scholarly journals A novel view of paroxysmal nocturnal hemoglobinuria pathogenesis: more motile PNH hematopoietic stem/progenitor cells displace normal HSPCs from their niches in bone marrow due to defective adhesion, enhanced migration and mobilization in response to erythrocyte-released sphingosine-1 phosphate gradient

Leukemia ◽  
2012 ◽  
Vol 26 (7) ◽  
pp. 1722-1725 ◽  
Author(s):  
J Ratajczak ◽  
M Kucia ◽  
K Mierzejewska ◽  
R Liu ◽  
C H Kim ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Progenitor Cells ◽  
Paroxysmal Nocturnal Hemoglobinuria ◽  
Hematopoietic Stem ◽  
Sphingosine 1 Phosphate

Blood Cell Replenishment and Bone Marrow Stem Cell Pool Renewal Are Regulated By Different Circadian Peaks Via Norepinephrine and TNFα/S1P Signaling

Blood ◽  
2013 ◽  
Vol 122 (21) ◽  
pp. 217-217
Author(s):  
Karin Golan ◽  
Aya Ludin ◽  
Tomer Itkin ◽  
Shiri Cohen-Gur ◽  
Orit Kollet ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Progenitor Cells ◽  
Conflicts Of Interest ◽  
Surface Expression ◽  
Daily Basis ◽  
Hematopoietic Stem ◽  
Activated Macrophage ◽  
Sphingosine 1 Phosphate ◽  
Stem And Progenitor Cells ◽  
Primitive State

Abstract Hematopoietic stem and progenitor cells (HSPC) are mostly retained in a quiescent, non-motile mode in the bone marrow (BM), shifting to a cycling, differentiating and migratory state on demand. How HSC replenish the blood with new mature leukocytes on a daily basis while maintaining a constant pool of primitive cells in the BM throughout life is not clear. Recently, we reported that the bioactive lipid Sphingosine 1-Phosphate (S1P) regulates HSPC mobilization via ROS signaling and CXCL12 secretion (Golan et al, Blood 2012). We hypothesize that S1P influences the daily circadian egress of HSPC and their proliferation. We report that S1P levels in the blood are increased following initiation of light at the peak of HSPC egress and are reduced towards the termination of light when circulating HSPC reach a nadir. Interestingly, mice with constitutively low S1P plasma levels due to lack of one of the enzymes that generates S1P (Sphingosine kinase 1), do not exhibit fluctuations of HSPC levels in the blood between day and night. We report that HSPC numbers in the BM are also regulated in a circadian manner. Unexpectedly, we found two different daily peaks: one in the morning, following initiation of light, which is accompanied by increased HSPC egress and the other at night after darkness, which is associated with reduced HSPC egress. In both peaks HSPC begin to cycle and differentiate via up-regulation of reactive oxygen species (ROS) however, the night peak had lower ROS levels. Concomitant with the peak of primitive stem and progenitor cells, we also observed (to a larger extent in the night peak), expansion of a rare activated macrophage/monocyte αSMA/Mac-1 population. This population maintains HSPC in a primitive state via COX2/PGE2 signaling that reduces ROS levels and increases BM stromal CXCL12 surface expression (Ludin et al, Nat. Imm. 2012). We identified two different BM peaks in HSPC levels that are regulated by the nervous system via circadian changes in ROS levels. Augmented ROS levels induce HSPC proliferation, differentiation and motility, which take place in the morning peak; however, they need to be restored to normal levels in order to prevent BM HSPC exhaustion. In the night peak, HSPC proliferate with less differentiation and egress, and activated macrophage/monocyte αSMA/Mac-1 cells are increased to restore ROS levels and activate CXCL12/CXCR4 interactions to maintain a HSPC primitive phenotype. Additionally, S1P also regulates HSPC proliferation, thus mice with low S1P levels share reduced hematopoietic progenitor cells in the BM. Interestingly S1P is required more for the HSPC night peak since in mice with low S1P levels, HSPC peak normally during day time but not at darkness. We suggest that the first peak is initiated via elevation of ROS by norepinephrine that is augmented in the BM following light-driven cues from the brain (Mendez-Ferrer at al, Nature 2008). The morning elevated ROS signal induces a decrease in BM CXCL12 levels and up-regulated MMP-9 activity, leading to HSC proliferation, as well as their detachment from their BM microenvironment, resulting in enhanced egress. Importantly, ROS inhibition by N-acetyl cysteine (NAC) reduced the morning HSPC peak. Since norepinephrine is an inhibitor of TNFα, upon light termination norepinephrine levels decrease and TNFα levels are up-regulated. TNFα induces activation of S1P in the BM, leading to the darkness peak in HSPC levels. S1P was previously shown also to induce PGE2 signaling, essential for HSPC maintenance by the rare activated αSMA/Mac-1 population. Indeed, in mice with low S1P levels, we could not detect a peak in COX2 levels in these BM cells during darkness. We conclude that S1P not only induces HSPC proliferation via augmentation of ROS levels, but also activates PGE2/COX2 signaling in αSMA/Mac-1 population to restore ROS levels and prevent HSPC differentiation and egress during the night peak. We hypothesize that the morning HSPC peak, involves proliferation, differentiation and egress, to allow HSPC to replenish the blood circulation with new cells. In contrast, the second HSPC night peak induces proliferation with reduced differentiation and egress, allowing the renewal of the BM HSPC pool. In summary, we identified two daily circadian peaks in HSPC BM levels that are regulated via light/dark cues and concomitantly allow HSPC replenishment of the blood and immune system, as well as maintenance of the HSPC constant pool in the BM. Disclosures: No relevant conflicts of interest to declare.

