Stimulus-dependent actin polymerization in bovine neutrophils

Abstract

Polymorphonuclear leukocytes (PMNs) are responsible for much of the first wave of leukocyte-mediated host defense against microbial pathogens. In order to migrate through the endothelium of vessel walls, undergo chemotaxis, and phagocytize microbes, PMNs must modulate their cytoskeletal elements and undergo change of cellular shape. We have used fluorescence flow cytometric analysis and cellular microscopic observations to demonstrate actin polymerization in bovine PMNs and to examine the kinetics of PMN actin polymerization utilizing different PMN stimuli. In addition, we compared temporal relationships between cellular shape and actin polymerization. Actin polymerization occurred rapidly, and the kinetics of actin polymerization were similar for each of the three PMN agonists used, ZAS (10%), PAF (10⁻⁶ M), and rhC5a (10⁻⁷ M). Actin polymerization was near-maximal by 10 sec poststimulation (95.4% of maximal F-actin content attained by 10 sec poststimulation with ZAS stimulation), and reached peak values by 30 sec. The maximal increase in F-actin content of agonist-stimulated cells as compared to resting cells was 2.8-fold with ZAS; 2.3-fold with PAF; and 2.3-fold with rhC5a. PMN shape change (pseudopodia, membrane raffles) was not as rapid, with only 22.4% of cells attaining visible membrane deformation by 10 sec and requiring 120 sec to reach peak shape-change values. After attaining peak values, the two events also differed. Whereas the percent of shape-changed PMNs remained plateaued up to 5 min poststimulation, the F-actin content gradually decreased after 30 sec, approaching F-actin values of unstimulated PMNs.