Unexpected Evidence That Inducible Nitric Oxide Synthase (iNOS) Is a Negative Regulator of Hematopoietic Stem/Progenitor Cell Mobilization

Background. Nitric oxide (NO) is gaseous free radical molecule involved in several biological processes related to inflammation, tissue damage, and infections. NO is synthesized by three isoforms of nitric oxide synthetase: two constitutively expressed isoforms, neuronal NOS (nNOS) and endothelial NOS (eNOS), and one isoform (iNOS) that is induced in response to inflammation. Interestingly, iNOS activity is enhanced during complement cascade (ComC) activation, which is a crucial element of innate immunity, and its cleavage fragments C3 and C5 (C3a and C5a, respectively) orchestrate the egress of HSPCs from BM into peripheral blood (PB). Mobilization studies in eNOS^-/- mice revealed that the lack of constitutively expressed eNOS in endothelium reduces vascular endothelial growth factor (VEGF)-induced mobilization of endothelial progenitor cells (EPCs) and increases murine mortality after myelosuppression (Nature Med. 2003, 9:1370-6). However, no mobilization studies have been performed so far to assess the role of inducible hematopoietic cell-expressed iNOS in the mobilization of HSPCs. Because of this research void, we became interested in the potential role of iNOS in mobilization of HSPCs. This interest had two motivations. First, it was demonstrated in a recent paper that NO inhibits migration of granulocytes and monocytes, and it is known that these cells are crucial in facilitating egress of HSPCs across the bone marrow (BM)-peripheral blood (PB) barrier. Second, NO, which promotes inflammation, may have anti-inflammatory activity, and inflammation itself is one of the driving forces in HSPC mobilization. Aim of the study. To fill in this knowledge gap, we focused on the role of iNOS in the trafficking of hematopoietic cells as well as mobilization and homing of HSPCs. Materials and Methods. iNOS was upregulated or downregulated in established hematopoietic cell lines to study migration and adhesion of these cells. HSPC mobilization studies were performed in iNOS^-/- mice, and, in parallel, homing and engraftment of iNOS^-/- BM cells was evaluated in wild type animals. Since activation of the ComC is negatively regulated by heme oxygenase 1 (HO-1), and the ComC may downregulate HO-1, we tested the effect of HO-1 on iNOS-mediated cell trafficking. Results. Our results indicate that iNOS is a negative regulator of hematopoietic cell migration and prevents egress of HSPCs into PB during the mobilization process. At the molecular level, downregulation of iNOS resulted in downregulation of HO-1, and, vice versa, upregulation of iNOS enhanced HO-1 activity. Since, as we observed recently, HO-1 is a negative regulator of cell migration, the inhibitory effects of iNOS on the mobilization of HSPCs can be at least partially explained by its enhancing the HO-1 level in BM cells. Moreover, our studies, in which we generated hematopoietic chimeras, revealed that the iNOS effect depends on its expression in hematopoietic cells. Conclusions. We identified iNOS as a negative regulator of cell trafficking and mobilization of HSPCs. Further studies are needed to establish the mutual relationship between activation of iNOS and activation of HO-1 in hematopoietic cells, as both inducible enzymes are potent negative regulators of HSPC trafficking and together and regulate stem cell mobilization and homing. Therefore, inhibition of iNOS or HO-1 in HSPCs by employing small molecular inhibitors could find practical clinical application.