Lymphocyte locomotion in three-dimensional collagen gels comparison of three quantitative methods for analysing cell trajectories

We evaluated three different quantitative evaluation methods for lymphocyte locomotion in three-dimensional collagen gels: (1) the length of the two-dimensional migration path (distance migrated) was compared to (2) the resulting average displacement from the starting to the end point and (3) the displacement of the furthest migrating population (cells with high displacement). Locomotion of immunomagnetically isolated human CD4 + and CD8 + peripheral blood lymphocytes suspended in type I collagen gels was recorded using time-lapse videomicroscopy. Paths of randomly selected locomoting cells were digitized, reconstructed and quantitatively analysed. For spontaneously locomoting CD4 + and CD8 + lymphocytes (90 min observation period) the mean total distance migrated was 10.0 ± 3.7 μ/m/min (CD4 +; n = 114 cells) and 5.6 ± 3.3 μm/min (CD8 +; n= 90 cells). The mean displacement from the individual starting point amounted to 1.3 ± 0.7 μm/m3n for CD4 + and 1.1 ± 0.7 μm/min for CD8 + cells, thus representing only 5–25% of the total migration path (index range displacement/distance migrated: 0.13–50%). Incubation with interleukin-8 and/or receptor blocking by monoclonal antibodies against VLA-2 (Gi9) or VLA-4 (HP2/1) integrins significantly altered the mean length of the migration paths for six out of ten different experimental conditions. Average displacement or displacement of the most active cells detected significant changes in two and three out of ten samples. Whereas the interleukin-8 induced locomotory changes were correctly represented by end point determination, relatively slight but significant modulation in lymphocyte behaviour by anti-integrin antibodies was revealed solely by analysis of the complete cell trajectory. In conclusion, the cell trajectory may represent a sensitive method for evaluating induced subtle changes in lymphocyte locomotory characteristics.