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Endothelial junctional membrane protrusions serve as hotspots for neutrophil transmigration

Janine J.G. Arts, Eike K. Mahlandt, Max L.B. Grönloh, Lilian Schimmel, Ivar Noordstra, Abraham C.I. van Steen, Simon Tol, Jos van Rijssel, Martijn A. Nolte, Marten Postma, Satya Khuon, John M. Heddleston, Eric Wait, Teng-Leong Chew, Mark Winter, Eloi Montanez, Joachim Goedhart, Jaap D. van Buul

Preprint posted on January 21, 2021 https://www.biorxiv.org/content/10.1101/2021.01.18.427135v1.full

Taking a dive; neutrophils utilise protrusions in endothelial junctional membranes to transmigrate

Selected by Jonny Coates

Categories: cell biology, immunology

Background

Neutrophils are innate immune cells that are primarily responsible for phagocytosing cellular debris and invading pathogens. Neutrophils are the most abundant white blood cell in human blood and have relatively short life-spans; they are released from the bone marrow and eliminated by macrophages within the span of a single day (1).

These specialised cells are often the first to traffic to sites of injury and infection. In order to reach sites of infection, neutrophils follow chemoattractant signals and then migrate out of the blood stream and into tissues (2). This migratory cascade is outlined in figure 1. Once neutrophils reach the target tissue they must adhere to the lumen wall, a process that begins as tethering and rolling before neutrophils finally adhere to the lumen wall. Following adhesion, neutrophils transmigrate between endothelial cells (diapedesis) and penetrate the basement membrane.

Figure 1. Neutrophil adhesion cascade.

Neutrophils have been observed to exit at particular sites, sometimes termed “hotspots”. However, why neutrophils choose to exit at these sites is not well known. The authors of this preprint examined the exit sites used by neutrophils to migrate across the endothelium; applying lattice light sheet microscopy to explore why neutrophils chose particular exit sites.

 

Key findings

  1.   Leukocytes show a preference for endothelial membrane protrusions at cell junctions for transmigration

Utilising light sheet microscopy and time-lapse movies, the authors discovered endothelial junctional membrane protrusions that were used as sites of diapedesis by neutrophils. As neutrophils transmigrate following inflammatory stimuli, the authors treated tissue with TNF-α. This treatment increased the junctional membrane protrusions, enabling more neutrophil trafficking into the tissues.

 

Movie 1. Movie demonstrating neutrophils utilising JMPs for transendothelial migration

  1.   Protrusions depend on Rac1 activity

Junctional membrane protrusions were shown to be regulated by the actin cytoskeleton. To investigate this further, the authors focussed on Rac1, a small GTPase that has known roles in regulating the actin skeleton and lamellipodia formation. Activating Rac1 resulted in an induction of junctional membrane protrusions. Neutrophils then utilised these artificially created exit sites for diapedesis.

Figure 2. Summary of the main findings from the preprint. Adapted from Fig 4I in the preprint under a CC-BY 4.0 license.

 

Importance of this work / Why I selected this preprint

This preprint beautifully demonstrates the power of microscopy in uncovering novel cell behaviours that we would otherwise miss – plus you can’t beat the amazing images and videos that accompany this preprint.

 

Open questions

 

  1. The authors show JMPs in cremaster muscles, did they investigate other tissues? As it is known that the mechanism of neutrophil entry can be tissue dependent it’s possible that these protrusions are unique to specific tissues.
  2. Why do certain endothelial cells form JMPs and not others?
  3. Do all pro-inflammatory stimuli increase JMPs? What role do anti-inflammatory signals play?

 

References

  1. Rosales C. Neutrophil: A Cell with Many Roles in Inflammation or Several Cell Types? Front Physiol [Internet]. 2018;9. Available from: https://www.frontiersin.org/articles/10.3389/fphys.2018.00113/full
  2. Voisin M-B, Nourshargh S. Neutrophil Transmigration: Emergence of an Adhesive Cascade within Venular Walls. J Innate Immun. 2013;5(4):336–47.

Tags: immunology, leukocyte migration, light-sheet microscopy, microscopy, protrusions, transmigration

Posted on: 19th February 2021 , updated on: 25th February 2021

doi: https://doi.org/10.1242/prelights.27391

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Author's response

Jaap van Buul shared

  1. The authors show JMPs in cremaster muscles, did they investigate other tissues? As it is known that the mechanism of neutrophil entry can be tissue dependent it’s possible that these protrusions are unique to specific tissues.

Answer: no, we did not look at other tissue yet. This is something we are aiming at doing.

  1. Why do certain endothelial cells form JMPs and not others?

Answer: To be frank, we do not know. What we do know is that endothelial cell monolayers show a large matter of heterogeneity. For example, von Willebrand factor or ICAM-1 are highly expressed in one endothelial cell whereas the expression is drastically reduced in the neighbouring one. Why this is, is not known, but perhaps something like this is also true for cell activation.

  1. Do all pro-inflammatory stimuli increase JMPs? What role do anti-inflammatory signals play?

Answer: We have tested several inflammatory mediators, TNF, IL1B, LPS and IFN. All these stimuli induced the JMPs. We dd not combine these stimuli yet, to see if they can reinforce each other. Also, we did not test if the length of stimulus changes JMP kinetics. Something to figure out!

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