Objective: To determine whether leukocytes show a preference to firmly adhere to the endothelium near the endothelial cell (EC) junction in their native environment, i.e., blood-perfused venules in situ.

Methods: Intravital confocal microscopy was used to determine the positions of firmly adherent leukocytes with respect to EC junctions in cremaster muscle venules in anesthetized mice. EC area and EC junction locations were identified using immunofluorescent labeling of platelet-endothelial cell adhesion molecule-1 (PECAM-1), an endothelial junction protein. Leukocytes were identified using transillumination through the same optical path.

Results: Seventy-five percent of firmly adherent leukocytes overlapped an EC junction (the average distance to the nearest EC junction was 2.4 +/- 0.1 micro m, n = 263). This distance was less in smaller diameter venules, hence the percentage of adherent leukocytes that overlapped an endothelial junction was larger. EC shape and size varied with venular diameter: in smaller venules (30-49 micro m diameter), ECs had significantly less area (316 +/- 19 micro m(2)) and were narrower (12.5 +/- 0.4 micro m) than in larger (70-89 micro m diameter) venules, which had 614 +/- 28 micro m(2) area and 17.8 +/- 0.5 micro m width, respectively (p < .001). Furthermore, the majority (73.0%) of firmly adherent leukocytes crawled an average distance of 29.4 +/- 2.8 micro m at a mean intralumenal migration velocity of 7.6 +/- 0.4 micro m/min before undergoing transmigration or detaching and reentering the free fluid stream.

Conclusions: Most adherent leukocytes in blood-perfused venules are located at or near EC junctions. This is primarily due to the size and shape of venular ECs. Further, most leukocytes that appear to be adherent are in fact motile, and migrate a significant distance along the lumenal wall. The authors speculate that this reflects movement to those EC junctional regions that support transendothelial migration.