Lymphocyte electrotaxis in vitro and in vivo

Abstract

Electric fields are generated in vivo in a variety of physiologic and pathologic settings, including penetrating injury to epithelial barriers. An applied electric field with strength within the physiologic range can induce directional cell migration (i.e., electrotaxis) of epithelial cells, endothelial cells, fibroblasts, and neutrophils suggesting a potential role in cell positioning during wound healing. In the present study, we investigated the ability of lymphocytes to respond to applied direct current (DC) electric fields. Using a modified Transwell assay and a simple microfluidic device, we show that human PBLs migrate toward the cathode in physiologically relevant DC electric fields. Additionally, electrical stimulation activates intracellular kinase signaling pathways shared with chemotactic stimuli. Finally, video microscopic tracing of GFP-tagged immunocytes in the skin of mouse ears reveals that motile cutaneous T cells actively migrate toward the cathode of an applied DC electric field. Lymphocyte positioning within tissues can thus be manipulated by externally applied electric fields, and may be influenced by endogenous electrical potential gradients as well.

Figure 1. Transwell-based electrotaxis assay

(A) Illustration of transwell assay for studying cell migration in an applied electric field. An electric field was applied to the transwell (2.5V across the transwell) by placing two platinum electrodes, which were connected to a DC power supply, to the top and bottom well of the transwell respectively. Cells were allowed to migrate from the top well to the bottom well through the membrane. The electrical potential in the transwell assay was simulated. (B) The simulated electrical potential was plotted as a function of normalized vertical coordinate Y for different normalized horizontal coordinate X within the membrane (−0.3–0.3). 1 = 7.8mm for the normalized coordinate X. 1 = 10mm for the normalized coordinate Y.

Figure 2. Migration of human blood lymphocytes and monocytes in an applied electric field

The migration of different subsets of hPBMC increased in transwell assay when the electric field was configured with the positive electrode in the upper well and the negative electrode in the bottom well (Top(+)/Bottom(−)) compared to it in the reversed electric field (Top(−)/Bottom(+)) or without the field. The error bars represent the standard error of the mean (s.e.m.) of multiple independent experiments (n = 9~11).

Figure 3. Microfluidic chamber for visualization of cell migration in an applied electric field

(A) Illustration of microfluidic system for studying cell migration in an applied electric field. (B) A picture of the microfluidic system. Two identical channels were configured side-by-side in a single device and can be used for migration studies separately. A nickle was placed next to the device as a scale reference.

Figure 4. Directional migration of memory T cells in an applied electric field in vitro

(A) Cell tracks of a representative experiment show that cells preferentially migrate toward the cathode of the electric field (to the left, 270°). An electric field (1V cm⁻¹) was applied and the migration of cells was recorded for 30 min at 6 frames min⁻¹. The solid circles indicate the end of the tracks. (B) The rose diagram shows the distribution of migration angles (the migration angles were calculated from x-y coordinates at the beginning and the end of the cell tracks, and were grouped in 20° intervals, with the radius of each wedge indicating cell number). The mean angle vector and the 95% confidence interval were also shown. Rayleigh uniformity test confirmed that the distribution of migration angles is not uniform. The Modified Rayleigh test (V test) showed that the deviation of the migration angles from the direction of electric field (270°) is not significant. (C) The percentage of cells migrated toward the cathode of the electric field, electrotactic index and the speed that were calculated using over 100 cell tracks from at least three independent experiments are presented in tabular form. Electrotactic Index and the speed are presented as the average value plus/minus the standard error of the mean (s.e.m.) of all cells. (D–F) Effects of field reversal. (D) Four representative cell tracks showing that cells migrated toward the cathode of the electric field and followed the reverse of the electric field (90° to 270° at the 60^th min). An electric field to the right (1V cm⁻¹) was applied for 1 hour and was reversed (to the left) for another hour. Migration of cells was recorded continuously for 2 hours at 6 frames min⁻¹. The circles indicate the end of the tracks and the arrows indicate when the electric field was reversed. Rose diagrams show the distribution of migration angles in the first hour (E) and the second hour (F) of the experiment (the migration angles were calculated from x-y coordinates at the beginning and the end of the cell tracks (n = 45), and were grouped in 20° intervals, with the radius of each wedge indicating cell number). The mean angle vector and the 95% confidence interval were also shown. Rayleigh uniformity test confirmed that the distribution of migration angles is not uniform. The Modified Rayleigh test (V test) showed that the deviations of the migration angles from the direction of electric field (90° in the first hour and 270° in the second hour) are not significant.

Figure 5. Signaling of human peripheral blood lymphocytes in an applied electric field

Phosphorylation of Erk1/2 (A) and Akt (B) is increased in an applied electric field as measured by phospho-flow cytometry. Cells were stimulated by an electric field (2.5V across ~1.3cm) for 60 min at room temperature in a 24-well plate. Stimulated cells, and unstimulated control cells incubated in parallel but without an electric field, were stained with anti-phospho-Erk1/2 antibody or anti-phospho-Akt-ser473 antibody. Each experiment was repeated at least 3 times with similar results

Figure 6. Migration of T cells in mouse ear pinna in response to an applied electric field

(A) Confocal image of CXCR6+ (GFP+) cells in the ear tissue of a CXCR6 GFP transgenic mouse (image selected from a single focal plane). The cells in circles are the migrating cells (these cells with lymphocytic morphology are motile and are typically outnumbered by sessile GFP+ cells of dendritic morphology in the ear tissue as visualized by confocal microscopy). (B) Distribution of motile lymphocytic cell displacement along the X and Y direction in the absence of an applied electric field. The large circle is the average cell displacement. The error bar represents standard deviation. (C) Distribution of motile lymphocytic cell displacement along the X and Y direction in an applied electric field (2.5V across ~5mm in the ear tissue). The large circle is the average cell displacement. The error bar represents standard deviation. Note that most cells were within the visible focal plane for only a fraction of the 1–2 hour visualization period. The electrostatic index (E.I.) is also shown as average ± s.e.m..