• Sickle RBC ROS production is mediated in part by NADPH oxidase activity.• Sickle RBC ROS production can be induced by plasma signaling molecules.Chronic inflammation has emerged as an important pathogenic mechanism in sickle cell disease (SCD). One component of this inflammatory response is oxidant stress mediated by reactive oxygen species (ROS) generated by leukocytes, endothelial cells, plasma enzymes, and sickle red blood cells (RBC). Sickle RBC ROS generation has been attributed to sickle hemoglobin auto-oxidation and Fenton chemistry reactions catalyzed by denatured heme moieties bound to the RBC membrane. In this study, we demonstrate that a significant part of ROS production in sickle cells is mediated enzymatically by NADPH oxidase, which is regulated by protein kinase C, Rac GTPase, and intracellular Ca 21 signaling within the sickle RBC. Moreover, plasma from patients with SCD and isolated cytokines, such as transforming growth factor b1 and endothelin-1, enhance RBC NADPH oxidase activity and increase ROS generation. ROS-mediated damage to RBC membrane components is known to contribute to erythrocyte rigidity and fragility in SCD. Erythrocyte ROS generation, hemolysis, vaso-occlusion, and the inflammatory response to tissue damage may therefore act in a positive-feedback loop to drive the pathophysiology of sickle cell disease. These findings suggest a novel pathogenic mechanism in SCD and may offer new therapeutic targets to counteract inflammation and RBC rigidity and fragility in SCD. (Blood. 2013;121(11):2099-2107 IntroductionVaso-occlusion and hemolysis from the rigid and concurrently fragile red blood cells (RBC) in patients with sickle cell disease (SCD) cause a variety of acute and chronic manifestations ranging from frequent and severe painful crises to stroke and chronic organ failure. Chronic inflammation has emerged as an important pathogenic mechanism in SCD, and oxidative stress is increasingly recognized as a component of this chronic inflammatory state, inducing damage to a variety of subcellular and tissue structures. 1,2Patients with SCD have decreased plasma levels of glutathione, vitamin C, and vitamin E, presumably due to consumption by increased oxidant production. [3][4][5] RBC and other cell types show evidence of lipid peroxidation and oxidative damage to structural proteins.6-8 Additionally, plasma from SCD patients has elevated levels of advanced glycation end products 9,10 and products of lipid peroxidation (F-2 isoprostanes, malonaldehyde, and 4-hydroxynonenal), [11][12][13] all of which are markers of oxidative stress. There are several postulated mechanisms for the increased oxidative stress in patients with SCD. Sickle (SS) RBC reactive oxygen species (ROS) generation has been attributed to sickle hemoglobin auto-oxidation and iron-mediated Fenton chemistry reactions catalyzed by denatured heme moieties bound to the RBC membrane.14 Plasma hemoglobin and free heme resulting from chronic hemolysis generate superoxide radicals via the same nonenzymatic mechanisms. 15 In...

To understand the role of cytoskeleton and membrane signaling molecules in erythroblast enucleation, we developed a novel analysis protocol of multiparameter high-speed cell imaging in flow. This protocol enabled us to observe F-actin and phosphorylated myosin regulatory light chain (pMRLC) assembled into a contractile actomyosin ring (CAR) between nascent reticulocyte and nucleus, in a population of enucleating erythroblasts. CAR formation and subsequent enucleation were not affected in murine erythroblasts with genetic deletion of Rac1 and Rac2 GTPases because of compensation by Rac3. Pharmacologic inhibition or genetic deletion of all Rac GTPases altered the distribution of Factin and pMRLC and inhibited enucleation. Erythroblasts treated with NSC23766, cytochalasin-D, colchicine, ML7, or filipin that inhibited Rac activity, actin or tubulin polymerization, MRLC phosphorylation, or lipid raft assembly, respectively, exhibited decreased enucleation efficiency, as quantified by flow cytometry. As assessed by high-speed flow-imaging analysis, colchicine inhibited erythroblast polarization, implicating microtubules during the preparatory stage of enucleation, whereas NSC23766 led to absence of lipid raft assembly in the reticulocyte-pyrenocyte border. In conclusion, enucleation is a multistep process that resembles cytokinesis, requiring establishment of cell polarity through microtubule function, followed by formation of a contractile actomyosin ring, and coalescence of lipid rafts between reticulocyte and pyrenocyte. (Blood. 2012;119(25): 6118-6127) IntroductionErythropoiesis in mammals concludes with the dynamic process of enucleation, by which the orthochromatic erythroblast generates a reticulocyte that will mature to become a red blood cell, and a pyrenocyte, a membrane-encased nucleus surrounded by a thin rim of cytoplasm. [1][2][3] After enucleation, the reticulocytes are released into the bloodstream, whereas the pyrenocytes expose apoptotic signals on their surface, resulting in engulfment and degradation by the central macrophage of the erythroblastic island. 3,4 The mechanism of enucleation has been a long-standing matter of investigation and remains controversial.Early electron microscopy studies suggested that enucleation may be analogous to cytokinesis, pointing to the resemblance of the cytoplasmic constriction between incipient reticulocyte and pyrenocyte to the cleavage furrow at the equatorial region of a mitotic cell. 5 Koury et al demonstrated with electron and immunofluorescent microscopy, using mouse splenocytes infected with the anemia-inducing strain of Friend virus, that F-actin bundles concentrate at the furrow behind the extruding nucleus, and that cytochalasin-D, a potent inhibitor of actin polymerization, inhibits enucleation. 6 In parallel, a quantitative study by Chasis et al showed that inhibition of microtubule polymerization by colchicine stalls enucleation in vivo and in vitro in rat bone marrow. 7 A recent study by Keerthivasan et al using primary mouse and human erythroblasts...

We assessed the effect of epinephrine on human monocytes. Monocytes were isolated from 16 healthy obese and 10 lean healthy subjects. Insulin sensitivity was assessed by euglycemic hyperinsulinemic clamp. Obese subjects were subdivided into 2 sub-groups, insulin sensitive (IS) and insulin resistant (IR). Monocyte properties [attachment to laminin 1, migration through laminin 1, oxidized-low density lipoprotein (oxLDL) phagocytosis] were assessed pre- and post-stimulation in vitro with epinephrine. Experiments were repeated after incubation with a Na(+)/H( +) exchanger-1 inhibitor (NHE-1) (cariporide). Epinephrine increased monocyte attachment to laminin in lean and obese IR subjects through involvement of NHE-1, PKC, NO synthase, NADPH oxidase and actin polymerization. In contrast, epinephrine did not affect monocyte migration. Epinephrine increased oxLDL phagocytosis in all groups studied. Incubation with cariporide attenuated oxLDL phagocytosis. Epinephrine induces monocyte dysfunction which may be atherogenic.