Probing the biomechanical contribution of the endothelium to lymphocyte migration: diapedesis by the path of least resistance

Abstract

Immune cell trafficking requires the frequent breaching of the endothelial barrier either directly through individual cells ('transcellular' route) or through the inter-endothelial junctions ('paracellular' route). What determines the loci or route of breaching events is an open question with important implications for overall barrier regulation. We hypothesized that basic biomechanical properties of the endothelium might serve as crucial determinants of this process. By altering junctional integrity, cytoskeletal morphology and, consequently, local endothelial cell stiffness of different vascular beds, we could modify the preferred route of diapedesis. In particular, high barrier function was associated with predominantly transcellular migration, whereas negative modulation of junctional integrity resulted in a switch to paracellular diapedesis. Furthermore, we showed that lymphocytes dynamically probe the underlying endothelium by extending invadosome-like protrusions (ILPs) into its surface that deform the nuclear lamina, distort actin filaments and ultimately breach the barrier. Fluorescence imaging and pharmacologic depletion of F-actin demonstrated that lymphocyte barrier breaching efficiency was inversely correlated with local endothelial F-actin density and stiffness. Taken together, these data support the hypothesis that lymphocytes are guided by the mechanical 'path of least resistance' as they transverse the endothelium, a process we term 'tenertaxis'.

Keywords: Actin; Barrier; Endothelium; Leukocyte; Migration; Stiffness.

Fig. 1.

Assessment of junctional integrity and diapedesis route preference in different vascular endothelial monolayers. Primary rat brain (rB), rat heart (rH), human lung (hL) and human heart (hH) MVECs were grown to confluence and stimulated with TNF-α (24 h) before (A) measuring basal TEER or (B–D) addition of rat or human T cells for 10 min (black bars; all endothelial cells) or 30 min (white bars; rat brain MVECs only), prior to fixation and staining with antibodies against VEC (blue), ICAM-1 (green) and LFA-1 (red). (B) Schematic (i) and representative confocal imaging (ii) of paracellular and transcellular diapedesis events. Scale bar: 5 µm. (C) Quantification of ‘transmigrating’ cells (see scheme in supplementary material Fig. S1A). (D) Quantification of relative paracellular (i) and transcellular (ii) diapedesis. Data show the mean±s.e.m. (at least four separate experiments); *P<0.05, ***P<0.001.

Fig. 2.

Modulation of junctional integrity in rat brain MVECs affects the route of diapedesis. Primary rat brain MVECs were grown to confluence and stimulated with TNF-α (24 h) before the addition of adrenomedullin (AM, 10 µM), 8-pCPT-2′-O-Me-cAMP (O-Me, 200 µM), histamine (His, 300 µM) or PP2 (10 µM). (A) Changes in TEER are shown following treatments. Data show the mean±s.e.m. (at least four separate experiments). (B) Immunofluorescence imaging of rat brain MVECs following treatment with adrenomedullin and O-Me for 30 min and histamine or PP2 for 10 min prior to fixation, permeabilization and staining for VEC (green) and F-actin (red). The areas indicated by dashed boxes and asterisks are shown at higher magnification in the lower panels. Ctl, control; white arrowheads, cortical actin; yellow arrowheads, gaps. Data are representative of at least five separate experiments. Scale bars: 10 µm. (C) Quantification of the number of gaps per field (i) and the percentage of total gap area per field (ii) in PP2-treated and histamine-treated cells. (D) Rat brain MVECs were treated as before prior to the addition of rat T cells for 30 min followed by fixation, staining and quantification of paracellular (i) and transcellular diapedesis (ii). Data show the mean±s.e.m. (at least four separate experiments); **P<0.01. (E) Comparison of basal TEER in primary rat brain MVECs at passage 1 (P1) and passage 4 (P4) (i) and quantification of the route of diapedesis at passage 4 (ii). Data show the mean±s.e.m. (at least three separate experiments).

Fig. 3.

