A Bistable Mechanism Mediated by Integrins Controls Mechanotaxis of Leukocytes

Abstract

Recruitment of leukocytes from blood vessels to inflamed zones is guided by biochemical and mechanical stimuli, with mechanisms only partially deciphered. Here, we studied the guidance by flow of primary human effector T lymphocytes crawling on substrates coated with ligands of integrins LFA-1 (L2) and VLA-4 (41). 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 integrins affinity, combined with an inhibiting crosstalk of LFA-1 towards VLA-4. At the cellular level, direction is determined by the passive, flow-mediated orientation of the non-adherent 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. STATEMENT OF SIGNIFICANCECell guidance is crucial to many biological functions, but the precise mechanisms remain unclear. We have analyzed here an original phenotype of flow-guided cells mimicking leukocytes crawling onto blood vessels, and show that the controlling parameter of cells decision to migrate upstream or downstream is the relative number of two specific adhesion molecules, the integrins LFA-1 and VLA-4.The spatial polarization of these integrins affinity plus a feedback loop between them creates a bistable system, where cells adhere either by their front or their tail to orient upstream or downstream, respectively. This mechanism proposes a complete chain of events from stimuli to cell orientation which differs strongly from the chemotaxis paradigm, because the external stimuli triggers no signaling.

LFA-1 signals to promote actin polymerization and upstream migration in T cells

“…In contrast, T cells migrating on VCAM-1 showed less contact with the stimulatory surface and seem to adhere mostly through the center and uropod of the cell (Fig 2E-F). This morphology is consistent with observations by others (14,15). Analysis of the movies revealed that while T cells migrating on ICAM-1 coated surfaces moved smoothly, showing sustained actin polymerization at the leading edge, T cells on VCAM-1 had periodic bursts of movement concomitant with brief actin flares at the front of the cell (Fig 2F-G, see Supplemental Movies 1 and 2).…”

“…We and others have previously shown that T cells preferentially migrate against the direction of shear on ICAM-1 and with the direction of shear on VCAM-1 (11)(12)(13)(14)(15)(16)(17). To verify that our primary mouse T cells display this behavior, we activated T cells for two days and rested them for an additional three days in the presence of IL-2, a procedure that generates highly motile T cell blasts expressing both LFA-1 and VLA-4.…”

Different integrins mediate haptotaxis of T lymphocytes towards either lower or higher adhesion zones

“…Figure 3 superimposes transmission images to localize cell body (grey), fluorescence microscopy images of the patterned stripes (red) and RICM images of the cell adhesion footprint (green). On homogeneously adherent substrates (Figure 3-A,D) , cells display adherent lamellipods and non-adherent rear on ICAM-1, and conversely adherent rear and detached lamellipods on VCAM-1, as previously described (28). On substrates with adherent and non-adherent stripes (Figure 3-B,E), adhesion (green) signal was bright on integrin-ligand stripes (bright red) and extinct on anti-adhesive stripes (light red), which confirms that cells crawl on adherent stripes and swim over non-adherent stripes.…”

“…Residual fluorescent intensity due to adsorbed antibodies was measured and then subtracted from the previous measurements. Previous study (28) showed a linear relation between the fluorescent intensity and the bulk concentration. We assume that the signal is given by the total number of molecules in the thin channel, then the volume concentration can be converted to a surface concentration for a channel of 41 µm in height.…”

“…However, calcium release experiments supported that no intracellular signaling was triggered by modulations of the adhesive stimulus (6). The sharp difference between haptotaxis mediated by integrins LFA-1 or VLA-4 may instead be linked to the strong differences in the dynamics of their spatial distribution and activation at the cell surface (28). Elucidation of these mechanisms would therefore benefit from precise characterization at single cell level of the strength of peripheral adhesion, and of the cartography of integrins localization and affinity state.…”