Molecular Accessibility: Microvilli and the Endothelial Glycocalyx

Leukocytes play a critical role in the body's response to infection, and an important step in leukocyte function is the extravasation cascade in which the leukocytes transmigrate through the endothelium to get to sites of infection. The balance of this interaction is crucial for proper tissue function as over-activation can lead to a number of inflammatory diseases. In order to understand the extravasation cascade we must first understand the mechanics of interaction between a leukocyte and the endothelium, and while many facets of this interaction are known, there is still a gap in understanding the molecular accessibility during leukocyte rolling, adhesion, and spreading on the luminal surface of the endothelium during extravasation. The focus of this thesis is on the role that physical factors, namely the surface structure of the cells and their deformability, play in regulating the interaction of leukocytes with the endothelium.;To assess how the surface structure of the leukocyte can influence molecular accessibility, images of leukocytes spreading on a glass surface were used to seed a computational model. These calculations predicted that the majority of integrins (LFA-1) and chemokine receptors (CXCR1) were localized in the valleys between microvilli where they are sequestered from the surface. When the leukocyte spreads onto the glass, the change in structure can lead to a greater than 1000-fold increase in the number of adhesion molecules and chemokine receptors capable of forming bonds with the surface.;The thickness and stiffness of the endothelial glycocalyx will influence leukocyte interaction with the endothelium by acting as a steric barrier. The amount of resistance to adhesive events the glycocalyx provides is the result of two properties: glycocalyx thickness and stiffness. The endothelial glycocalyx was directly measured with an atomic force microscope to have a thickness of 150 +/- 10 nm and a modulus of 0.08 +/- 0.01 kPa. When the endothelium was stimulated with TNFalpha to activate the cells, the thickness of the glycocalyx was reduced by 68% and the modulus of the glycocalyx was reduced by 38%. This loss of glycocalyx indicates that there is a significant increase in molecular accessibility once the cells are activated.;Using these measured values, an analytic model of a leukocyte interacting with the endothelium was developed to quantify the force required to engage adhesion molecules. The model predicts that, under normal conditions, a force of 1.8 nN would be required to engage the integrins in the microvilli valleys with the adhesion molecules on the endothelial surface because of the steric resistance of the glycocalyx. This is an order of magnitude more force than the cell is expected to exert when circulating through the vasculature. When the thickness and modulus of the glycocalyx are reduced after TNFalpha stimulation, this force requirement drops by 90% to only 130 pN, a value well within the expected range of normal physiological forces.;The results of these studies describe how the leukocyte and endothelium work in tandem to limit adhesive events under homeostatic conditions and increase adhesion when activated. Under homeostatic conditions, the leukocyte sequesters adhesion molecules in the valleys of its microvilli and the endothelium expresses a robust glycocalyx to act as a steric barrier. When the cells are activated, the increase in the number of accessible adhesion molecules on the leukocyte and the loss of the glycocalyx layer act to increase adhesion and promote extravasation.