Abstract
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-1α (MIP-1α/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 Cx3CR1gfp/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.
Highlights
The multistep cascade of leukocyte migration into injured or inflamed tissue consists of sequential intercellular adhesion events with endothelial cells [1]
In order to establish an optimal protocol for the perivenular microinjection of chemoattractants, we first analyzed the extent of local inflammation in the cremasteric tissue after microinjection of the chemokine macrophage inflammatory protein-1a (MIP-1a) performed at three different distances from a venule: 25–50 mm, 75–100 mm, and 175–200 mm
The highest number of adherent and transmigrated leukocytes was found when the microinjection was performed at a distance of 25–50 mm (Fig. 1)
Summary
The multistep cascade of leukocyte migration into injured or inflamed tissue consists of sequential intercellular adhesion events with endothelial cells [1]. The mechanisms underlying these initial steps of leukocyte rolling and firm adhesion have been studied in detail [1,2,3]. We suggest perivenular microinjection of chemoattractants or bacteria into the murine M. cremaster using a microinjection technique in order to induce target-oriented leukocyte migration. Using in vivo near-infrared reflected-light oblique transillumination (RLOT) microscopy, we analyzed leukocyte adhesion, transmigration, and interstitial migration upon microinjection with relevant chemoattractants including MIP-1a, PAF, or fluorescent-labeled E. coli. We combined in vivo RLOT and fluorescence microscopy in order to evaluate migration patterns of neutrophils and monocytes in Cx3CR1gfp/gfp mice
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