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Stimulus-dependent actin polymerization in bovine neutrophils
P N Bochsler1, N R Neilsen, D F Dean
1Department of Pathobiology, College of Veterinary Medicine, University of Tennessee, Knoxville 37901.
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 ruffles) 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.
Insights
Polymorphonuclear leukocytes (PMNs) rapidly polymerize actin within seconds of stimulation, but cellular shape changes occur more slowly. This study quanties actin polymerization and shape change kinetics in bovine PMNs.
Area of Science:
- Immunology
- Cell Biology
Background:
- Polymorphonuclear leukocytes (PMNs) are critical for host defense against pathogens.
- PMN function, including migration and phagocytosis, requires cytoskeletal modulation and shape changes.
Purpose of the Study:
- To demonstrate and quantify actin polymerization in bovine PMNs.
- To examine the kinetics of actin polymerization and cellular shape change in response to different stimuli.
- To compare the temporal relationship between actin polymerization and shape change.
Main Methods:
- Fluorescence flow cytometry was used to analyze actin polymerization.
- Cellular microscopic observations were employed to assess PMN shape change.
- Bovine PMNs were stimulated with ZAS, PAF, and rhC5a.
Main Results:
- Actin polymerization was rapid, near-maximal by 10 seconds and peaking by 30 seconds post-stimulation.
- Maximal increases in F-actin content ranged from 2.3- to 2.8-fold compared to resting cells.
- Cellular shape change was slower, with peak values reached at 120 seconds, and differed temporally from actin polymerization.
Conclusions:
- Actin polymerization is a rapid early event in PMN activation.
- The kinetics of actin polymerization and cellular shape change are distinct.
- These findings provide insights into the dynamic cellular processes underlying PMN function.