Mechanical Forces in Endothelial Cells during Firm Adhesion and Early Transmigration of Human Monocytes

Abstract

Transmigration of leukocytes across the endothelial barrier is a tightly controlled process involving multiple steps, including rolling adhesion, firm adhesion, and then penetration of leukocytes through the endothelial monolayer. While the key molecular signals have been described in great detail, we are only just beginning to unveil the mechanical forces involved in this process. Here, using a microfabricated system that reports traction forces generated by cells, we describe forces generated by endothelial cells during monocyte firm adhesion and transmigration. Average traction force across the endothelial monolayer increased dramatically when monocytes firmly adhered and transmigrated. Interestingly, the endothelial cell that was in direct contact with the monocyte exhibited much larger traction forces relative to its neighbors, and the direction of these traction forces aligned centripetally with respect to the monocyte. The increase in traction force occurred in the local subcellular zone of monocyte adhesion, and dissipated rapidly with distance. To begin to characterize the basis for this mechanical effect, we show that beads coated with anti-ICAM-1 or VCAM-1 antibodies bound to monolayers could reproduce this effect. Taken together, this study provides a new approach to examining the role of cellular mechanics in regulating leukocyte transmigration through the endothelium.

FIGURE 1

Approach to measure traction force in endothelial monolayers during monocyte adhesion and early transmigration. (a) Endothelial cells grown on mPADs (monolayer size: 150 μm X 150 μm). Immunofluorescence staining indicates cell nucleus (blue), actin filaments (green), microposts (gray), and β-catenin (magenta), respectively. (b) Endothelial cell monolayers grown on mPADs with monocytes transmigrating through them. Images taken from focal plane 5.75 μm above, 0 μm, and 4.83 μm below the monolayer. Immunofluorescence staining indicates endothelial cell nucleus (dark blue), monocytes (bright cyan), microposts (red), and β-catenin (green) (monolayer size: 100 μm 3 100 μm). (c) Schematic figure for monocyte firm adhesion and early transmigration on endothelial cells on posts. The fluorescence images on the right are cell-tracker green staining at different focal planes of the monocyte transmigrating on an endothelial monolayer. (d) Array of endothelial monolayers showing each monolayer has 1 or 0 monocytes transmigrating on it. (e) Histogram showing most of the endothelial monolayers have 1 or 0 monocyte on it. Scale bars indicate 10 μm.

FIGURE 2

Traction forces reported during firm adhesion and early transmigration. (a) Fluorescence images showing endothelial monolayers at baseline (Ctrl), TNFα-treated (TNF), and with monocyte transmigrating on it (TEM), respectively. Immunofluorescence staining indicates β-catenin (green); and monocyte (bright cyan); nucleus (blue); microposts (red). Scale bars indicate 10 μm; white arrows in figures indicate the vector of traction forces with scaled arrow bar indicating 32 nN. (b) Bar graph indicating increase in average traction force in endothelial monolayers with monocytes transmigrating on them. *p < 0.05, indicates comparison against Ctrl; # p < 0.05, indicates comparison against TNF. (c) Definition of the relative angle (Rel. Angle): the angle (absolute value, in degrees) between the traction force vector of each location on the endothelial monolayer and the centripetal line connecting the center of the monocyte to that location; and also the definitions of Ct Endo and NCt Endo, as the endothelial cell directly contacting the monocyte, or not, respectively. For monocytes spanning more than one endothelial cell, we defined the Ct cell as the one with the largest contacting area with the monocytes. (d) Histograms showing the distribution of Rel. Angle for Ctrl, TNF, and TEM conditions. The TEM condition was fit with gamma distribution (see “Materials and Methods” section). See “Materials and Methods” for how ghost monocyte locations were generated for Ctrl and TNF conditions. (e) Bar graph indicating a significant difference in average traction forces between Ct and NCt cells in the TEM condition. *p < 0.05, indicates comparison against Ct. (f) Histograms showing the distribution of Rel. Angle in both Ct and NCt cells for Ctrl, TNF, and TEM conditions. Ctrl and TNF are fit with uniform distribution and TEM is fit with gamma distribution. All error bars indicate standard deviation.

FIGURE 3

Spatial distribution of traction forces in endothelial monolayer during firm adhesion and early transmigration. (a) The magnitude of traction forces in the transmigration monolayer in Fig. 2a was re-plotted in pseudo-color, filtered and smoothed with a bi-cubic 2D spatial filter. (b) Zones from “Local” to “Distant” relative to the location of monocyte. Z1 is the zone of posts in the closest vicinity around the monocyte, Z2 next closest, Z3… and so on. The segmentation was performed on the transmigrating monolayer in Fig. 2a. (c) Average traction force in each zone as defined in (b) compared across all conditions. * or #, p < 0.05, indicates comparison against Ctrl at each zone. (d) Zones from “Edge” to “Interior.” “Edge” is defined as the outmost zone, and “Interior” is defined as the combination of all the rest of the zones. (e) Bar graph indicating no significant difference in average traction forces between “Edge” and “Interior” zones as defined in (d) in all conditions. All error bars indicate standard deviation.

FIGURE 4

Activation of endothelial ICAM-1/VCAM-1 is enough to trigger increase in traction forces. (a) Fluorescence images showing endothelial monolayers at baseline (Ctrl), TNFα-treated (TNF), and with monocyte arrested on it (TEM), respectively. Immunofluorescence staining indicates beta-catenin (green); and monocyte (bright cyan); nucleus (blue); microposts (red). In the last two images, the bright green circular dots are the ICAM-1 and VCAM-1-coated beads. Scale bars indicate 10 μm; white arrows in figures indicate the vector of traction forces with scaled arrow bar indicating 32 nN. (b) Bar graph indicating increase in average traction force in ICAM-1-treated endothelial monolayers. *p < 0.05, indicates comparison against Ctrl. (c) Bar graph indicating no significant difference in average traction forces between Ct and NCt cells in ICM or VCM condition. (d) Average traction force in each zone from “Local” to “Distant” relative to the location of ICAM-1/VCAM-1 beads, as defined in Fig. 3b, compared across all above conditions. * or #, p < 0.05, indicates comparison against Ctrl at each zone. All error bars indicate standard deviation.