Cone-plate viscometric studies of neutrophil adhesion to the endothelial ligands E-selectin and ICAM-1
McIntire, Larry V.
Doctor of Philosophy
Neutrophil adhesion to endothelium is prerequisite for extravasation and localization at inflammatory sites. In the vasculature this process occurs under conditions of hydrodynamic shear requiring that bonds formed between neutrophils and endothelium withstand forces imposed by flowing blood. In this work we investigated interactions of neutrophils with the endothelial ligands ICAM-1 and E-selectin. Detailed examination of the effects of hydrodynamic forces and molecular mechanisms of neutrophil adhesion to cell lines expressing these molecules was performed using cone-plate viscometry and two-color flow cytometry. The relative contributions of LFA-1 and Mac-1 to the dynamics and strength of neutrophil adhesion to ICAM-1 varied with hydrodynamic conditions, activation state, and ICAM-1 expression. Target cells expressing ∼1000 ICAM-1 sites/mum2 were captured with an efficiency of 0.15 at 100s-1. An eight-fold increase in ICAM-1 surface density doubled adhesion efficiency to ∼0.3 at low shear and increased the shear range over which adhesion occurred. The collisional contact duration required for successful capture was predicted to be ∼9ms for Ilow and ∼4ms for Ihigh. Shear alone was sufficient for LFA-1 mediated adhesion to ICAM-1 and fMLP stimulation boosted capture efficiency four-fold. Mac-1 was one-third as efficient in capture, but stabilized aggregates over several minutes of shear and at stresses exceeding 5 dyn/cm2. beta 2-integrin mediated adhesion to ICAM-1 appears to be a cooperative and sequential process of LFA-1-dependent capture followed by Mac-1-mediated stabilization. Studies with selectin transfectants indicated that tethering through E-selectin was more efficient than L-selectin in promoting beta2-integrin mediated adhesion. fMLP-stimulated neutrophils bound E-selectin at two-fold higher levels than L-selectin. Shear with E-selectin transfectants over a discrete range of shear rates (∼400 to 600s-1) induced adhesion of ∼30% of unstimulated neutrophils. This correlated with a 50% increase in Mac-1 expression within one minute. Treatment of neutrophils with a specific inhibitor of p38 MAP kinase decreased both of these processes to background levels, implicating a p38 pathway in transduction of intracellular signals induced by E-selectin binding. Together these studies provide evidence that neutrophil binding to endothelial ligands functions optimally under discrete hydrodynamic conditions, providing an intrinsic mechanism regulating the transition from transient tethering to stable but reversible adhesion.
Biomedical engineering; Chemical engineering; Computer science