The role of sphingosine 1-phosphate in neutrophil trans-migration

Abstract

Sphingosine 1-phosphate (S1P), a bioactive lipid mediator and ligand of 5 G-protein coupled receptors, is involved in many cellular processes including cell survival and proliferation, lymphocyte migration, and endothelial barrier function. As neutrophils are major mediators of inflammation, neutrophil trans-endothelial migration could be the target of therapeutic approaches to many inflammatory conditions. The aim of this project was to assess whether S1P can protect against inflammation by affecting neutrophil trans-endothelial migration, either by acting on neutrophils directly or indirectly through the endothelial cells. The direct effects of S1P on isolated human neutrophils from healthy volunteers were assessed. It was shown that S1P signals in neutrophils mainly through the receptors S1PR1 and S1PR4 and it induces phosphorylation of ERK1/2. Moreover, S1P pretreatment enhances IL-8 induced phosphorylation. However, in chemotaxis assays, S1P pre-treated neutrophils showed no altered migration towards IL-8 in comparison to untreated neutrophils. Additionally, in an in vitro flow-based adhesion assay, S1P pretreatment did not have a significant effect on IL-8 induced neutrophil adhesion to VCAM-1 and ICAM-1. Next, the effects of S1P on endothelial cells were measured. When HMEC-1 endothelial cell line and HUVEC primary endothelial cells were treated with S1P or S1P receptor agonists CYM5442 and CYM5541, the production of the chemokine IL-8 was induced. On the other hand, this treatment inhibited neutrophil trans-endothelial migration through HMEC-1 and HUVEC endothelial cells. This indicates S1P enhances endothelial barrier integrity, with a mechanism involving reduction of VE-cadherin phosphorylation. Finally, S1P treatment caused upregulation of the adhesion molecules VCAM-1 and ICAM-1, but inhibition of TNF-α induced upregulation, also shown as reduced neutrophil adhesion to endothelial cells under in vitro flow conditions. To investigate the in vivo effects of S1P, two mouse models of cell recruitment were used, the peritoneum cell recruitment and the air pouch model. In the peritoneum cell recruitment model, S1P administration could successfully inhibit neutrophil recruitment at the peritoneum induced by IL-8. In conclusion, functional assays indicated no direct effect of S1P on neutrophil migration, although S1P receptor signalling in neutrophils can activate MAPK/ERK iii signalling pathways and enhance IL-8 signalling. However, S1P can affect neutrophil migration indirectly, either by inducing IL-8 and adhesion molecules expression by endothelial cells, or by enhancing endothelial barrier integrity leading to inhibition of trans-endothelial migration of neutrophils. The latter effect appears to be more pronounced in vivo.