Identification of signalling pathways regulating neutrophil homeostasis

In man 10e11 neutrophils are released from the bone marrow each day and these cells have a half life of 6 - 8hrs in the blood. While the dogma is that senescent neutrophils are cleared by the liver and spleen, the Imperial group has shown that as neutrophils age they up-regulate their expression of the chemokine receptor CXCR4 and as such 30% of senescent neutrophils are cleared by the bone marrow. Trafficking of senescent neutrophils back to the bone marrow in vivo is CXCR4-dependent. CXCR4 is a G protein coupled receptor and SDF its sole ligand. The SDF/CXCR4 chemokine axis is critical for the retention of mature neutrophils within the bone marrow. Thus CXCR4 antagonists rapidly mobilise neutrophils from the bone marrow While the signalling pathways regulating SDF stimulated chemotaxis have been studied in T cells and T cell lines, SDF/CXCR4 signalling has not previously been studied in neutrophils. We are particularly interested to investigate whether the signalling pathways responsible for the CXCR4-dependent retention of neutrophils in the bone marrow are the same as those driving the homing of senescent neutrophils back to the bone marrow. Our working hypothesis is that different kinases are involved in CXCR4-dependent neutrophil retention and clearance. We shall therefore use Cellzomes unique kinome technology to analyse SDF-induced activation of kinases in neutrophils isolated from the bone marrow reserve (CXCR4lo) as compared with senescent blood neutrophils (CXCR4hi). It has been reported that CXCR4 is expressed by bone marrow endothelium. We have developed unique expertise in integrative physiology with respect to investigating bone marrow biology. Specifically we have developed an in situ perfusion system of the mouse femoral bone marrow. In this system, in an anaesthetised mouse, the femoral artery is cannulated such that the bone marrow vasculature can be perfused directly with buffer, neutrophils exiting the bone marrow are collected via cannulation of the femoral vein and thus can be quantified. Infusion of specific kinase inhibitors generated by Cellzome will identify the signalling pathways involved in the SDF/CXCR4 mediated retention of neutrophils in the bone marrow. The CXCR4 antagonist will be used as a positive control for these experiments. Of note using specific inhibitors, we have previously used the in situ perfusion system to demonstrate a role for PI3 kinase in IL-5-stimulated mobilisation of eosinophils from the bone marrow. The role of specific kinases in neutrophil clearance via the bone marrow will be assessed either by a) infusion of fluorescently-labelled senescent CXCR4hi neutrophils into the mouse femoral bone marrow using the in situ perfusion system and b) i.v. injection of 111In-labelled neutrophils in mice. Neutrophils will be pre-treated with vehicle of kinase inhibitors to assess their role in CXCR4-dependent clearance.This project should lead to the identification of specific kinases downstream of CXCR4 that play a role in neutrophil homeostasis. Cellzome has state of the art capabilities in mass spectrometry based proteomics which it uses to quauntitate the effects of drugs on signalling pathways. The project will use these techniques to analyse, in depth, the status of multiple pathways in neutrophils as the are generated, released and removed. The Imperial group has world leading expertise in neutrophil pharmacology and physiology. Thus, this project brings together integrative physiology and quantitative proteomics and offers a highly valuable training programme for the student.