Immunocytochemical Localization of Neuronal Nitric Oxide Synthase (nNOS) in Neutrophils: Hydroxyurea Therapy Induced Nucleo-Cytoplasmic Translocation in Sickle Cell Disease

Sickle cell disease (SCD) is an inherited hemoglobinopathy leading to the deformability of red blood cells, enhanced cellular adhesion, and blockage in microcirculation. “Sickle Neutrophils” cause oxidative stress and decreased bioavailability of nitric oxide (NO) due to enhanced generation of reactive oxygen species (ROS). The mechanism of action of the common medication of SCD, hydroxyurea (HU), has not yet been thoroughly examined. This investigation examines the subcellular localization of neuronal nitric oxide synthase (nNOS) using immunoelectron microscopy, and confocal microscopy, as well as NO production and ROS generation by flow cytometry in neutrophils from steady-state SCD patients, and SCD patients on HU therapy. nNOS was increased and prominently distributed in the cytoplasmic compartment, with no nNOS staining in the nucleus of neutrophils in SCD-HU patients. ROS was found to be increased in SCD patients and was significantly reduced after HU treatment. An increase in NO production and decrease in ROS are linked to nNOS’s nucleo-cytoplasmic shuttling in SCD neutrophils after HU therapy that provides a novel cellular mechanism through which HU regulates neutrophil function. In conclusion, this study establishes for the first time that HU therapy induces a favorable nucleo-cytoplasmic translocation of nNOS in SCD neutrophils, indicating a key mechanism behind the therapeutic effectiveness of HU in SCD.