The Multistep Process of Homotypic Neutrophil Aggregation: A Review of the Molecules and Effects of Hydrodynamics

Abstract

Homotypic adhesion of neutrophils stimulated with chemoattractant is analogous to capture on vascular endothelium in that both processes are supported by L-selectin and β₂-integrin adhesion receptors. Under hydrodynamic shear, cell adhesion requires that receptors bind sufficient ligand over the duration of intercellular contact to withstand the hydrodynamic stresses. Using cone and plate viscometry to apply a uniform linear shear field to suspensions of neutrophils and flow cytometry to quantitate the size distribution of aggregates formed over the time course of formyl peptide stimulation, we conducted a detailed examination of the affect of shear rate and shear stress on the kinetics of cell aggregation. The efficiency of aggregate formation was fit from a mathematical model based on Smoluchowski's two-body collision theory. Over a range of venular shear rates (400–800 s^-1), β90% of the single cells are recruited into aggregates ranging from doublets to groupings larger than sextuplets. Adhesion efficiency fit to the kinetics of aggregation increased with shear rate from β20% at 100s^-1 to a maximum level of β80% at 400 s^-1. This increase to peak adhesion efficiency was dependent on L-selectin and β₂-integrin, and was resistant to shear stress up to β7 dyn/cm². When L-selectin was blocked with antibody, β₂-integrin (CD11a, b) supported adhesion at low shear rates (< 400 s^-1). Aggregates formed over the rapid phase of aggregation remain intact and resistant to shear up to 120 s. At the end of this plateau phase of stability, aggregates spontaneously dissociate back to singlets. The rate of cell disaggregation is linearly proportional to the applied shear rate. The binding kinetics of selectin and integrin appear to be optimized to function within discrete ranges of shear rate and stress, providing an intrinsic mechanism for the transition from neutrophil tethering to firm but reversible adhesion.

Key Words:

• Neutrophil aggregation
• shear
• L-selectin
• β₂-integrin