RESEARCH SUMMARY

Chemokines are chemotactic cytokines that act through plasma membrane-tethered receptors. They are involved in cell functions including regulation of immune defence as well as in tumour growth, atherosclerosis and asthma. Migration of immune cells from blood into tissues is crucial in immune surveillance and host defence. Our group uses cell biology and knockout animal models to study the chemokine response of monocytic cells.

Cell migration is a complex biological function triggered by integrins, growth factors and chemokines. To evaluate the importance of membrane trafficking during cell migration towards a chemokine gradient, we monitored the number of chemokine receptor CCR2 molecules at the cell surface. Cells that did not migrate needed higher concentrations of the chemokine CCL2 for CCR2 internalization to occur, suggesting that internalization occurs during chemotaxis and that it is reduced in non-migrating cells. We manipulated clathrin/dynamin-mediated endocytosis to understand the mechanisms implicated in CCL2-stimulated cell migration. Sucrose treatment of cells interferes with endocytosis mediated by clathrin, a protein involved in transfer of material between cell organelles. Sucrose treatment impaired CCR2B internalization and migration of monocytic cells. When RNA interference was used to knock down clathrin, CCR2B internalization and transferrin uptake were prevented, as was cell migration. Dynamin is an endocytic pinchase implicated in vesicle scission; this function can be inhibited with the help of dynasore. When cells were dynasore-treated, cell migration was reduced. Our findings indicate that hindering membrane trafficking reflects deficient cell migration. The relevance of membrane trafficking in the cell migration process needs further evaluation using chemokinesis (wound assay) models and live-imaging techniques.

We also found that the nuclear receptor RXR (retinoid X receptor) regulates transcription of the chemokines CCL6 and CCL9 in vivo and in vitro. Mice lacking RXR in macrophages showed lower blood levels of CCL6 and CCL9. When these mice suffered from peritonitis, they showed less inflammation and fewer leukocytes were attracted; they were also less prone to sepsis. Our results suggest RXR as a target for treatment of inflammatory processes.

Our studies established CCR2 endocytic routes and their importance in monocytic cell migration. These insights into the molecular mechanisms of desensitization and signalling define potential therapeutic targets in diseases such as asthma and allergic inflammation.

In a collaborative study, we devised a method that assigns the topology of membrane proteins using fluorescent proteins (FP). This pH exchange assay (PEA) takes advantage of the pH sensitivity of GFP and YFP. Major advantages of the PEA are its technical simplicity, since it requires only imaging on a widefield or confocal microscope, its applicability to live, undisturbed cells, and the ability to quantify the proportion of inside/outside orientation for proteins with multiple topologies. We believe that this assay will interest groups working with membrane proteins.