Kinetics of granulocyte deformability following exposure to chemotactic stimuli

In order to quantitate the kinetics of granulocyte deformability changes consequent to chemotactic stimulation, transit times (TT) of neutrophils through 8-microns diameter pores were studied via a modified cell transit analyzer (CTA). Cells isolated from normal human blood were tested at 20-second intervals for 5 minutes following stimulation with N-formyl-methionyl-leucyl-phenyl-alanine (fMLP) or zymosan-activated plasma (ZAP). The effects of cytochalasin B (CB), N-ethylmaleimide (NEM), and ibuprofen were also evaluated. Salient results included: (1) greater TT with increasing fMLP concentration (0.01-100 nM) with a peak response at 40-60 seconds followed by a return to or toward control at 5 minutes; (2) longer TT at 60 seconds for at least 75% of the cells at all fMLP concentrations, yet for 1 nM at 5 minutes nearly one half had TT less than unstimulated cells; and (3) similar temporal responses during a 5-minute period to ZAP simulation, with a nonlinear relation between cell rigidity and F-actin content. CB (20 microM) and NEM (1 mM) caused an immediate 30% to 35% decrease of TT for unstimulated cells; ibuprofen (10-1000 micrograms/ml) did not affect unstimulated TT, yet significantly reduced the response to fMLP and ZAP. Cell volume, as judged by CTA pulse height, decreased following fMLP stimulation, thus indicating that cell swelling does not contribute to the longer pore transit times of activated granulocytes. Our results, combined with literature reports describing microvascular occlusion by neutrophils, strongly suggest the importance of kinetic rather than static studies of granulocyte deformability; further, they indicate the usefulness of the modified CTA for such measurements.