Leukocyte selectin and integrin dependent adhesion under fluid flow
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Cell rolling and adhesion are important features that regulate leukocyte trafficking in human vasculature. Various adhesion molecules belonging to the selectin, integrin and immunoglobulin gene superfamilies act in synergy to control the kinetics of these processes. In order to gain insight into the mechanisms regulating cell rolling and adhesion, this thesis develops a biophysical computation scheme that delineates between the roles of the diverse adhesion molecules in regulating the rolling and adhesion steps. Since integrins play an important role in firm cell adhesion, the role of diverse cations in regulating integrin binding efficiency is also examined. Firstly, a mathematical model is developed to quantify the efficiency of cell-substrate attachment in the parallel-plate flow chamber. This model decouples the physical features of the system that affect cell-substrate collision rates from the biological features, which influence cellular adhesivity. Thus, experimental data on cell rolling and adhesion density are converted into "frequency" parameters that quantify the "efficiency" with which cells in the flow chamber progress from the free stream to roll, and then transition from rolling to firm arrest. Secondly, this model is partially validated by comparing simulation results with experiments where neutrophils roll and adhere on substrates composed of cotransfected cells bearing E-selectin and ICAM-1. Results provide information regarding contact time needed for cells prior to rolling and adhesion, the effect of experiment inlet cell concentration on secondary capture and the influence of shear stress to cell rolling and adhesion. The frequency parameters are further used to estimate selectin on-rate. Finally, the ability of gadolinium and manganese to regulate leukocyte integrin is examined in neutrophil aggregation system. Addition of 20μM gadolinium or 1mM manganese could significantly prevent neutrophil aggregation breakup after IL-8 stimulation and therefore induce high integrin adhesion efficiency. Antibody blocking experiments confirm gadolinium regulates leukocyte adhesivity through both LFA-1 and Mac-1, while manganese mainly affects Mac-1 function. It further shows both cations augment CD18 active epitope presence, especially, compete with calcium in 327C antibody binding at the same time. A possible mechanism is pointed out which indicates gadolinium appears to affect integrin specifically while manganese plays other roles too.