Direct observation and quantitative analysis of spatiotemporal dynamics of individual living monocytes during transendothelial migration.
OBJECTIVE
To visualize and quantitatively analyze spatiotemporal dynamics of individual living monocytes during transendothelial migration (TEM).


RESULTS
We developed an in vitro new experimental system using confocal laser scanning microscope with following two improvements: (1) ultra thin collagen gel layer (30-50 microm thick) constructed under human umbilical vein endothelial cell layer for three-dimensional observation with high magnification; (2) appropriate fluorescent labeling of living monocytes and endothelial cells to keep highest cell activity. Individual monocytes behaved quite diversely. Approximately 70% of adhered monocytes directionally crawled to intercellular junction, and started invasion. Time from adhesion to start of invasion was 8.6 +/- 5.4 min (mean +/- S.D., n=61 monocytes). Approximately 80% of such invading monocytes completed TEM, but remaining 20% of once invading monocytes hesitated transmigration, and returned onto the endothelial surface. Time from start to finish of invasion was 6.3 +/- 3.2 min (mean +/- S.D., n=53 monocytes).


CONCLUSIONS
Using our collagen gel-based newly-developed system, we visualized and quantitatively analyzed detailed spatiotemporal, three-dimensional dynamics of individual living monocytes during TEM. We revealed that monocytes encountered at least two hurdles, at starting invasion, and leaving endothelium, to achieve complete TEM. Approximately 56% (80% of 70% of adhered monocytes) passed both hurdles.
Department of Physiology, Kawasaki Medical School, 577 Matsushima, Kurashiki, Okayama 701-0192, Japan. khashimo@med.kawasaki-m.ac.jp
Ken
K
Hashimoto
Noriyuki
N
Kataoka
Emi
E
Nakamura
Hiroko
H
Asahara
Yasuo
Y
Ogasawara
Katsuhiko
K
Tsujioka
Fumihiko
F
Kajiya
D002465:Cell Movement; D002478:Cells, Cultured; D004730:Endothelium, Vascular / Q000166:cytology; D006801:Humans; D009000:Monocytes / Q000166:cytology*