Mechanisms of homotypic lymphocyte aggregation and cell motility characteristics induced by activation of VLA integrins
Doctor of Philosophy
Cell Adhesion may be modulated at the plasma membrane by intracellular signals or antibodies which confer an active ligand-binding conformation. Lymphocyte homotypic aggregation measured in tissue culture constitutes a model system in which to study the mechanism of $\beta\sb1$ integrin dependent function. First, a quantitative assay for homotypic cellular aggregation has been described. This procedure applies a digital image analysis technique to measure aggregation kinetics both in terms of evolution of aggregate size and changes in cell stacking. The method allows us to automate the image processing and data analysis steps in order to minimize user intervention and to improve the reproducibility of the measurements. Second, we studied the mechanism of homotypic aggregation induced by various monoclonal antibodies to the $\beta\sb1$ integrin. Our findings indicate that the rate and extent of adhesion in Jurkat cells can be modulated by the stoichiometry of sites occupied by the aggregation inducing antibody, 33B6, and the inhibiting antibody, 18D3. We also hypothesize that homotypic aggregation is induced by a change in the conformation of the $\beta\sb1$ integrin. Further, this conformation of the VLA integrin mediating aggregation is novel and distinct from the conformation recognized by mAb 15/7 which supports cell binding to VCAM-1 and fibronectin. Third, image analysis techniques were applied to track the motion of individual Jurkat cells to study the effect of cell activation and substrate modifications on cell motility characteristics. The data was analyzed using stochastic models based on the persistent random walk model and the Markov chain analysis. Cell activation by monoclonal antibody 33B6 reduced both the speed and persistence time of Jurkat cells. 18D3 did not affect the motility characteristics. When the substrate was modified by a fibronectin coating, activation of the Jurkat cells by 33B6 increased the speed and diffusion coefficient of Jurkat cells in comparison with the OKT11 control and anti-$\beta\sb1$ mAb 18D3. Last, a mathematical model based on the Smoluchowski's theory of flocculation was developed to model homotypic aggregation. The model parameters were measured using independent experiments described earlier. Comparison of simulation results with experimental findings allowed us to correctly interpret the experimental data and to identify important physico-chemical parameters involved in aggregation. Results indicate that aggregation rates strongly depend upon the motility of cells and cell aggregates, the frequency of cell-cell collision and the strength of inter-cellular bonds.