Association among filamentous actin content, CD11b expression, and membrane deformability in stimulated and unstimulated bovine neutrophils

David J. McClenahan Department of Veterinary PathoBiology, College of Veterinary Medicine, University of Minnesota, St Paul, MN 55108.

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Oral A. Evanson Department of Veterinary PathoBiology, College of Veterinary Medicine, University of Minnesota, St Paul, MN 55108.

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Bruce K. Walcheck Department of Veterinary PathoBiology, College of Veterinary Medicine, University of Minnesota, St Paul, MN 55108.

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Douglas J. Weiss Department of Veterinary PathoBiology, College of Veterinary Medicine, University of Minnesota, St Paul, MN 55108.

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Abstract

Objective—To investigate rheologic properties of bovine neutrophils that may result in adhesion molecule- independent sequestration of neutrophils in inflamed lungs of cattle.

Animals—Healthy 2- to 4-week-old male Holstein calves.

Procedures—Neutrophil deformability, filamentous actin (F-actin) content, and CD11b expression was determined for unstimulated bovine neutrophils and bovine neutrophils incubated with the inflammatory mediators tumor necrosis factor-α (TNF), platelet-activating factor (PAF), interleukin-8 (IL-8), zymosan-activated plasma (ZAP), Pasteurella haemolytica-derived lipopolysaccharide (LPS), and P haemolytica leukotoxin. Neutrophils were separated into 3 subpopulations on the basis of size. The F-actin content and CD11b expression were evaluated by use of flow cytometry. Leukocyte deformability was evaluated by filtration of dilute whole blood.

Results—The subpopulation of the smallest-sized neutrophils (> 90% of neutrophils) contained little F-actin. A subpopulation of slightly larger neutrophils had a profound increase in F-actin content and CD11b expression. The subpopulation of the largest neutrophils had increased F-actin content and CD11b expression, compared with those for both subpopulations of smaller neutrophils. Incubation of neutrophils with PAF and ZAP, but not TNF, IL-8, LPS, or leukotoxin, resulted in decreased neutrophil deformability and increased F-actin content. Incubation with PAF and TNF induced an increase in size of neutrophils.

Conclusions and Clinical Relevance—Size can be used to identify subpopulations of large and rigid neutrophils in blood samples from healthy calves. Platelet-activating factor and activated complement fragments are potent inducers of F-actin formation and neutrophil rigidity. Physical changes in neutrophils may impede their transit through lung microvasculature and result in leukocyte trapping independent of adhesion molecule interactions with endothelial cells. (Am J Vet Res 2000;61:380–386)

Abstract

Objective—To investigate rheologic properties of bovine neutrophils that may result in adhesion molecule- independent sequestration of neutrophils in inflamed lungs of cattle.

Animals—Healthy 2- to 4-week-old male Holstein calves.

Procedures—Neutrophil deformability, filamentous actin (F-actin) content, and CD11b expression was determined for unstimulated bovine neutrophils and bovine neutrophils incubated with the inflammatory mediators tumor necrosis factor-α (TNF), platelet-activating factor (PAF), interleukin-8 (IL-8), zymosan-activated plasma (ZAP), Pasteurella haemolytica-derived lipopolysaccharide (LPS), and P haemolytica leukotoxin. Neutrophils were separated into 3 subpopulations on the basis of size. The F-actin content and CD11b expression were evaluated by use of flow cytometry. Leukocyte deformability was evaluated by filtration of dilute whole blood.

Results—The subpopulation of the smallest-sized neutrophils (> 90% of neutrophils) contained little F-actin. A subpopulation of slightly larger neutrophils had a profound increase in F-actin content and CD11b expression. The subpopulation of the largest neutrophils had increased F-actin content and CD11b expression, compared with those for both subpopulations of smaller neutrophils. Incubation of neutrophils with PAF and ZAP, but not TNF, IL-8, LPS, or leukotoxin, resulted in decreased neutrophil deformability and increased F-actin content. Incubation with PAF and TNF induced an increase in size of neutrophils.

Conclusions and Clinical Relevance—Size can be used to identify subpopulations of large and rigid neutrophils in blood samples from healthy calves. Platelet-activating factor and activated complement fragments are potent inducers of F-actin formation and neutrophil rigidity. Physical changes in neutrophils may impede their transit through lung microvasculature and result in leukocyte trapping independent of adhesion molecule interactions with endothelial cells. (Am J Vet Res 2000;61:380–386)

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