CHARACTERISING ARAP3-MEDIATED INTEGRIN INACTIVATION IN THE NEUTROPHIL

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Immune cells need to generate inflammation to kill pathogens when they fight infections. This is a tightly regulated process, but occasionally the regulation goes wrong and immune cells generate inflammation even in the absence of infection. This happens in chronic inflammatory conditions, such as rheumatoid arthritis or chronic obstructive pulmonary disease. Chronic inflammation causes much suffering and poses an important drain on health services world-wide. No known drugs can cure chronic inflammation. Neutrophils, the most common immune cells in the blood, are important players in the generation of inflammation. Neutrophils leave the blood stream to travel to sites of inflammation. They can only do this if they can leave the blood stream by crawling through the vessel wall into the surrounding tissue. This relies on specialised adapters on their molecules, the integrins. Integrins exist in a "sticky" (active) and a "non-sticky" (inactive) state. Many labs have studied how integrins become active: this depends on several intracellular regulators. Not having integrins, or not being able to activate them stops neutrophil recruitment to inflammatory sites. This stops inflammation but also immunity and leads to recurrent infections. Only few studies have analysed integrins that are always active. It turns out that this also reduces neutrophil recruitment to inflammatory sites. We have shown that an intracellular regulator called 'ARAP3' is required for inactivating neutrophil integrins. Without ARAP3 neutrophils are not efficiently recruited to inflammatory sites, but recruitment is not abolished. ARAP3 is itself regulated by at two other regulators (called PI3-kinase and Rap), and it in turn regulates two further regulators (called Arf6 and RhoA). We will study how Rap, RhoA and Arf6 are working together with ARAP3 in the neutrophil to regulate integrin inactivation. We will do this by introducing small molecular changes into ARAP3 so that it can no longer talk to one of the other regulators in turn. We will use stem cells from the bone marrow of mice that have no ARAP3, and engineer them to have one of the slightly changed ARAP3s instead. The modified stem cells will then be placed into culture. When we change their culture medium, they will transform into neutrophils. These neutrophils' integrins will be analysed in the test tube. This will allow us to learn how the intracellular regulators work together with ARAP3 to regulate integrin inactivation. This work may build the foundations for new therapeutic approaches that target chronic inflammation.