Spontaneous apoptosis of neutrophils in whole blood and its relation to apoptosis gene proteins.
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.
Majewska
E
E
Department of Pathophysiology and Clinical Immunology, Military Medical Academy, Lodz, Poland. pbuk@achilles.wam.lodz.pl
Sulowska
Z
Z
Baj
Z
Z
D017209:Apoptosis / Q000235:genetics*; D001769:Blood / Q000276:immunology*; D002478:Cells, Cultured; D005434:Flow Cytometry; D006801:Humans; D009504:Neutrophils / Q000166:cytology / Q000276:immunology*; D019253:Proto-Oncogene Proteins c-bcl-2 / Q000096:biosynthesis; D016159:Tumor Suppressor Protein p53 / Q000096:biosynthesis; D019014:fas Receptor / Q000096:biosynthesis
D019253:Proto-Oncogene Proteins c-bcl-2; D016159:Tumor Suppressor Protein p53; D019014:fas Receptor