Modulation of junctional integrity in rat heart MVECs affects the route of diapedesis. Primary rat heart MVECs were grown to confluence and stimulated with TNF-α (24 h) before the addition of adrenomedullin (AM, 10 µM), 8-pCPT-2′-O-Me-cAMP (O-Me, 200 µM), histamine (His, 300 µM) or PP2 (10 µM). (A) Changes in TEER are shown following treatments. Data show the mean±s.e.m. (at least four separate experiments). (B) Rat heart MVECs were treated as above prior to the addition of rat T cells for 10 min followed by fixation, staining and quantification of paracellular (i) and transcellular diapedesis (ii). Ctl, control. Data show the mean±s.e.m. (at least four separate experiments); *P<0.05. (C) Immunofluorescence imaging of rat heart MVECs following treatment with adrenomedullin and O-Me for 30 min and histamine or PP2 for 10 min prior to fixation, permeabilization and staining for VEC (green) and F-actin (red) (i) and quantification of the number of gaps per field (ii) and the percentage of total gap area per field (iii) in PP2-treated and histamine-treated cells (data show the mean±s.e.m.). In immunofluorescence images, the areas indicated by dashed boxes and asterisks are shown at higher magnification in the lower panels. Yellow arrowheads, gaps. Data are representative of at least five separate experiments. Scale bars: 10 µm.

Fig. 4.

Effect of shear flow, genetic modulation of Rho GTPases and substrate stiffness on route of migration. (A) Human lung MVECs were grown to confluence, stimulated with TNF-α (24 h) and exposed to short (S) or long-term (LT) shear (30 min or >36 h, respectively; 10 dyne/cm²). (i) Samples were fixed and stained for actin, or subjected to fluorescent tracer permeability (ii, iii) or diapedesis (iv) assays. (ii) Representative images of paracellular ‘leakage’ of fluorescein–streptavidin that was ‘captured’ on the biotin-coated substrate underlying the endothelium (upper panels) near VEC-stained junctions (lower panels). Scale bars: 10 µm. (iii) Quantification of the permeability under static (control, Ctl), short or long-term shear conditions. (iv) Human T cells were added for 10 min to allow for migration, followed by fixation, staining and quantification of total diapedesis (see supplementary material Fig. S4Aii) and route of diapedesis. (B) Human lung MVECs were transfected with constitutively active (CA) Rho, Rap or Rac or dominant negative (DN) Rho or Rac, followed by fixation, staining and quantification of the route of diapedesis. (C) Human lung MVECs were grown to confluence on either glass (>10 GPa) or elastic substrates of 28 kPa and 1.5 kPa elastic modulus. Cells were stimulated with TNF-α (24 h) before the addition of human T cells for 10 min followed by fixation, staining and quantification of total diapedesis (left) and transcellular route usage (right). (D) Human lung MVEC monolayers were prepared as above on glass (>10 GPa; control; white bars) or an elastic substrate (1.5 kPa; black bars) and then treated with adrenomedullin (AM, 10 µM) or histamine (His, 300 µM) before the addition of human T cells for 10 min and quantification of the route of diapedesis. Quantitative data show the mean±s.e.m. [at least four (A,C,D) or three (B) separate experiments]; *P<0.05, ***P<0.001.

Fig. 5.

Lymphocytes ‘feel’ the vascular endothelium through invadosome-like protrusions. (A) (i) Orthogonal schematic view of a lymphocyte forming invadosome-like protrusions (ILPs) against the endothelial surface. (ii) Representative ultrastructural views of primary lymphocyte (blue) ILP (blue arrowhead) deforming the surface of activated endothelium (green). Scale bars: 300 nm. (B) Examples of ILPs (blue arrowheads) probing the junctions and endothelial cell (EC) body. (i) Note two separate ILPs on either side of an adherens junction (AJ) and one ‘burrowing’ ILP or pseudopod (cyan asterisk). (iii) Note one ILP protruding at the adherens junction (red asterisk) and one protruding at an adjacent highly tenuous non-junctional region (green asterisk), as well as a burrowing ILP (cyan asterisk). Data are representative of >100 images. Scale bars: 500 nm. (C) Human lung MVECs were transfected with MemDsRed (magenta) and soluble GFP (green; a cytoplasm volumetric marker), stimulated with TNF-α (24 h) and subjected to live-cell imaging in the presence of T cells. Upper panels show a time-point shortly after lymphocytes have settled on the endothelium, but before the formation of ILPs (relative time = 0 min). Lower panels show a time-point after lymphocyte spreading and formation of ILPs (relative time 1.15 or 1.45 min). Note that for each MemDsRed ring, a circular region of diminished GFP signal is formed, indicating cytoplasm displacement by ILPs (blue arrowheads). These can be seen pushing into the MVEC at the cell–cell junctions (i) or in the cell body (ii). See also corresponding supplementary material Movie 1. Data are representative of >50 separate experiments. Scale bars: 5 µm.

Fig. 6.

