Monitoring Scavenging Activity of Chemokine Receptors.

Abstract

Migration and positioning of cells is fundamental for complex functioning of multicellular organisms. During an immune response, cells are recruited from remote distances to a distinct location. Cells that are passively transported leave the circulation stimulated by locally produced signals and follow chemotactic cues to reach specific destinations. Such gradients are short (<150 μm) and require a source of production where the concentration is the highest and a sink in apposition where the attractant dissipates and the concentration is the lowest. Several straight forward methods exist to identify in vitro and in vivo cells producing chemoattractants. This can be achieved at the transcriptional level and by measuring secreted proteins. However, to demonstrate the activity of sinks in vitro and in vivo is more challenging. Cell-mediated dissipation of an attractant must be revealed by measuring its uptake and subsequent destruction. Elimination of chemoattractants such as chemokines can be monitored in vitro using radiolabeled ligands or more elegantly with fluorescent-labeled chemoattractants. The latter method can also be used in vivo and enables to monitor the process in real time using time-lapse video microscopy. In this chapter, we describe methods to produce fluorescently labeled chemokines either as fusion proteins secreted from insect cells or as recombinant bacterial proteins that can enzymatically be labeled. We discuss methods that were successfully used to demonstrate sink activities of scavenger receptors. Moreover, fluorescent chemokines can be used to noninvasively analyze receptor expression and activity in living cells.