scholarly journals Sphingosine-1-phosphate-Mediated Mobilization of Hematopoietic Stem/Progenitor Cells during Intravascular Hemolysis Requires Attenuation of SDF-1-CXCR4 Retention Signaling in Bone Marrow

2013 ◽  
Vol 2013 ◽  
pp. 1-5 ◽  
Author(s):  
Kasia Mierzejewska ◽  
Yuri M. Klyachkin ◽  
Janina Ratajczak ◽  
Ahmed Abdel-Latif ◽  
Magda Kucia ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Steady State ◽  
Progenitor Cells ◽  
Blood Cells ◽  
Blocking Agent ◽  
Intravascular Hemolysis ◽  
Hematopoietic Stem ◽  
Sphingosine 1 Phosphate ◽  
Retention Signal ◽  
Synergistic Increase

Sphingosine-1-phosphate (S1P) is a crucial chemotactic factor in peripheral blood (PB) involved in the mobilization process and egress of hematopoietic stem/progenitor cells (HSPCs) from bone marrow (BM). Since S1P is present at high levels in erythrocytes, one might assume that, by increasing the plasma S1P level, the hemolysis of red blood cells would induce mobilization of HSPCs. To test this assumption, we induced hemolysis in mice by employing phenylhydrazine (PHZ). We observed that doubling the S1P level in PB from damaged erythrocytes induced only a marginally increased level of mobilization. However, if mice were exposed to PHZ together with the CXCR4 blocking agent, AMD3100, a robust synergistic increase in the number of mobilized HSPCs occurred. We conclude that hemolysis, even if it significantly elevates the S1P level in PB, also requires attenuation of the CXCR4-SDF-1 axis-mediated retention in BM niches for HSPC mobilization to occur. Our data also further confirm that S1P is a major chemottractant present in plasma and chemoattracts HSPCs into PB under steady-state conditions. However, to egress from BM, HSPCs first have to be released from BM niches by blocking the SDF-1-CXCR4 retention signal.