AFM-enabled nanoindentation to assess the stiffness of endothelial junctions. (A) Comparison between the size and shape of a single ILP (i) and the AFM probe used (ii). (B) Representative AFM height (upper panels) and corresponding Young's Elastic Modulus (lower panels) micrographs taken at the junction of human lung MVECs before and after the indicated treatments. (C) Quantification of the percentage change in Young's Elastic Modulus. Data show the mean±s.e.m. (at least four separate experiments); ***P<0.001.

Fig. 7.

Assessing the contribution of endothelial nuclear lamina and cytoskeleton in determining sites of lymphocyte diapedesis. (A) Electron microscopy micrographs of lymphocyte ILPs (blue arrowheads) formed above the endothelial cell (EC) nuclei (green). Note that individual (i) or clustered ILPs (ii) (and burrowing ILPs or pseudopods; cyan asterisks) can be seen exerting sufficient force to locally deform the nuclear lamina. (iii) Example of concomitant formation of both shallow ‘frustrated’ ILPs above the nucleus (blue arrowheads) and a deeply cytoplasm-penetrating ILP immediately adjacent to the nucleus (green asterisk). Scale bars: 500 nm. (B) Live-cell imaging showing a T cell avidly probing with multiple ILPs (white arrowheads) in the area above the endothelial cell nucleus (dashed blue line) before forming a transcellular pore adjacent to the nucleus. DIC, differential interference contrast. Scale bar: 5 µm. See also corresponding supplementary material Movie 2. (C) Human lung MVECs were transfected with MemDsRed and either actin–GFP, tubulin–GFP or vimentin–GFP, stimulated with TNF-α (24 h) and subjected to live-cell imaging during T cell diapedesis. The density of actin, tubulin and vimentin at the sites of barrier breach were quantified as described in Material and Methods. (i) Experiment type 1: a low density of T cells was added to the MVEC monolayer and each individual lymphocyte was tracked until the initiation of diapedesis. (ii) Experiment type 2: a high (saturating) density of T cells was added to the MVEC monolayer and all sites of diapedesis initiation were identified at a fixed time-point of 15 min. Data show the mean±s.e.m. (at least seven separate experiments). (iii) Representative images of experiment type 2: upper panels show merged DIC and fluorescence images of a singular actin–GFP-transfected human lung MVEC in the context of a confluent monolayer at time 0 and 15 min after addition of T cells. Lower panels show the corresponding fluorescence intensity heat maps of actin distribution. Data are representative of at least seven separate experiments. Scale bars: 10 µm.

Fig. 8.

Tenertaxis: lymphocytes actively probe the endothelial cell surface and underlying cytoskeleton and prefer to migrate in areas of low F-actin. (A) Human lung MVECs were transfected with actin–GFP (green) and MemDsRed (magenta) and stimulated with TNF-α (24 h) before the addition of T cells and live-cell imaging. (i) In areas of high-density actin filaments, T cells are seen to avidly palpate the MVEC with numerous frustrated ILPs (arrowheads) without initiation of diapedesis (cell 1). Alternatively, T cells that initiate breach in areas of dense actin are often unable to complete diapedesis (cell 2). See also corresponding supplementary material Movie 4. (ii) In areas of lower actin density, ILP can be seen readily driving barrier breach between actin fibers. See also corresponding supplementary material Movie 6. DIC, differential interference contrast. (B) In areas of modest actin density, ILPs can be seen to dynamically bend the actin fibers (white arrowheads) during lateral migration, which ultimately allows for diapedesis upon reaching a region of relatively lower actin density (see heat map panels). See also corresponding supplementary material Movie 5. (C) (i) Representative electron microscopy micrograph of frustrated ILPs failing to deform thick F-actin bundles (green). Red arrowheads, actin filaments. (ii) Representative electron microscopy micrograph of ILPs readily bending or distorting individual actin filaments. The area outlined in red is shown at higher magnification in the lower image. (D) (i) Human lung MVECs were transfected with actin–GFP (green) and MemDsRed (red), stimulated with TNF-α (24 h) and treated with cytochalasin D (200 mM, 30 min at 37°C) before the addition of T cells and live-cell imaging. (ii) Following 10–15 min of imaging, samples were fixed and stained for VEC (blue) to identify the adherens junctions. ILP probing (white arrowheads) can be seen to readily form transcellular breaches throughout the cell body (i), as well as adjacent to intact VEC junctions (ii, yellow arrowheads). See also corresponding supplementary material Movie 7. Scale bars: 5 µm (for A,B,D), 200 nm (for C). (E) Quantification of the route of migration following cytochalasin D treatment. Data show the mean±s.e.m. (at least five separate experiments); ***P<0.001.