Regulation of the Ras cycle in neutrophils

White blood cells called neutrophils play an important role within the immune system fighting infections. They are able to migrate towards sites of infection and destroy germs. We have proven, using genetically modified mice, that a special class of protein, Ras proteins, found inside neutrophils is essential for the processes of migration and destruction. At the moment it is not known how Ras proteins are controlled in neutrophils. Establishing the mechanism is an important part of the bigger process of attempting to understand migration and germ-killing and hence more clearly appreciate what goes wrong with these processes in a number of diseases such as arthritis and familial weakness to infection. The attractants, that drive migration, and the germs initiate the processes of killing and migration by binding to specialised molecules found on the cell surface, called receptors. Once activated the receptors stimulate further signalling through networks of molecules inside cells, including Ras proteins, to the machinary that causes migration, found at the front and back of the cell, and germ-killing, found inside the cell. We hypothesise that Ras proteins play an important role in focusing the signals down to the correct locations in the cell where the relevant cell machinary is found. At the moment it is not clear how Ras is activated in neutrophils, neither in terms of the identity of the molecules responsible or the mechanism. We plan to use an approach that enables us to create mice specifically lacking Ras or specific Ras activating molecules in their white blood cells and then to test whether the white blood cells from those mice work normally. If we have removed Ras or the correct Ras-acivator then we would expect to see a loss of Ras activation and also certain of the white blood cells normal responses. We then know we have correctly identified the real ras activating molecule. We know about all of the possible ras activating molecules (about 18 possibilities) that could do the job by looking in the DNA-sequence data bases created by sequencing of the mouse chromosomes. Once we know the identity of these molcules that regulate Ras then it is possible in the future for drug or Biotech companies to test the possibility that they might represent good targets for drugs designed to treat diseases like arthritis.

Neutrophils play a critical role in the immune system's defence against infection through their ability to migrate to sites of inflammation and destroy potential pathogens. These processes are regulated through the concerted actions of a variety of cell surface receptors and a network of intracellular signalling pathways. Murine genetics has proven that PI3K-gamma is a master kinase at the head of these signalling cascades. The dogmatic view of the field is that PI3K-gamma is directly activated by G-protein beta gamma subunits, We have unpublished genetic data showing that Ras is a major regulator of PI3K-gamma downstream of heptahelical receptors in neutrophils. These results establish that Ras, which has long known to be rapidly activated by these receptors but considered to have a relatively minor role on the basis of the impact of MAPK inhibitors, has an important role in neutrophil physiology. We, and others, have data indicating Ras activation in this context is not dependent on PLC or Src activity; these results rule out what would generally be seen as the natural candidates for the role of Ras-activator in this situation. Our hypothesis is that acute activation of Ras signalling plays an important role in coordinating the functional responses of fully differentiated neutrophils to inflammatory mediators. Our primary aims are to define the mechanisms regulating Ras activation in neutrophils, the spatio-temporal properties of that activation and the full range of roles for Ras in neutrophil physiology. We plan to utilise a variety of techniques to address these issues in vivo, but without the need to create mice with germ-line genetic modifications. We propose to use Lenti-virus to deliver RNAis, to explanted, murine, bone-marrow stem cells prior to repopulation of irradiated recipients, to suppress expression of potential RasGEFs in neutrophils (approx 18 in the genome, of which only a subset are credible in this context, RasGAPs are unlikely to be involved, see later). This approach will identify those molecules required for rapid activation of Ras. Any constructs that block neutrophil production, which therefore have an important role in that process, will be subjected to tet-inducible expression to by-pass this problem (see later for detail). We will analyse recombinant versions of proteins identified by the above procedure to define the mechanism of activation, which is likely to be novel, eg, via direct G-protein activation. If this screen fails to deliver candidates we will attempt to purify RasGEF activities from neutrophils using classical purification techniques, as we did to identify the P-Rex family of RacGEFs. It is now clear that the intracellular sites at which PI3K-gamma is activated are crucial to its roles in gradient-sensing, leading chemotaxis and regulating superoxide formation. Currently, the mechanisms underpinning the targeted activation of PI3K-gamma in these contexts are undefined. We will attempt to define the spatio-temporal properties of the Ras signals in neutrophils that organise activation of PI3K-gamma. To do this we will make use of new evolved versions of eGFP-RafRBD reporters, which have been shown to detect endogenous Ras activation, co-expressed with a contrastingly tagged marker of the plasma membrane to enable precise deconvolution of translocation. They will be delivered, using Lenti-virus, as above. Neutrophils from these mice, expressing the above reporters, will be imaged during adhesion, phagocytosis and in both uniform and gradient stimulation. Finally, we shall approach the process of defining the scale of the roles of Ras proteins by inducibly suppressing (RNAi) or interfering (dominant negatives) with the function of N and K-Ras proteins found in neutrophils. We will study a range of functional responses of mature neutrophils derived from these chimeras, specifically, superoxide formation, chemotaxis, adhesion and microbial killing.