A Novel View of Paroxysmal Nocturnal Hemoglobinuria (PNH) Pathogenesis: Do Pathologic PNH Hematopoietic Stem/Progenitor Cells (HSPCs) Displace Normal HSPCs From Their Niches in Bone Marrow Because They Are More Motile Due to Defective Adhesion and Enhanced Migratory Properties?

Blood ◽  
2011 ◽  
Vol 118 (21) ◽  
pp. 732-732
Author(s):  
Janina Ratajczak ◽  
Rui Liu ◽  
Nagendra Natarajan ◽  
Jaroslaw P. Maciejewski ◽  
Vivek R. Sharma ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Progenitor Cells ◽  
Lipid Rafts ◽  
Lipid Raft ◽  
Peripheral Blood ◽  
Paroxysmal Nocturnal Hemoglobinuria ◽  
Conflicts Of Interest ◽  
Gpi Anchor ◽  
Hematopoietic Stem ◽  
Cd34 Antigen

Abstract Abstract 732 Background . Paroxysmal nocturnal hemoglobinuria (PNH) is an uncommon acquired hemolytic anemia that results from the expansion of hematopoietic stem cells with a mutation in one of the enzymes (PIG-A) responsible for glycosylphosphatidylinositol (GPI anchor) biosynthesis, which is a post-translation modification of proteins associated with lipid rafts on the cell membrane surface. Some of these proteins are involved in the resistance of erythrocytes to lysis by the final product of complement cascade (CC) activation, C5b-C9, also known as the membrane attack complex (MAC). As we reported, the CXCR4 receptor, which binds a-chemokine stromal derived factor-1 (SDF-1) in regulating the trafficking of hematopoietic stem/progenitor cells (HSPCs), is also associated with lipid rafts (Blood 2005;105:40-8). In addition, we recently demonstrated that the bioactive lipid sphingosine-1-phosphate (S1P), which is a major chemoattractant directing egress of HSPCs from bone marrow (BM) into peripheral blood (PB) during mobilization, is released from erythrocytes by C5b-C9/MAC (Leukemia 2010;24:976-85). Hypothesis. Based on this finding, we hypothesized that HSPCs are continuously mobilized from the BM of PNH patients due to the susceptibility of PIG-A-deficient erythrocytes to CC activation, which elevates the free S1P level in plasma, as well as to their defective adhesion in the BM microenvironment due to impaired lipid raft formation. Experimental strategies. To address this hypothesis, peripheral blood mononuclear cells (PBMNC) were isolated from 6 PNH patients and stained with the fluorescent variant of aerolysin (FLAER), which binds GPI anchor and thus identifies normal, but not PNH, cells in FACS analysis. PNH patient-derived cells were tested for i) the level of CD34 antigen expression, ii) chemotaxis in response to SDF-1 and S1P, and iii) adhesion to fibronectin and bone marrow stromal cells. Results. We observed in PNH patients ∼3-fold higher expression of CD34 antigen on FLAER– cells circulating in PB than FLAER+ cells, which suggests that PNH-mutated HSPCs are preferentially released/mobilized into PB. Next, in Transwell chemotaxis assays followed by in vitro clonogenic assays with cells collected from the lower Transwell chambers, we observed that FLAER– cells responding to SDF-1 are ∼20 times more enriched in migrating clonogenic BFU-E and CFU-GM progenitors than their normal FLAER+ counterparts. Moreover, in parallel experiments, FLAER– CFU-GM that were plated over BM-derived fibroblasts or fibronectin in the presence of SDF-1 and S1P (known activators of VLA-4–VCAM-1-mediated cell adhesion) exhibited impaired adhesion in comparison to normal FLAER+ CFU-GM cells. Conclusions. Based on these observations, we propose a novel view of the pathogenesis of PNH and the expansion of PNH-affected cells in BM. Accordingly, the lack of PIG-A protein, which plays an important role in lipid raft formation, confers an advantage to PNH-affected HSPCs, which become more mobile. These cells are preferentially mobilized into PB in response to S1P released from C5b-C9/MAC-lysed erythrocytes. Thus, PNH-mutated HSPCs over time may outcompete normal HSPCs for their niches in BM, due to their increased motility, and contribute to the PNH type of hematopoiesis. Disclosures: No relevant conflicts of interest to declare.

