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. 2020 Feb 4;118(3):565-577.
doi: 10.1016/j.bpj.2019.12.013. Epub 2019 Dec 18.

A Bistable Mechanism Mediated by Integrins Controls Mechanotaxis of Leukocytes

Alexander Hornung  1 , Thomas Sbarrato  1 , Nicolas Garcia-Seyda  1 , Laurene Aoun  1 , Xuan Luo  1 , Martine Biarnes-Pelicot  1 , Olivier Theodoly  1 , Marie-Pierre Valignat  2
Affiliations

A Bistable Mechanism Mediated by Integrins Controls Mechanotaxis of Leukocytes

Alexander Hornung et al. Biophys J. .

Abstract

Recruitment of leukocytes from blood vessels to inflamed zones is guided by biochemical and mechanical stimuli, with the mechanisms only partially deciphered. Here, we studied the guidance by the flow of primary human effector T lymphocytes crawling on substrates coated with ligands of integrins lymphocyte function-associated antigen 1 (LFA-1) (αLβ2) and very late antigen 4 (VLA-4) (α4β1). We reveal that cells segregate in two populations of opposite orientation for combined adhesion and show that decisions of orientation rely on a bistable mechanism between LFA-1-mediated upstream and VLA-4-mediated downstream phenotypes. At the molecular level, bistability results from a differential front-rear polarization of both integrin affinities, combined with an inhibiting cross talk of LFA-1 toward VLA-4. At the cellular level, direction is determined by the passive, flow-mediated orientation of the nonadherent cell parts, the rear uropod for upstream migration, and the front lamellipod for downstream migration. This chain of logical events provides a comprehensive mechanism of guiding, from stimuli to cell orientation.

