Quantification of neutrophil spreading and migration and the development of a novel model for neutrophil motility

Neutrophils are a key element in the body\u27s innate immune system and are the first line of defense against pathogens and infections, but are also implicated in several autoimmune diseases. In order to design drugs to aid in neutrophils\u27 natural immune ability or impair undesirable functions, an intense effort is needed to understand how neutrophils can translate cues from their physical environment and chemical stimuli into directed migration to the source of inflammation. To gain insights into how the endothelium can regulate neutrophil migration by the presentation of various adhesive ligands, we performed a systematic study of how adhesion molecules and shear rate are intertwined in guiding neutrophils to their final destination (Chapter 2). Reflection Interference Contrast Microscopy was employed with an image processing algorithm to study the initial dynamics of adhesion and spreading following activation. We found that neutrophil spreading is anisotropic from the point of activation and occurs through a slow and fast growth regime, in which, the latter occurs in union with the formation of a tight adhesion (Chapter 3). We used traction force microscopy to measure the traction stresses that neutrophils create during chemokinesis and chemotaxis to develop a novel method of neutrophil motility where the rear of the neutrophil is both anchoring the cell and where the contractile forces are concentrated (Chapter 4). This model was further investigated with the use of pharmalogical inhibitors to determine the effect of F-actin and microtubules on force generation (Chapter 5). Finally, the feasibility of using transgenic mice models was investigated. These mice models will be needed to better elucidate mechanistic details of various proteins involved in force creation allowing for directed migration (Chapter 6)