Novel Evidence That Hematopoietic Stem/Progenitor Cells (HSPCs) Are Mobilized During Hemolysis in an Erythrocyte Lysis-Derived, Sphingosine-1-Phosphate (S1P)-Dependent manner—the Crucial Involvement of Complement Cascade (CC) Activation and Attenuation of CXCR4 Retention Signaling

Blood ◽  
2012 ◽  
Vol 120 (21) ◽  
pp. 3189-3189
Author(s):  
Kasia Mierzejewska ◽  
Magdalena Kucia ◽  
Janina Ratajczak ◽  
Mariusz Z Ratajczak
Keyword(s):  
Progenitor Cells ◽  
Paroxysmal Nocturnal Hemoglobinuria ◽  
Molecular Mechanisms ◽  
Distal Part ◽  
Complement Cascade ◽  
Dependent Manner ◽  
Hematopoietic Stem ◽  
Sphingosine 1 Phosphate ◽  
Deficient Mice ◽  
Chemotactic Gradient

Abstract Abstract 3189 Background. Hemolytic syndromes, such as sickle cell anemia and paroxysmal nocturnal hemoglobinuria, are characterized by an increased number of hematopoietic stem/progenitor cells (HSPCs) circulating in peripheral blood (PB). However, the molecular mechanisms responsible for this effect are unclear. In our previous work we have demonstrated that sphingosine-1-phosphate (S1P) released from lysed erythrocytes and activated platelets is a strong chemottractant for bone marrow (BM)-residing HSPCs (Leukemia 2010;24:976–85). Hypothesis. We hypothesized that S1P released from lysed erythrocytes is a major factor responsible for egress of HSPCs from BM into PB in hemolytic syndromes. Experimental approach. To test this hypothesis, normal mice were injected with phenylhydrazine (PHZ), a compound known to induce hemolysis, and we evaluated the number of Sca-1+Kit+Lin– (SKL) HSPCs circulating in PB as well as the number of clonogenic CFU-GM progenitors mobilized into PB. In parallel, we evaluated the blood plasma levels of S1P and stromal derived factor-1 (SDF-1) by sensitive ELISA, the free hemoglobin (Hb) level, as well as complement cascade (CC) activation by measuring the C5b-C9 (membrane attack complex, MAC) level. In some of the experiments, we combined PZH treatment with injection of the CXCR4 antagonist AMD3100. To better assess the role of CC activation, we also performed mobilization in C5-deficient mice, which do not activate the distal part of the CC and thus do not generate C5b-C9/MAC. Results. We found that hemolysis increases the PB plasma level of S1P but does not affect the SDF-1 level. Furthermore, while PHZ-induced hemolysis mobilizes HSPCs into PB with a peak at 6 h after infusion, this mobilization effect is significantly potentiated by administration of AMD3100, which attenuates CXCR4–SDF-1-mediated retention of HSPCs in the BM microenvironment. Of note, PHZ-induced hemolysis together with AMD3100 mobilized twice as many HSPCs as AMD3100 alone. The degree of mobilization of HSPCs correlated with the free Hb level in plasma and activation of the CC (by an increase in MAC level), and, more importantly, mobilization was not seen in C5-deficient mice. Conclusions. We confirmed our previous observation that with the steady-state S1P level, the PB has already established a strong chemotactic gradient for BM-residing HSPCs (Leukemia 2010;24:976–85), which are actively retained in BM niches in a CXCR4–SDF-1-dependent manner. Hemolysis alone, even if it elevates the S1P level in PB significantly, requires two important events i) attenuation of CXCR4–SDF-1 axis-mediated retention in BM niches and ii) simultaneous activation of the CC, which is crucial for induction of permeabilization of the BM–PB barrier. Furthermore, our data also support the notion that the S1P but not the SDF-1 level in PB establishes the critical chemotactic gradient for HSPCs and is responsible for egress of these cells. Based on the observation that C5-deficient mice are poor mobilizers, inactivation of the distal part of the CC should be considered as a therapeutic approach, not only in paroxysmal nocturnal hemoglobinuria but also in other hemolytic syndromes. Disclosures: No relevant conflicts of interest to declare.

