Neutrophil chemotaxis in moving gradients of fMLP.

In this study the fluid gradient chamber, a modified version of the Boyden chamber that enables mobile gradients, was used to study the migration of human granulocytes in gradients of fMLP. Temporal chemotactic gradients were created by moving density-stabilized spatial gradients at different velocities in relation to migrating cells. Random and directed cell migration was quantified by applying a theoretical population distribution model to experimental cell distributions obtained from cell counts at different depths in the filters. Rates of random and directed migration generally increased with gradient velocity. At negative gradient velocities, i.e., when the gradients were moved in a direction opposite to that of cell migration to decrease fMLP concentration over time, random and directed migration was inhibited. At positive gradient velocities, migration rates were not significantly different from those seen in immobile gradients. The fact that the rate of directed migration was smaller at negative gradient velocities indicates that negative temporal gradients reduced the average speed and/or orientation of the chemotactically migrating cells. In immobile gradients, the cells generated a small concentration increase over time when they migrated in the up-gradient direction. Consequently, a positive temporal gradient as perceived by the cells may act as a positive feedback signal to maintain chemotactic migration.

The initiation of the neutrophil chemotactic response in filters.

The fluid gradient chamber was used to study the migration of human neutrophils in preformed gradients of N-formyl-methionyl-leucyl-phenylalanine. After 60 min, the chemotactic gradient was replaced by a new one of identical steepness but opposite direction. In a control group of experiments, the first gradient was retained. Migration was assessed from cell distributions in filter sandwiches after 30, 60, and 90 min. Filters obtained after 5, 15, and 30 min of migration were stained with fluorescent phalloidin for microscopic evaluations of cell polarity. At 30 min most cells had polarized in vertical directions and invaded the filters. The distance of chemotactic migration was similar during the second and the third 30-min periods (although the direction of migration was reversed in the new gradient) and significantly greater than during the first 30 min. In conclusion, the initial slow response to the chemotactic gradient represents an adaptation of the cells that later respond promptly to changes in gradient direction.

A subpopulation analysis of f-MLP stimulated granulocytes migrating in filters.

Leukocyte migration in vitro has been studied extensively during many years without providing satisfactory theoretical models for the different migratory behaviors (chemotaxis and chemokinesis) of leukocyte populations. The present study utilized the fluid gradient chamber, which is a new method to study leukocyte migration in filters. Human neutrophils were applied between two stacked filters and migrated in all directions under the influence of constant concentrations or chemotactic gradients of f-MLP, maintained in fluid phase density gradients. The distributions of the granulocytes over filter depth were fitted to theoretical functions composed by 1-3 Gaussian distributions, representing subpopulations. The results showed that the neutrophils migrated as two discrete subpopulations during chemokinetic stimulation (a constant concentration of f-MLP). One of the subpopulations showed less active and passive (slow sedimentation under the influence of gravity) translocation. The most mobile subpopulation was divided into two new subpopulations when exposed to chemotactic stimulation (concentration gradient of f-MLP), one of which responded chemotactically and one of which migrated in random directions. The properties of the different subpopulations where characterized in terms of diffusion coefficient (random migration), convection velocity (chemotactic migration) and sedimentation coefficient (passive translocation).

Migration of human granulocytes in filters: effects of gravity and movable gradients of f-MLP.

The Boyden chamber technique for chemotaxis uses a mesh filter that constitutes a matrix for cell locomotion and, at the same time, creates a local restriction for convective fluid movements that allows the establishment of a diffusive concentration gradient of chemotactic substance in the filter. In the present study, the Boyden chamber was modified by the introduction of a filter sandwich that allowed cell migration both upwards and downwards and by the use of a fluid density gradient controlling cell buoyancy and mechanically supporting a movable chemotactic gradient. This method was used to study chemotaxis and random migration of human granulocytes under the influence of gravitational forces and movable gradients of f-MLP. The results show that gravity affected cell motion significantly during random migration but not during chemotaxis. The rate of chemotactic migration was dependent on the steepness of the spatio-temporal f-MLP gradients. A stationary spatial gradient produced less migration than a gradient that was slowly moved through the filter sandwich in a direction opposite to that of the cell migration. The presence of f-MLP at constant concentration caused a minor, statistically insignificant, increase of the rate of random migration.