Not all (cells) who wander are lost: Upstream migration as a pervasive mode of amoeboid cell motility

Abstract

Leukocytes possess the ability to migrate upstream-against the direction of flow-on surfaces of specific chemistry. Upstream migration was first characterized in vitro for T-cells on surfaces comprised of intracellular adhesion molecule-1 (ICAM-1). Upstream migration occurs when the integrin receptor α_Lβ₂ (also known as lymphocyte function-associated antigen-1, or LFA-1) binds to ICAM-1. LFA-1/ICAM-1 interactions are ubiquitous and are widely found in leukocyte trafficking. Upstream migration would be employed after cells come to arrest on the apical surface of the endothelium and might confer an advantage for both trans-endothelial migration and tissue surveillance. It has now been shown that several other motile amoeboid cells which have the responsibility of trafficking from blood vessels into tissues, such as Marginal zone B cells, hematopoietic stem cells, and neutrophils (when macrophage-1 antigen, Mac-1, is blocked), can also migrate upstream on ICAM-1 surfaces. This review will summarize what is known about the basic mechanisms of upstream migration, which cells have displayed this phenomenon, and the possible role of upstream migration in physiology and tissue homeostasis.

Keywords: ICAM-1; LFA-1; T-cells; hematopoietic stem cells; inflammation; leukocytes; migration.

FIGURE 1

Angle histogram for one representative experiment in four different leukocyte cell populations (primary T cell, effector T cells, HSB2 T cells, and neutrophils) in static (upper panels) and flow (lower panels) conditions showing the distribution of θ in ≤40 angle bins. The length of each bin reflects the fraction of cells with a given angle that fall within a group. Under the flow condition, the shear stress was set to a value of 8 dyn cm⁻² and the direction of the flow was along the Y-axis from top to bottom, as indicated by the arrow. The numbers of cells analyzed were respectively 192, 234, 148, and 134 in the static condition and 23, 73, 93, and 60 under the flow condition for primary T cells, effector T cells, HSB2 T cells, and neutrophils. Reproduced from Valignat et al., Biophys J, 2013 January 22; 104(2):322-31.

FIGURE 2

(A) Biochemical signals downstream of LFA-1 after engagement with ICAM-1. (B) Flow histograms and (C) Migration Indices of WT and Crk DKO mouse CD4⁺ T cells migrating on ICAM-1 surface at 800s^-1 shear rate. (D) CD4⁺ T cells from Cas9-expressing mice were transduced with the indicated gRNAs, selected for 3 d in puromycin, and then lysed and immunoblotted for the indicated proteins. NT, non-targeting gRNA control. GAPDH was used as a loading control. (E) Migration index of T cells expressing the indicated gRNAs migrating on ICAM-1 under shear flow (shear rate 800 s⁻¹). (F) Percentage of cells migrating upstream from experiments shown in panel (E) (G) Cells expressing the indicated gRNAs were allowed to migrate on ICAM-1-coated surfaces, fixed, and stained with fluorescent phalloidin. Representative images, Scale bar: 10 μm. Adapted from Roy et al., J Cell Sci, 2020 September 9; 133(17):jcs248328.

FIGURE 3

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. Adapted from Hornung et al., Biophys J, 2020 February 4; 118(3):565-577.

FIGURE 4

Diagram explains the key players in neutrophil upstream migration. VLA-4-VCAM-1 interactions lead always to downstream migration. Neutrophils have 2 cell surface ligands for ICAM-1: LFA-1 and Mac-1. When both Mac-1 and LFA-1 are allowed to engage ICAM-1, downstream migration is seen. Blocking Mac-1 function with monoclonal antibodies allows for LFA-1-ICAM-1 mediated upstream migration.

FIGURE 5

(A) Plot of migration index over time of activated CD4⁺ T cells on activated HUVEC and ICAM-1 surfaces. Upstream migration is indicated by a negative migration index, downstream migration by positive values, and random migration by values near zero. Blockade of LFA-1 prevents upstream migration on stimulated HUVECS, while cells with unblocked LFA-1 initially migrate upstream before reverting to downstream migration. ICAM-1-only recombinant protein surface data is provided for comparison. Data presented mean ± SEM, n = 4 independent experiments. (B) Plot showing the remaining fraction of tracked cells at each time point. Cells on HUVEC monolayers were tracked from initial migration to transmigration or the end of the experiment, whichever is sooner. (C) Comparison of fraction of cells which migrated upstream on HUVEC monolayers with or without LFA-1 blockade. Data presented mean ± SEM, n = 4 independent experiments. (D) Comparison of the time from arrest to transmigration on HUVEC monolayers with or without LFA-1 blockade. Data presented mean ± SEM, n = 4 independent experiments. *p < 0.05, **p < 0.005. Reproduced from Anderson et al., Cell Adh Migr, 2019 December; 13(1):163-168.