My group is interested in the intracellular mechanisms regulating NADPH oxidase function in neutrophils and the control of neutrophil and eosinophil survival by hypoxia and inflammatory cytokines. This work has a particular focus on the role of phosphoinositide 3-kinase and HIF-alpha isoforms in these processes. We also have a translational research programme, which uses radiolabeled autologous granulocytes (both neutrophils and eosinophils) combined with SPECT-CT to examine granulocyte kinetics, function and fate in vivo. For more details regarding the PIs research background read more below.
My interest in cell signalling developed during my MRC Training Fellowship with Professor Nahorski in the Department of Cell Physiology and Pharmacology at Leicester. Using bovine trachealis as a model system I provided the first direct pharmacological proof of an inositol 1,4,5-trisphosphate (Ins(1,4,5)P3) receptor in smooth muscle and demonstrated the importance of agonist-stimulated metabolism of Ins(1,4,5)P3 in generating the rapid and transient Ins(1,4,5)P3 ‘spike’ seen following contractile agonist stimulation. During this work I also identified a novel pathway for Ins(1,4,5)P3 metabolism to Ins(4,5)P2 and helped develop the first radio-receptor assay for measuring Ins(1,4,5)P3 and PtdIns(4,5)P2 mass.
Following my move to Edinburgh as a Lecturer and then Wellcome Trust Senior Clinical Fellow working with Professor Chris Haslett, I applied my training in lipid-derived second messengers to investigate the mechanisms linking the processes of granulocyte priming, activation and apoptosis. These studies continued follwing my move to Cambridge. Neutrophil priming is an event whereby the response of the cell to an activating stimulus is augmented greatly by prior exposure to a priming agent (eg. LPS, TNFalpha) and is a critical determinant of the pathogenic capacity of these cells in vivo. I demonstrated a cardinal role for phosphoinositide 3-kinase (PI3-kinase) activation and PtdIns(3,4,5)P3 generation in triggering superoxide anion release in neutrophils and that up-regulation of this pathway is the central mechanism underlying priming. In collaboration with Drs Hawkins and Stephens at the Babraham Institute, we demonstrated that the myeloid restricted Gbeta/gamma-regulated PI3-kinase (p101/PI3-kinase-gamma) is the principal source of agonist-stimulated PtdIns(3,4,5)P3 in neutrophils, work that involved the generation of a unique p101/ PI3-kinase-gamma mouse knockout. These studies also revealed the mechanism whereby PtdIns(3,4,5)P3 activates the NADPH oxidase namely via a direct interaction between PtdIns(3)P, a metabolite of PtdIns(3,4,5)P3, and the PX domain of the p40phox oxidase subunit.
My group revealed for the first time that neutrophil priming is reversible and that these cells can participate in a complete cycle of priming, de-priming and re-priming. This observation refuted the view that priming is a terminal event and suggests that neutrophils primed in vivo can be retained in the pulmonary circulation and allowed to de-prime before re-entering the circulation. This has been confirmed using laser trapping of neutrophils where we have preliminary data to suggest that de-priming can be accelerated by repetitive stretch. To test the above hypothesis we have developed techniques using 111-Indium and 99m-Technicium-labelled autologous granulocytes, which have allowed us to make the first measurements of single pass neutrophil transit across the lung in man; these confirm major initial retention of ex-vivo primed cells by the pulmonary circulation followed by slow but complete release. These studies have now been extended into studying human eosinophil kinetics in vivo, where we hope to develop clinically useful eosinophil scanning and techniques to quantify lung-specific uptake.
In collaboration with Dr Sarah Walmsley and Professor Moira Whyte at Sheffield University my group has also demonstrated that priming and hypoxia have profound effects on neutrophil longevity. We have generated evidence in both human and murine cells that Class I PI3-kinases and NF-kappaB play dominant roles in this survival effect and have provided the first demonstration of hypoxic sensing in neutrophils together with a molecular explanation for the profound survival effect of hypoxia in neutrophils.
Together, these studies have provided novel insights into the roles of Ins(1,4,5)P3 in pharmaco-mechanical coupling and PI3-kinase and hypoxia in neutrophil priming and survival; this latter work predicts that the myeloid restricted PI3-kinase-gamma and the hypoxia-PHD-HIF-1alpha axis represent important pharmacological targets in granulocytic inflammation.
Scanning EM showing the striking differences in the appearance of apoptotic (top left and top right) and non-apoptotic human neutrophils.
Eosinophil accumulation within a bronchial wall blood vessel
SPECT image across the upper thorax of a human subject demonstrating the distribution of 99mTc-labelled autologous neutrophils 4 hours post-injection. The vertebrae, sternum and ribs are evident peripherally together with activity in the central mediastinal blood vessels; the red ROI represent the lung fields.