Micro-PTV measurement of the fluid shear stress acting on adherent leukocytes in vivo.

Leukocyte adhesion is determined by the balance between molecular adhesive forces and convective dispersive forces. A key parameter influencing leukocyte adhesion is the shear stress acting on the leukocyte. This measure is indispensable for determining the molecular bond forces and estimating cell deformation. To experimentally determine this shear stress, we used microparticle tracking velocimetry analyzing more than 24,000 images of 0.5 microm fluorescent microbeads flowing within mildly inflamed postcapillary venules of the cremaster muscle in vivo. Green fluorescent protein, expressed under the lysozyme-M promoter, made leukocytes visible. After applying stringent quality criteria, 3 of 69 recordings were fully analyzed. We show that endothelial cells, but not leukocytes, are covered by a significant surface layer. The wall shear rate is nearly zero near the adherent arc of each leukocyte and reaches a maximum at the apex. This peak shear rate is 2-6-fold higher than the wall shear rate in the absence of a leukocyte. Microbead trajectories show a systematic deviation toward and away from the microvessel axis upstream and downstream from the leukocyte, respectively. The flow field around adherent leukocytes in vivo allows more accurate estimates of bond forces in rolling and adherent leukocytes and improved modeling studies.

Event-tracking model of adhesion identifies load-bearing bonds in rolling leukocytes.

OBJECTIVES: P-selectin binding to P-selectin glycoprotein ligand-1 (PSGL)-1 mediates leukocyte rolling under conditions of inflammation and injury. The aims of this study were to develop an efficient, high temporal resolution model for direct simulation of leukocyte rolling and conduct a study of load-bearing bonds using the model. MATERIALS AND METHODS: A stochastic pi-calculus-driven event-tracking model of adhesion (ETMA) was developed and compared with experimental data. Multiple simulations for each case were conducted to obtain high-confidence numerical characteristics of leukocyte rolling. RESULTS: Leukocyte rolling and the underlying P-selectin-PSGL-1 bonds were studied under low wall shear rate (25-50 s(-1)) conditions from measured parameters of leukocyte rolling and bond properties. For the first time, the location, number, lifetime, history, and kinetics of load-bearing bonds and their influence on cell rolling were identified and instantaneous cell displacements, translational and rotational velocities, and cell-substrate distances derived. The model explains the commonly observed "stop-start" type rolling behavior and reveals that a few load-bearing bonds are sufficient to support rolling, while a large number of bonds dissociate before becoming load bearing. CONCLUSIONS: ETMA provides a method for more precise, direct simulation of leukocyte rolling at low wall shear rates and sets a foundation upon which further refinements can be introduced.