High availability of NO, oxidative stress and neutrophil extracellular trap (NETs) contents are often noticed at the site of inflammation/infection. Studies from this lab and others have reported NO mediated free radical generation from neutrophils; role of NO in NETs formation however remains undefined so far. The present study was therefore undertaken to explore the effect of NO donors on NET release from human neutrophils (PMNs), using confocal/scanning microscopy, measuring the extracellular DNA content and NET-bound elastase activity. Addition of NO donors (SNAP and SNP) to adhered PMNs led to a time and concentration dependent NETs release, which was blocked by N-acetyl cysteine, suggesting involvement of free radicals in NETs formation. Free radical formation by NO donors was assessed by using DCF-DA, DMPO-nitrone antibody and by p47 phox migration to the neutrophils membrane. NO mediated formation of free radicals and NETs was significantly reduced by the pretreatment of neutrophils with diphenyleneiodonium (DPI), a NADPH-oxidase inhibitor and 4-aminobenzoic acid hydrazide (ABAH), a myeloperoxidase inhibitor, suggesting role of enzymatic free radical generation by NO donors. We thus demonstrate that NO by augmenting free radical formation in human neutrophils mediates NETs release.

Nitric oxide synthase (NOS) expression and catalytic status in human peripheral blood mononuclear cells (PBMCs) is debatable, while its sub-cellular distribution remains unascertained. The present study characterizes NOS transcripts by real time PCR, NOS protein by immunoprecipitation (IP)/Western blot (WB), nitric oxide (NO) generation by DAF-2DA and NOS sub-cellular distribution by immunogold electron microscopy in resting PBMCs, monocytes and lymphocytes obtained from healthy donors. We observed constitutive expression of full length NOS isoforms (nNOS, iNOS and eNOS) in PBMCs: with the highest expression of iNOS in comparison to nNOS and eNOS. Isolated monocytes expressed more eNOS transcript and protein as compared to nNOS and iNOS. Lymphocytes however had more iNOS transcripts and protein than nNOS and eNOS. NOS was catalytically active in PBMCs, monocytes as well as in lymphocytes as evident by NO generation in the presence of substrate and cofactors, which was significantly reduced in the presence of NOS inhibitor. Immunogold electron microscopy and morphometric analysis revealed the distinct pattern of NOS distribution in monocytes and lymphocytes and also exhibited differences in the nuclear-cytoplasmic ratio. nNOS localization was much more in the cytosol than in the nucleus among both monocytes and lymphocytes. Interestingly, iNOS distribution was comparable in both cytosol and nucleus among monocytes, but in lymphocytes iNOS was predominantly localized to the cytosol. The present study exhibits constitutive presence of all the NOS isoforms in PBMCs and reports the distinct pattern of NOS distribution among monocytes and lymphocytes.

Nitric oxide (NO), a multifaceted signaling molecule, regulates a wide array of cell functions, including proliferation, differentiation, cytostasis, and apoptosis, which depend on the cell type and redox status. This study systematically explores the effects of NO donors on promyelocytic HL-60 cell proliferation and apoptosis. The NO donor DETA-NO modulated the HL-60 cell cycle in a biphasic manner. DETA-NO in lower concentrations (1-100 microM) had a proliferative effect as investigated by [(3)H]thymidine incorporation, BrdU labeling, and cell cycle analysis, whereas cells treated with higher concentrations (250 microM-1 mM) showed cytostasis, apoptosis, mitochondrial membrane potential loss, caspase-3 activity, and dUTP nick-end labeling. The proliferative effect of DETA-NO was NO dependent and redox sensitive, as the effect was abolished by cPTIO and DTT pretreatment, respectively. Expression of various cell cycle regulators such as Cdk2, cyclin B, and cyclin E was significantly augmented in cells treated with 10-50 microM DETA-NO. The proliferative effect of NO was blocked by roscovitine, a Cdk2 inhibitor. S-nitrosylation of Cdk2 and an increase in the Cdk2-associated kinase activity was observed for the first time in DETA-NO-treated cells. This study demonstrates that the DETA-NO-mediated biphasic effect was dependent on Cdk2 nitrosylation/activation and the loss of mitochondrial potential at low and high concentrations, respectively.