Ryschich Eduard, Kerkadze Vachtang, Lizdenis Paulius, Paskauskas Saulius, Knaebel Hanns-Peter, Gross Wolfgang, Gebhard Martha Maria, Büchler Markus W, Schmidt Jan
Department of Surgery, University of Heidelberg, Heidelberg, Germany.
J Surg Res. 2006 Oct;135(2):291-6. doi: 10.1016/j.jss.2006.02.020. Epub 2006 Apr 21.
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.
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 (anti-CD18 antibody) adhesion molecules. Additionally, the migratory activity of isolated leukocytes in collagen gel was analyzed and compared with their intravascular locomotion.
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 mum/min that was not significantly different from crawling velocity of extravascular leukocytes (8.9 +/- 4.5 mum/min). The maximum distance of leukocyte crawling observed was 150 microm. The maximum time of crawling was 15 min. Intraluminal crawlers traveled over a mean distance of 35 +/- 17 mum 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.
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.
主动运动能力是白细胞的一个重要特征。在此，我们使用一种结合了活体显微镜检查和数字延时视频显微镜检查的混合技术，来研究血管内白细胞运动的生理学和分子机制。
在雄性Wistar大鼠中，使用数字视频记录和延时图像压缩技术对肠系膜小静脉进行活体显微镜检查。在局部应用肿瘤坏死因子-α（TNF-α）后，以及在内皮细胞（抗细胞间黏附分子-1抗体）和白细胞（抗CD18抗体）黏附分子被阻断后，分析白细胞的运动和渗出情况。此外，分析并比较分离的白细胞在胶原凝胶中的迁移活性与其在血管内的运动情况。
黏附的白细胞沿着内皮细胞层呈现出活跃的管腔内爬行。大多数永久性黏附细胞（84±13%）在小静脉的管腔内部位积极爬行。白细胞爬行速度的基线测量结果显示，平均速度为9.0±1.8μm/分钟，这与血管外白细胞的爬行速度（8.9±4.5μm/分钟）没有显著差异。观察到的白细胞爬行的最大距离为150微米。最大爬行时间为15分钟。管腔内的爬行细胞平均移动距离为35±17μm，平均持续时间为5.4±1.4分钟。在未受刺激的条件下，几乎所有爬行的白细胞都从内皮细胞上脱离，并且没有穿过血管壁迁移。TNF-α导致白细胞渗出显著增加。抗细胞间黏附分子-1和抗CD18抗体显著降低白细胞的爬行。分离的白细胞在胶原凝胶中的迁移比例（87%±6%）与体内血管内爬行白细胞的百分比没有显著差异。
数字延时活体显微镜检查方法是研究白细胞在血管内、跨内皮和血管外迁移的一种有利技术。使用该技术，我们表明白细胞与内皮细胞的相互作用是一个活跃且动态的过程。这个过程涉及白细胞沿着内皮细胞长时间（几分钟）的爬行，最终从内皮细胞上脱离。血管内白细胞的爬行反映了循环白细胞的迁移潜力，并且强烈依赖于黏附分子的表达。对于渗出，还需要额外的促炎刺激。