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Figures

Figure 5
Figure 5
Imaging of high affinity LFA-1 and VLA-4 in cell contact zone by TIRF reveals complex regulation mechanism of their affinity. (A) Shown are representative microscopic images in bright field (top) and TIRF (center) of crawling T cells fixed under a shear stress of 8 dyn/cm−2 and stained for high affinity LFA-1 with mAb24 (green) and high affinity VLA-4 with mAb B44 (magenta). Scale bars, 10 μm. (B) Intensity profiles performed for each fluorescent channel highlight integrin distribution along the cell axis. Values were normalized to the highest value recorded on either condition. White regions indicate the cell body area. (Additional representative profiles are reported in Fig. S5). To see this figure in color, go online.
Figure 1
Figure 1
Flow reveals different migration modes on mixed ICAM-1/VCAM-1 substrates. (A) Shown is the percentage of migrating cells and (B) speed versus substrate composition in shear-free conditions. XI/YV stands for X% ICAM-1/Y% VCAM-1. (C) Shown is the direction of T lymphocytes under shear flow expressed as the MI (positive values indicating motion upstream and negative values indicating motion downstream) versus substrates composition at shear stresses of 4 dyn/cm−2. The MI was calculated for all cells as the ratio between the end-to-end displacement and the cumulative curvilinear traveled distance. All data are mean ± SE; n = 6 independent experiments with at least 500 cells in each experiment, ∗p < 0.05, ∗∗p < 0.001, ∗∗∗p < 0.001 with respect to substrate composition, 100% ICAM-1/0% VCAM-1, one-way ANOVA with post hoc Dunnett’s test. (D) Shown are bright-field images sequence of cells crawling on pure ICAM-1, pure VCAM-1, and mixed ICAM-1/VCAM-1 substrates. Scale bars, 10 μm, with time laps of 40 s. (E) Shown are trajectories of mobile cells on pure ICAM-1, pure VCAM-1, and mixed ICAM-1/VCAM-1 substrates, with a color code for cells crawling upstream (blue), crawling downstream (red), and rolling (black). Time span is 17 min. Scale bars, 100 μm. To see this figure in color, go online.
Figure 2
Figure 2
ICAM-1 imposes strong adhesion and upstream crawling, whereas VCAM-1 allows transient adhesion and downstream crawling/rolling. (A) Shown is the adhesion strength of mobile cells, measured as the percentage of cells resistant to a shear stress of 4 dyn/cm−2 with respect to the initial number of mobile cells on the substrate. XI/YV stands for X% ICAM-1/Y% VCAM-1. (B) Shown are percentages of cells crawling upstream and rolling downstream with respect to the total number of cells migrating on the surface, under a shear stress of 4 dyn/cm−2 and for a different substrate composition. (C) Shown are rose plots of cell directions at different substrate compositions. (D) Shown is the percentage of upstream (blue) and downstream (red) crawling cells, determined here by cumulating data in the blue and red quadrant of the rose plots of cell migration for, respectively, upstream and downstream crawling cells. All data are mean ± SE; n = 6 independent experiments with at least 500 cells in each experiment. ∗p < 0.05, ∗∗p < 0.001, ∗∗∗p < 0.001 with respect to substrate composition, 100% ICAM-1/0% VCAM-1, one-way ANOVA with post hoc Dunnett’s test. To see this figure in color, go online.
Figure 3
Figure 3
Speed increases with flow for rolling cells but remains constant for upstream crawling cells. (A) Shown is speed versus substrate composition of all cells in shear-free condition and of upstream crawling cells and downstream crawling cells and rolling cells under a shear stress of 4 dyn/cm−2. X I/Y V stands for X% ICAM-1/Y% VCAM-1. All data are mean ± SE; n = 6 independent experiments with at least 500 cells in each experiment. ∗p < 0.05, ∗∗p < 0.001, ∗∗∗p < 0.001, one-way ANOVA with post hoc Dunnett’s test. (B) Shown are cumulative distance traveled by individual crawling cells on ICAM-1 (left), mixed ICAM-1/VCAM-1 (center), and VCAM-1 (right) substrates. The color of each curve indicates the migration mode of the corresponding cell tracked—blue for upstream and red for downstream crawling cells. Black solid lines represent the mean, and black dotted lines represent the SD. To see this figure in color, go online.
Figure 4
Figure 4
Cell rear is detached for upstream-bound cells and cell front for downstream-bound cells. Shown is the representative image sequence of crawling cells under a flow of 8 dyn/cm−2 in phase contrast (top) and reflection interference contrast microscopy (RICM) (center). On the merged images, the RICM image is contrast inverted and colored in red. The black arrow indicates flow direction, and the white arrow indicates the direction of cell migration. The adhesion zone (dark in RICM, red in merge) is positioned in cell front for upstream crawling cells and in cell rear for downstream crawling cells. Scale bars, 10 μm. To see this figure in color, go online.
Figure 6
Figure 6
Perturbation experiments support that integrin expression level dictates the decision of orientation versus flow. (A) Shown are two-dimensional cytometry graphs of activated T cells versus expression of heterodimer αL (Ab α -CD11a) for LFA-1 and β2 (Ab α-CD29) for VLA-4 (stained cells, Blue; unstained cells, red). (B) Shown are percentages of available integrins on effector T lymphocytes versus the concentration of blocking antibodies in solution, as determined by cytometry. Blocking antibodies were TS1/22 for LFA-1 and natalizumab for VLA-4. (C) Shown are percentages of upstream crawling cells on mixed ICAM-1/VCAM-1 substrates with and without the addition of blocking antibodies TS1/22, against integrins LFA-1 (left), and natalizumab, against VLA-4 (right). Blocking of LFA-1 displaces phenotype distribution toward the downstream phenotype, and blocking of VLA-4 displaces phenotype distribution toward the upstream phenotype. To see this figure in color, go online.
Figure 7
Figure 7
A bistable mechanism of cell adhesion spatial regulation explains integrin control of T cell flow mechanotaxis. On pure substrates of ICAM-1 or VCAM-1, the T cell population has homogeneous phenotypes with an opposite orientation on ICAM-1 and VCAM-1. On mixed substrates of ICAM-1 or VCAM-1, T cells distribute in two populations with opposite orientations and characteristics similar to phenotypes on pure substrates. Decisions of orientation on mixed substrates are controlled by the expression level of integrins LFA-1 and VLA-4 via a bistable polarization of cell adhesion; a higher LFA-1 expression leads to a LFA-1-dominated adhesion of cell front (very similar to upstream crawling cells on ICAM-1), whereas a higher expression of VLA-4 leads to adhesion of cell rear and center (very similar to downstream crawling cells on VCAM-1). Inhibiting cross talk of LFA-1 toward VLA-4 reinforces adhesion polarization toward cell front, which favors wind vane mechanism and upstream phenotype. Activating cross talk of VLA-4 toward LFA-1 reinforces the adhesion of cell uropod, which hampers the wind vane mechanism and favors the downstream phenotype. To see this figure in color, go online.
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