ESAM supports neutrophil extravasation, activation of Rho, and VEGF-induced vascular permeability

Endothelial cell–selective adhesion molecule (ESAM) is specifically expressed at endothelial tight junctions and on platelets. To test whether ESAM is involved in leukocyte extravasation, we have generated mice carrying a disrupted ESAM gene and analyzed them in three different inflammation models. We found that recruitment of lymphocytes into inflamed skin was unaffected by the gene disruption. However, the migration of neutrophils into chemically inflamed peritoneum was inhibited by 70% at 2 h after stimulation, recovering at later time points. Analyzing neutrophil extravasation directly by intravital microscopy in the cremaster muscle revealed that leukocyte extravasation was reduced (50%) in ESAM−/− mice without affecting leukocyte rolling and adhesion. Depletion of >98% of circulating platelets did not abolish the ESAM deficiency–related inhibitory effect on neutrophil extravasation, indicating that it is only ESAM at endothelial tight junctions that is relevant for the extravasation process. Knocking down ESAM expression in endothelial cells resulted in reduced levels of activated Rho, a GTPase implicated in the destabilization of tight junctions. Indeed, vascular permeability stimulated by vascular endothelial growth factor was reduced in ESAM−/− mice. Collectively, ESAM at endothelial tight junctions participates in the migration of neutrophils through the vessel wall, possibly by influencing endothelial cell contacts

In vivo imaging and quantitative analysis of leukocyte directional migration and polarization in inflamed tissue

Directional migration of transmigrated leukocytes to the site of injury is a central event in the inflammatory response. Here, we present an in vivo chemotaxis assay enabling the visualization and quantitative analysis of subtype-specific directional motility and polarization of leukocytes in their natural 3D microenvironment. Our technique comprises the combination of i) semi-automated in situ microinjection of chemoattractants or bacteria as local chemotactic stimulus, ii) in vivo near-infrared reflected-light oblique transillumination (RLOT) microscopy for the visualization of leukocyte motility and morphology, and iii) in vivo fluorescence microscopy for the visualization of different leukocyte subpopulations or fluorescence-labeled bacteria. Leukocyte motility parameters are quantified off-line in digitized video sequences using computer-assisted single cell tracking. Here, we show that perivenular microinjection of chemoattractants [macrophage inflammatory protein-1alpha (MIP-1alpha/Ccl3), platelet-activating factor (PAF)] or E. coli into the murine cremaster muscle induces target-oriented intravascular adhesion and transmigration as well as polarization and directional interstitial migration of leukocytes towards the locally administered stimuli. Moreover, we describe a crucial role of Rho kinase for the regulation of directional motility and polarization of transmigrated leukocytes in vivo. Finally, combining in vivo RLOT and fluorescence microscopy in Cx3CR1(gfp/gfp) mice (mice exhibiting green fluorescent protein-labeled monocytes), we are able to demonstrate differences in the migratory behavior of monocytes and neutrophils.Taken together, we propose a novel approach for investigating the mechanisms and spatiotemporal dynamics of subtype-specific motility and polarization of leukocytes during their directional interstitial migration in vivo

Leukocyte interstitial migration.

<p>Interstitially migrating leukocytes were visualized using near-infrared RLOT microscopy (objective magnification 20×) and tracked during 5 min in digitized video recordings using an imaging software. A - leukocyte interstitial migration after microinjection of MIP-1α, B - after intrascrotal injection of PAF, C – upon intrascrotal injection of MIP-1α, and D - after microinjection of MIP-1α followed by superfusion of the Rho kinase inhibitor Y27632. a - red numbers on the intravital microscopic images shows the position of each analyzed leukocyte at the beginning of cell tracking; b - location of the same leukocytes at the end of the cell tracking, respectively; c - green lines on intravital microscopic images and colored lines on the panels (d) show the migration tracks of single leukocytes; asterisks show the site of microinjection, scale bar 20 µm.</p

Morphological changes and polarization of interstitially migrating leukocytes.

<p>Freeze-frames from <i>in vivo</i> microscopy video recordings obtained 60 min after microinjection of PAF (A), 60 min intrascrotal injection of PAF (B), and 60 min microinjection of PAF in combination with superfusion of the Rho kinase inhibitor Y27632 for 30 min (C; objective magnification 40×). A: directional locomotion of a leukocyte towards the attractant with protrusions of lamellipodia at the front and a trailing uropod after microinjection of PAF. B: polarized leukocyte during random migration in the interstitial tissue after intrascrotal injection of PAF. C: blocking effect of Rho kinase inhibitor Y27632 on leukocyte polarization induced by microinjection of PAF. D: leukocyte polarization was measured as cell eccentricity: long axis (shown in black line)/short axis (depicted as interrupted line). Quantitative data of cell eccentricity after microinjection of saline, MIP-1α, PAF, <i>E. coli</i>, intrascrotal injection of PAF, as well as after microinjection of PAF with superfusion of Y27632; mean±SEM; *p<0.05 vs. saline, # p<0.05 vs. Y27632; n = 15.</p

Effect of Rho kinase inhibition on leukocyte motility.

