surveillance lymphocytes recirculate from the blood, through LNs, into lymphatics, and back to the blood (Kehrl, 2004; Young, 1999). In a non-inflamed state, millions of naive lymphocytes enter mammalian LNs daily via high endothelial venules (HEVs) and exit via lymphatics (Kehrl, 2006; von Andrian and Mempel, 2003). TEM occurs through micro-wide gaps in HEVs generated by transmigrating lymphocytes where lymphocytes provide the mechanical force needed to overcome the endothelial cell (EC) barriers allowing their cell body to squeeze through EC gaps and pores (Carman and Springer, 2008; Muller, 2003). Recent studies suggest that the endothelial actin cytoskeletal network maintains the EC cell shape creating a mechanical barrier (Barzilai et al, 2017; Renkawitz and Sixt, 2010). Engagement of transmigrating leukocytes with the endothelium can trigger extensive modifications of EC actin cytoskeleton, including EC contraction and gap openings (Barzilai et al, 2017)

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