Chapter 37 : Chemokine and Chemokine Receptor Analysis

Chemokine receptor signal transduction. Chemokine receptors are a subfamily of G protein-coupled seven transmembrane-spanning cell surface receptors. They are coupled to heterotrimeric G proteins of the Gi subclass, which are distinguished by their pertussis toxin sensitivity. Chemokine receptor activation leads to the stimulation of multiple signal transduction pathways, including the activation of phosphatidylinositol 3-kinase (PI3K) and phospholipase C (PLC), leading to generation of inositol triphosphates, intracellular calcium release, and protein kinase C (PKC) activation. Chemokine signaling also induces the upregulation of integrin affinity and the activation of Rho, leading to cytoskeletal reorganization. Agonist-stimulated receptors also activate G protein receptor kinases, which leads to receptor phosphorylation, arrestin binding, G protein uncoupling (desensitization), and clathrin-mediated receptor endocytosis (internalization).

Transmigration assay system. Positive, negative, and absent gradients of a chemokine are established in order to assess chemotaxis (movement towards a chemokine) and chemokinesis (random movement of cells in response to a chemokine in the absence of a gradient). Cells are plated into the upper chamber of the transwell system or Boyden chamber and the proportion of migrating cells determined by accurate counting of cells that migrate to the lower chamber. The upper and lower chambers are separated by a polycarbonate membrane of standard pore size (3 μm to 8 μm), depending on the migrating cell type.

Digitized time-lapse photography of cells moving in the presence of a gradient of chemokine. Positive and negative gradients of a chemokine can be established in methylcellulose, as previously described ( 49 ). Cells are plated into methylcellulose and a gradient is established by inoculating the methylcellulose at a fixed point with the chemokine. Cells are then visualized migrating in response to the gradient using time-lapse video microscopy.

In vivo analysis of cell motility by multiphoton IVM in popliteal lymph node. The basic multiphoton microscope system consists of an infrared laser to deliver two-photon excitation; a laser intensity adjuster to decrease the laser power; a beam translation optical system to convey the laser beam to the back aperture of the objective; a laser scan head to raster scan the field of view with the microscope objective by rapid synchronized movement of dichroic steering mirrors; a low-magnification high-numerical-aperture water immersion objective; and external nondescanned detectors to concurrently acquire multiple fluorescent channels. The popliteal lymph node is visualized by making a skin incision in the knee, which is immersed in saline and sealed with a cover slip and vacuum grease after percutaneous clamps are used to prevent tissue movement. A thermistor is used to monitor temperature close to the lymph nodes, and the temperature is adjusted with a heating coil. Fluorescence is induced only in the focal plane; vertical image stacks are repetitively acquired that are then transformed into 3D images. Figure adapted from Mempel et al. ( 73 ).