BME MS Defense: Tejas Khire

Mobility Analysis of CXCR1 Receptor in Resting and Activated Human Neutrophils

Co-supervised by Professor Jim McGrath and Professor Richard Waugh

Abstract

Chemokine signaling is an important process that results into neutrophil activation and subsequent transmigration through the endothelium. Chemokine receptors mediate the extracellular signal and facilitate cell adhesion and extravasation. Understanding the mobility of chemokine receptor, thus, is an important step in understanding the dynamic characteristics of these receptors.

We used the technique of Fluorescence Recovery After Photobleaching (FRAP) technique to measure the diffusion coefficient of CXCR1 in resting neutrophils under physiological conditions. We estimated the diffusion coefficient of CXCR1 as 0.9+/-0.2*10-10 cm2/s at room temperature, and observed that the mobility of CXCR1 is faster than that of L-selectin, but is slower than that of β2 integrins. The comparison of these observed values with theoretical models attributes the differences in mobility partly to the difference in the size of transmembrane domains, and partly to the other factors such as difference in cytoskeletal organizations, and presence of homo and/or heterodimers. To study CXCR1 mobility in activated neutrophils, we used the microfluidic system optimized in our laboratory to observe directional neutrophil migration either by chemotaxis or haptotaxis. We observed that fibronectin is not a very good choice for neutrophil haptotaxis and the surface immobilization of fibronectin on to glass is potentially damaged due to unknown reasons. We further developed and optimized fMLP mediated neutrophil chemotaxis on ICAM-1 coated surfaces. We observed that neutrophils shed their membrane upon their initial spreading and this membrane residue consists of CXCR1 and LFA-1 receptors. Further immuno-labeling experiments indicated that activated neutrophils also internalize CXCR1 receptors although kinetics of these individual processes is unknown. This is the first report, up to our knowledge, to demonstrate the internalization of IL-8 receptor in the presence of fMLP stimulation. The simultaneous shedding, expression, and internalization and/or recycling provide greater insights about CXCR1 receptor trafficking in neutrophil undergoing chemotaxis. This knowledge about various transport mechanisms can serve as a platform for further biophysical modeling of these events and increase our understanding about different processes governing and influenced by chemokine signaling.