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.
Khandoga
Alexander Georg
AG
Walter Brendel Centre of Experimental Medicine, Ludwig-Maximilians-Universität München, Munich, Germany.
Khandoga
Andrej
A
Reichel
Christoph Andreas
CA
Bihari
Peter
P
Rehberg
Markus
M
Krombach
Fritz
F
D000577:Amides / Q000494:pharmacology; D000818:Animals; D002448:Cell Adhesion; D054405:Chemokine CCL3 / Q000008:administration & dosage; D002634:Chemotaxis, Leukocyte / Q000187:drug effects; D007962:Leukocytes / Q000166:cytology*; D051379:Mice; D008853:Microscopy / Q000379:methods; D011725:Pyridines / Q000494:pharmacology
D000577:Amides; D054405:Chemokine CCL3; D011725:Pyridines; C108830:Y 27632