Spontaneous Apoptosis of Neutrophils in Whole Blood and its Relation to Apoptosis Gene Proteins

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Abstract
Apoptosis of neutrophils limits their pro‐inflammatory potential. We tested the ability of fresh and cultured whole blood neutrophils to undergo spontaneous apoptosis and expression of p53, Fas/Apo‐1, bcl‐2 protein in the cells using flow cytometry. Neutrophil apoptosis was estimated using Annexin V and propidium iodide binding and verified under light microscopy. The percentage of early and late apoptotic neutrophils in the blood samples increased significantly after 20 h culture from 12.3 ± 14.2% and 4.3 ± 4.2% to 39.5 ± 14% and 15.3 ± 9.6%, respectively. The majority of late apoptotic neutrophils had altered morphology in FSC/SSC dot plot compared to alive or early apoptotic neutrophils. Cultured neutrophils presented markedly lower expression of bcl‐2 protein compared to fresh blood cells: 211 ± 321 median of fluorescence intensity (MFI) and 787 ± 1152 MFI, respectively. The increased percentage of late apoptotic cells after culture paralleled the increase in the Fas/Apo‐1 expression and negatively correlated with bcl‐2 expression. We noted intracellular expression of p53 protein in neutrophils, although the expression did not correlate neither to the percentage of the apoptotic neutrophils, nor to the Fas/Apo‐1 or bcl‐2 expression. Our results suggested that neutrophil apoptosis is gene regulated, moreover, we present a possibility to assess the neutrophil apoptosis and cellular expression of the proteins of apoptosis related genes in whole blood samples.
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