Active leukocyte crawling in microvessels assessed by digital time-lapse intravital microscopy

Objective. The ability of active movement is an important feature of leukocytes. Here, we used a hybrid technique that combines intravital microscopy and digital time-lapse video microscopy to investigate the physiology and molecular mechanisms of intravascular leukocyte movement. Methods. Intravital microscopy of mesenteric venules was performed in male, Wistar rats using digital video recording and time-lapse image compression. The leukocyte movement and extravasation were analyzed after local application of TNF-alpha, after blockade of endothelial (anti-ICAM-1 antibody) and leukocyte (antiCD18 antibody) adhesion molecules. Additionally, the migratory activity of isolated leukocytes in collagen gel was analyzed and compared with their intravascular locomotion. Results. Adherent leukocytes showed an active intraluminal crawling along the endothelial lining. Most permanent stickers (84 +/- 13%) crawled actively on the intraluminal site of venules. Baseline measurement of leukocyte crawling velocity yielded an average 9.0 +/- 1.8 mu m/min that was not significantly different from crawling velocity of extravascular leukocytes (8.9 +/- 4.5 mu m/min). The maximum distance of leukocyte crawling observed was 150 mu m. The maximum time of crawling was 15 min. Intraluminal crawlers traveled over a mean distance of 35 17 gm with the average duration of 5.4 +/- 1.4 min. Under unstimulated conditions, almost all crawling leukocytes detached from the endothelium and did not migrate through the vascular wall. TNF-alpha induced a significant increase of leukocyte extravasation. Anti-ICAM-1 and anti-CD18 antibodies significantly reduced leukocyte crawling. The proportion of isolated migrating leukocytes in collagen gel (87% +/- 6%) was not significantly different from the percentage of intravascular crawling leukocytes in vivo. Conclusions. The method of digital time-lapse intravital microscopy represents an advantageous technology for the investigation of intravascular, transendothelial, and extravascular migration of leukocytes. Using this technology, we showed that leukocyte-endothelial-interactions are an active and dynamic process. This process involves long-time (several minutes) crawling of leukocytes along the endothelium and, finally, detachment from the endothelium. Intravascular leukocyte crawling reflects the migratory potential of circulating leukocytes and strongly depends on the expression of adhesion molecules. For extravasation, an additional pro-inflammatory stimulus is required.