Nitric oxide synthesis in neutrophil precursor cells and effect of nitric oxide modulators on their maturation

Abstract

Neutrophil maturation in bone marrow and their release in circulation have been shown to be modulated by nitric oxide (NO). Present study was undertaken to further assess the role of NO in neutrophil maturation in vitro. Neutrophil precursor cells were isolated from Sprague Dawley rat (200-250 g) bone marrow by Percoll density gradient in myeloblasts/promyelocytes rich band 3, myelocytes/metamyelocytes rich band 2 and band cells/segmented Neutrophils rich band 1, purity was confirmed by Giemsa staining/CD markers, NADPH oxidase and myeloperoxidase activity. Intracellular NO level (assessed by DAF-2DA), total nitrite (measured by Griess reagent) content, NOS activity (using ³H-L-arginine) and nNOS protein (Western blotting) were highest in band 1 and lowest in band 3. We also investigated effect of NO donor (SNP 1µM-1mM) and NOS inhibitor (L-NAME 1mM) on cell cycle by using Propidium iodide and BrdU staining during their culture for 24 hrs. SNP treated cells exhibited an increase in apoptosis, while viable cells showed a marginal increase in the S phase in band 2 and band 3. NOS inhibitor (L-NAME) however increased the survival of cells without any modulation of cell cycle. Results obtained indicate that NO content augment with neutrophil maturation and seems to play an important role in the maturation of neutrophils.