Sequential contribution of L- and P-selectin to leukocyte rolling in vivo.

Abstract

Leukocyte recruitment into inflammatory sites is initiated by a reversible transient adhesive contact with the endothelium called leukocyte rolling, which is thought to be mediated by the selectin family of adhesion molecules. Selectin-mediated rolling precedes inflammatory cell emigration, which is significantly impaired in both P- and L-selectin gene-deficient mice. We report here that approximately 13% of all leukocytes passing venules of the cremaster muscle of wild- type mice roll along the endothelium at < 20 min after surgical dissection. Rolling leukocyte flux fraction reaches a maximum of 28% at 40-60 min and returns to 13% at 80-120 min. In P-selectin-deficient mice, rolling is absent initially and reaches 5% at 80-120 min. Rolling flux fraction in L-selectin-deficient mice is similar to wild type initially and declines to 5% at 80-120 min. In both wild-type and L- selectin-deficient mice, initial leukocyte rolling (0-60 min) is completely blocked by the P-selectin monoclonal antibody (mAb) RB40.34, but unaffected by L-selectin mAb MEL-14. Conversely, rolling at later time points (60-120 min) is inhibited by mAb MEL-14 but not by mAb RB40.34. After treatment with tumor necrosis factor (TNF)-alpha for 2 h, approximately 24% of all passing leukocytes roll in cremaster venules of wild-type and P-selectin gene-deficient mice. Rolling in TNF- alpha-treated mice is unaffected by P-selectin mAb or E-selectin mAb 10E9.6. By contrast, rolling in TNF-alpha-treated P-selectin-deficient mice is completely blocked by L-selectin mAb. These data show that P- selectin is important during the initial induction of leukocyte rolling after tissue trauma. At later time points and in TNF-alpha-treated preparations, rolling is largely L-selectin dependent. Under the conditions tested, we are unable to find evidence for involvement of E- selectin in leukocyte rolling in mice.

Leucocyte interactions with the mouse cremaster muscle microcirculation in vivo in response to tumour-conditioned medium

“…However, in vivo studies have demonstrated a role for P-and Lselectin but not E-selectin in mouse leucocyte rolling, during trauma and cytokine stimulation (Ley et al, 1995). Furthermore, tumours are known to produce cytokines such as interleukin 1 (IL-1) and TNF-a.…”

Trafficking of activated lymphocytes into the RENCA tumour microcirculation in vivo in mice

“…This may be due to the disorganized flow characteristics of the tumour enabling firm adhesion without initial rolling. The adhesion molecules P-selectin, E-selectin, L-selectin (CD62P and L) and VCAM-1 are all involved in regulating in vivo leucocyte rolling and adhesion (Ley et al, 1995). As rolling of effector cells was not observed in our studies, it is possible that there is down-regulation of adhesion molecules on the tumour endothelium, and this is currently being investigated in our model.…”

Deformable gas-filled microbubbles targeted to P-selectin

“…Capture efficiencies of non-deformable microbeads targeted with physiological capture ligands on purified selectin substrates are on the order of ∼ 10-35% [20,47] under similar conditions. Leukocytes roll on cytokine-stimulated endothelium with an efficiency of ∼20-30% in mouse venules [38], although the in vivo situation presents a substantially different target geometry and flow conditions. Although the wrinkled microbubbles described here achieved a degree of deformation similar to activated leukocytes [18,43], our results suggest that deformation alone does not necessarily compensate for inefficient binding biomechanics.…”

“…Trachea intubation to promote spontaneous respiration was accomplished with PE 90 tubing, and microbubbles were introduced into the right jugular vein through a PE 20 cannula. A cremaster muscle was exteriorized and continuously superfused with an isothermic bicarbonate-buffered solution as described [38]. The exteriorized cremaster was pinned to a custom-made microscope stage and placed beneath a saline immersion objective for intravital microscopy (Zeiss SW 40/0.8 NA).…”