Abstract
We investigated the changes in neutrophil phenotype and function after transendothelial migration, and the roles played by integrin receptors in their behaviour. Neutrophils were tracked microscopically as they migrated through endothelial cells into collagen gels, and were retrieved at desired times. When endothelial cells were treated with increasing doses of tumour necrosis factor-α, neutrophils not only migrated in greater number, but also to a greater depth in the gel. Apoptosis was barely detectable in neutrophils retrieved after 24h, and many remained viable and motile at 48h. Neutrophils retrieved after 1h had increased oxidative capacity and at 24h had similar capacity as freshly-isolated neutrophils. However, by then they had impaired ability to phagocytose bacteria. Compared to fresh neutrophils, total mRNA was halved by 24h, but while β2-integrin expression decreased, β1- and β3-integrin increased along with ICAM-1. Studies of integrin blockade indicated that while β2-integrins were needed to cross the endothelial barrier, no integrins were required for migration within the gel. β2-integrins also contributed to phagocytosis, but their binding was not required for prolonged survival. These results demonstrate a model for integrated analysis of neutrophil migration and function, and describe development of effector functions and the roles of integrins in human neutrophils for the first time.
Highlights
During inflammation, neutrophils are recruited from the blood stream and migrate through vascular endothelium to destroy pathogens and remodel damaged tissue
We previously reported a marked delay in apoptosis for neutrophils migrating through endothelial monolayers that had been treated with tumour necrosis factor-α (TNF) [32], suggesting that functional studies would be possible over prolonged periods if migrated cells could be tracked or retrieved
The foregoing raises questions regarding how the motility and effector functions of neutrophils change after they migrate across endothelium, how such changes are linked to changes in expression of integrins, and especially, whether integrins remain necessary for migration once cells have entered the interstitium. These questions could not be answered using existing in vitro experimental models available to study human cells, and so we developed a model in which endothelial cells (EC) were cultured on collagen gels and treated with TNF as a well-characterised initiator of neutrophil recruitment in other systems [33,34]
Summary
Neutrophils are recruited from the blood stream and migrate through vascular endothelium to destroy pathogens and remodel damaged tissue. The neutrophils undergo apoptosis and are themselves cleared by phagocytic macrophages to enable resolution of the inflammatory response [1]. The initial stages of adhesion from flow, and migration through endothelial monolayers have been studied and reviewed exhaustively [2,3], as have the abilities of neutrophils to release oxidants and proteolytic enzymes, and to phagocytose bacteria [4,5,6]. Cell migration and functional responses have rarely been studied together or after neutrophils have crossed the endothelial barrier. PLOS ONE | DOI:10.1371/journal.pone.0118593 February 23, 2015
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