FLOW CYTOMETRIC PROCEDURE TO DETECT APOPTOSIS OF BOVINE POLYMORPHONUCLEAR LEUKOCYTES IN WHOLE BLOOD

Authors: OOSTVELDT, K; DOSOGNE, H; BURVENICH, C; Paape, Max; BROCHEZ, V; EECKHOUT, E

Submitted to: Veterinary Immunology and Immunopathology
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 5/5/1999
Publication Date: N/A
Citation: N/A

Interpretive Summary: Currently there is much interest and excitement in the understanding of how cells undergo the process of apoptosis or programmed cell death. Scientists in the Immunology and Disease Resistance Laboratory, Beltsville, teamed up with scientists at the University of Ghent, Belgium and developed a procedure for measuring apoptosis of bovine neutrophils, a specialized first-line-of-defense white blood cell. Neutrophils migrate from the bloodstream directly into the mammary gland in response to bacterial invasion. Once contact is made with the bacterial horde, these cells release chemicals, including potent enzymes. They also combat the bacteria directly. But for the neutrophils it is a kamikaze exercise. After releasing their chemicals nearly all the neutrophils perish. Understanding how, why and when neutrophils are instructed to die may provide insight into the ability of these cells to protect the mammary gland of cows from getting mastitis.

Technical Abstract: A flow cytometric technique was developed to detect apoptosis and necrosis of bovine polymorphonuclear neutrophil leukocytes (PMN) using fluorescein isothiocanate labeled annexin-V and propidium iodide. Isolation of PMN from the blood resulted in false positive results for apoptosis. Therefore, a new method wass developed to identify living, apoptotic and necrotic PMN simultaneously in a single 100 ul whole bovine blood sample. In order to establish a positive control for bovine PMN apoptosis, the effect of several compounds that induce apoptosis by various mechanisms was tested. Only actinomycin D induced a significant increase of the percentage apoptotic PMN after 2 hours. Incubation of whole blood for 6 hours with cycloheximide, actinomycin D and buthionine sulfoximine resulted in a significant increase of apoptotic PMN compared to control values. With sodium arsenate, mainly necrosis could be detected after 6 hours of incubation. These flow cytometric results were confirmed by microscopic evaluation. Incubation of whole blood for 6 hours with Et-18-OCH3 induced annexin-V positive cells by effects on the lasma membrane but apoptosis was not observed by light microscopy. Therefore, it is recommended to confirm flow cytometric results of the described technique with additional (light microscopic) evaluation. Actinomycin D was demonstrated to be the most potent apoptosis inducing molecule and is recommended for use in further experiments as a positive control.