Augmentation of iNOS expression in myeloid progenitor cells expedites neutrophil differentiation

Abstract

Neutrophils play important role in immunity and inflammation through diverse mechanisms. Reports from this lab and others have demonstrated involvement of NO in neutrophil adhesion, chemotaxis, bacterial killing, reactive oxygen species generation, neutrophil extracellular traps’ formation, and apoptosis. Constitutive expression of iNOS in human neutrophils has also been documented. The role of NO‐iNOS in neutrophil differentiation however remains ill‐defined. The present study was undertaken to understand the role of NO generated from iNOS in the neutrophil differentiation by using iNOS‐overexpressing K562 cells (K562iNOS) and iNOS‐deficient murine progenitor cells (lineage negative cells; lin‐ve). We observed that iNOS overexpression led to increased neutrophilic differentiation in K562 cells; more specifically an early and accelerated neutrophilic differentiation was spotted in K562iNOS. These observations were further validated using iNOS knockout lin‐ve cells or hematopoietic progenitor cells that exhibited delayed neutrophil differentiation in comparison to its wild‐type counterpart. In addition, a significant increase in the gene expression of iNOS during neutrophilic differentiation of CD34+ hematopoietic stem and progenitor cells derived from human bone marrow further substantiates importance of iNOS in neutrophil differentiation. Moreover, a significant increase in NO generation during neutrophil differentiation was observed and enhanced neutrophil differentiation with NO donor was also observed, implying the importance of NO in neutrophil differentiation. Collectively, using alternative approaches, we demonstrated that neutrophil differentiation is significantly influenced by iNOS or NO, suggesting the possibility of exploiting this novel link for therapeutic aspects of NO generated from iNOS and neutrophil differentiation in hematopoiesis‐related disorders.

Expression of inducible NOS is indispensable for the antiproliferative and proapoptotic effect of imatinib in BCR–ABL positive cells

“…In a recent study, we have demonstrated that the expression of both iNOS transcript and protein is significantly less in PMNs from newly diagnosed CML patients, 17 whereas its overexpression expedites neutrophilic differentiation of CML cells 27 . To further identify its significance in BCR–ABL leukemogenesis, we examined the effect of imatinib, a BCR–ABL kinase inhibitor on NO generation and iNOS expression.…”

“…3A–3D). Since, PMNs from imatinib responders are more mature than those from drug naive and imatinib nonresponders, and it has been shown that the expression of iNOS was augmented with maturity of neutrophils, 27 we next assessed iNOS expression in neutrophils progenitors and mature cells obtained from drug naive and imatinib nonresponders CML patients. Reduced nitrite level and iNOS expression were evident in the progenitors and mature differentiated PMNs of drug naive and imatinib‐resistant CML patients and no significant differences were noted among the cells exhibiting various stage of neutrophils differentiation (Supplemental Figs.…”

“…Stably transfected neomycin resistance positive clones were selected 48 h after transfection by addition of 250 μg/ml G418 (Sigma). The concentration of G418 was increased at every 48 h up to 550 μg/ml for the cell selection over 3 weeks 26,27 …”

Systemic Insulin Resistance and Metabolic Perturbations in Chow Fed Inducible Nitric Oxide Synthase Knockout Male Mice: Partial Reversal by Nitrite Supplementation

“…The animals were sacrificed to retrieve the tissues (liver, epididymal white adipose tissues, and hind limb skeletal muscle). Total nitrite (nitrate and nitrite) was estimated in serum (100 μL) and tissues (liver, eWAT, and skeletal muscle, 50 mg) using Griess reagent by reducing nitrate to nitrite using pre-activated cadmium pellets followed by deproteinization in tissue homogenates with 3% trichloroacetic acid [ 46 ].…”

“…Quantitative densitometry was performed using Image J software for Windows 64bit (NIH, Bethesda, MA, USA). Real time PCR was performed as described previously [ 46 ] with primers listed in Table S2 and normalized with 18S rRNA.…”