The role of sphingosine 1- phosphate in neutrophil trans-migration Eirini Giannoudaki Thesis submitted in partial fulfilment of the requirements for the degree of Doctor of Philosophy Institute of Cellular Medicine Newcastle University September 2015 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 pre- treatment 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 pre- treatment 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 ii 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. iii Acknowledgements First of all I would like to share my appreciation for the generous funding by British Heart Foundation, without which there would be no project. Thank you to the charity and its donors. Next, I would like to thank my supervisors Prof. John Kirby and Prof. Simi Ali who supported me throughout this project. Without their guidance and patience I would not have been able to accomplish this study. Moreover, I would like to express my graditute for every person on the lab that was there when I needed something and especially Dr Fatmah Naemi who assisted me when I began, and shared her knowledge and expertise on many laboratory techniques. I would like to thank the members from Prof. John Simpson’s lab, specifically Dr Marie Ruchaud-Sparagano, Jonathan Scott and Jim Macfarlane, that shared their neutrophil isolation technique with me, and occasionally provided me with isolated neutrophils or donated blood for my studies. With that I would also like to thank the healthy volunteers that provided the blood for neutrophil isolation. Furthermore I would like to thank the staff from the Comparative Biology Center, especially Christopher Huggins that assisted in the mouse work and the staff from the flow cytometry facility for their help in flow cytometry. Finally, I would like to thank my parents for their continuous support throughout my life that led to my current path in science. And of course Dr Achilleas Floudas who was there to assist and support me through all the good and bad moments of this project. iv Table of contents Abstract ............................................................................................................................ ii Acknowledgements ......................................................................................................... iv Table of contents ............................................................................................................. v List of figures and tables ................................................................................................ x Abbreviations ............................................................................................................... xiv Chapter 1. Introduction .................................................................................................. 1 1.1 Immune system ................................................................................................... 2 1.1.1 Innate immunity .......................................................................................... 2 1.1.2 Adaptive immunity ..................................................................................... 5 1.2 Neutrophils ......................................................................................................... 9 1.2.1 Neutrophil activation ................................................................................. 10 1.2.2 Neutrophil – endothelial cell interactions ................................................. 11 1.2.3 Neutrophil degranulation .......................................................................... 13 1.2.4 Neutrophil respiratory burst ...................................................................... 14 1.2.5 Neutrophil phagocytosis............................................................................ 15 1.2.6 Neutrophil Extracellular Traps .................................................................. 16 1.3 Ischemia-reperfusion injury.............................................................................. 19 1.4 Endothelial cells ............................................................................................... 20 1.4.1 Endothelial cell junctions .......................................................................... 23 1.5 Sphingosine 1-phosphate .................................................................................. 27 1.5.1 S1P metabolism ......................................................................................... 27 1.5.2 S1P receptor signalling ............................................................................. 28 1.5.3 S1P as a second messenger ....................................................................... 31 1.5.4 S1P signalling on immune cell trafficking ................................................ 32 1.5.5 Cardiovascular effects of S1P ................................................................... 34 1.5.6 S1P in ischemia-reperfusion injury and other conditions ......................... 35 1.6 Project aims ...................................................................................................... 38 v Chapter 2. Materials and Methods .............................................................................. 39 2.1 Tissue culture ................................................................................................... 41 2.1.1 Cell counting ............................................................................................. 41 2.1.2 Cryopreservation ....................................................................................... 41 2.1.3 Dissociation of adherent cells for subculturing ......................................... 42 2.1.4 Cell lines ................................................................................................... 42 2.1.5 Primary cells.............................................................................................. 45 2.2 Reconstitution of S1P Receptor Ligands .......................................................... 48 2.2.1 S1P ............................................................................................................ 48 2.2.2 CYM-5442 ................................................................................................ 48 2.2.3 CYM-5541 ................................................................................................ 49 2.2.4 SEW2871 .................................................................................................. 49 2.2.5 FTY720-P .................................................................................................. 49 2.3 Flow cytometry ................................................................................................. 51 2.3.1 Staining protocol ....................................................................................... 51 2.4 Chemotaxis assays ............................................................................................ 54 2.4.1 Trans-membrane chemotaxis ...................................................................