<p>Leukocyte curve-line velocity (A), straight-line velocity (B), and directionality (C) were analyzed in the cremaster muscle at 60 min after microinjection of MIP-1α (black bars) followed by superfusion of the Rho kinase inhibitor Y27632 (gray bars) for either 0–5 min or 30–35 min. In control experiments (black bars), the cremaster muscle was superfused with saline upon microinjection of MIP-1α; mean±SEM; *p<0.05 vs. control, # p<0.05 vs. control at 60–65 min; n = 15.</p

Tissue distribution of rhodamine 6G after microinjection.

<p>A-C: <i>In vivo</i> microscopy images show tissue distribution of rhodamine 6G at 1 (A), 30 (B), and 60 min after microinjection (C). Three regions of interests 100×75 µm (depicted in B) within the cremasteric interstitial tissue were analyzed: on the vessel side ipsilateral to the microinjection site, on the contralateral side, and in the interstitial tissue at a distance of 350 µm from the site of microinjection (considered as background). Images are shown with different original color scales to emphasize the fluorescent intensity within 60 min after microinjection. The fluorescence intensity was determined within 60 min; the quantitative data are presented in D; n = 7.</p

Migration patterns of GFP-positive and GFP-negative cells.

<p>A: representative <i>in vivo</i> microscopy images show transmigration of GFP-positive (GFP-image) and GFP-negative cells (RLOT-image) at 60 min after microinjection of MCP-1 in Cx3CR1^gfp/gfp mice (objective magnification 20×; asterisk shows the site of microinjection). B–E: The motility parameters, curve-line migration velocity (B), straight-line migration velocity (C), and directionality (D) of interstitially migrating, GFP-positive (monocytes, gray bars) and GFP-negative cells (neutrophils, black bars) were analyzed at 60, 70, and 80 min after microinjection of MCP-1 for 5 min, respectively; mean±SEM; *p<0.05 vs. GFP-negative cells; # p<0.05 vs. 60–65 min; n = 5. E: polarization of GFP-negative cells (black bars) and GFP-positive cells (gray bars) analyzed at 70 min after microinjection of MCP-1; mean±SEM, n = 15.</p

Leukocyte adhesion and transmigration.

<p>Numbers of adherent (A) and transmigrated (B) leukocytes at 60 minutes after microinjection of saline, MIP-1α, PAF, or <i>E. coli</i>, and after intrascrotal injection of PAF (PAF i.s.) were counted on the vessel side ipsilateral to the microinjection site (black bars) and on the contralateral side (white bars); mean±SEM; n = 6; *<0.05 vs. ipsilateral side, #<0.05 vs. PAF- contralateral side; <i>E. coli</i> n = 3; PAF i.s. n = 3. C–F: intravital microscopic images from the murine cremaster muscle demonstrate adherent and emigrated leukocytes (arrows) after microinjection (C and E, respectively) and intrascrotal injection of PAF (D and F, respectively); asterisk shows the site of microinjection; objective magnification 20×; scale bar 25 µm.</p

Leukocyte motility.

<p>Parameters of leukocyte motility such as curve-line migration velocity (A), straight-line migration velocity (B), and directionality (C) were determined in digitized <i>in vivo</i> microscopy video sequences 60 min after microinjection of MIP-1α, PAF or <i>E. coli</i> as well as upon intrascrotal application of PAF (PAF i.s.). Interstitially migrating leukocytes (n = 15) were analyzed during 5 min using SimplePCI software. Parameters of leukocyte motility are presented for the vessel side ipsilateral to the microinjection site (black bars), on the contralateral vessel side (white bars), and after intrascrotal application of PAF – (gray bars); mean±SEM; *p<0.05 vs. saline, § p<0.05 vs. PAF i.s., & p<0.05 vs. PAF, $ p<0.05 vs. contralateral side; n = 15; <i>E. coli</i> n = 8.</p

Dependency of leukocyte adhesion and transmigration on the distance of microinjection from the vessel.

<p>Leukocyte adhesion (A) and transmigration (B) were analyzed 60 min after microinjection of MIP-1α performed at distances of 25–50, 75–100, and 175–200 µm from the postcapillary venule. Both parameters were observed in ROIs (100×50 µm) along the venule (mean±SEM; n = 3).</p