Novel Evidence That Sphingosine-1-Phosphate-Mediated Mobilization Of Hematopoietic Stem/Progenitor Cells (HSPCs) During Intravascular Hemolysis Requires Attenuation Of The SDF-1–CXCR4 Retention Axis Of HSPCs In Bone Marrow Niches – Implications For Paroxysmal Nocturnal Hemoglobinuria-Induced Mobilization of HSPCs

Blood ◽  
2013 ◽  
Vol 122 (21) ◽  
pp. 2477-2477
Author(s):  
Kasia Mierzejewska ◽  
Ahmed Abdel-Latif ◽  
Gabriela Schneider ◽  
Janina Ratajczak ◽  
Magdalena Kucia ◽  
...  
Keyword(s):  
Steady State ◽  
Plasma Level ◽  
Progenitor Cells ◽  
Paroxysmal Nocturnal Hemoglobinuria ◽  
Conflicts Of Interest ◽  
Specific Inhibitor ◽  
Complement Cascade ◽  
Hematopoietic Stem ◽  
Sphingosine 1 Phosphate ◽  
Cxcr4 Axis

Abstract Background We have recently reported that hematopoietic stem/progenitor cells (HSPCs) that harbor mutations of the PIG-A gene are preferentially mobilized into peripheral blood (PB) during hemolytic events in paroxysmal nocturnal hemoglobinuria (PNH) patients (Leukemia 2012;26:1722). This effect has been explained by i) an increase in the plasma level of sphingosine-1-phosphate (S1P), which at physiological doses is a major chemoattractant for HSPCs and is released from lysed erythrocytes (Leukemia 2010;24:976), and ii) the fact that PNH-cloned HSPCs, in contrast to normal HSPCs, show defective SDF-1–CXCR4-mediated retention in BM niches. It is known that under steady-state conditions the concentration of S1P in PB is already 25x higher than its concentration in the BM microenvironment and increases additionally during hemolysis. Aim of the study Since erythrocytes are a major source of plasma S1P, we asked whether massive hemolysis of erythrocytes leading to an additional increase in plasma S1P level would trigger mobilization of HSPCs. Furthermore, to shed more mechanistic light on the mobilization of HSPCs in PNH patients and to distinguish the effect of an increase in the S1P chemotactic gradient in PB plasma from the effect of defective retention of HSPCs in the BM microenvironment, we performed mobilization studies in mice exposed to the hemolysis-inducing agent phenhlhydrazine (PHZ) ± blockade of the BM-retaining SDF-1–CXCR4 axis by AMD3100. Experimental approach Normal C57Bl6 mice were injected with i) PHZ to induce hemolysis and S1P release from erythrocytes, ii) AMD3100 to perturb the SDF-1–CXCR4-mediated retention of HSPCs in BM niches, or iii) both PHZ and AMD3100. Subsequently, we evaluated the number of circulating Sca-1+Kit+Lin– (SKL) HSPCs as well as the number of clonogenic CFU-GM progenitors in PB. In parallel, we evaluated the S1P blood plasma levels by liquid chromatography electrospray ionization tandem mass spectrometry (HPLC ESI MS/MS) and the SDF-1 level by ELISA. In addition, we measured complement cascade (CC) activation by measuring the C5b-C9 (membrane attack complex, MAC) levels. Results We found that hemolysis doubles the PB plasma level of S1P (from 1 to 2 mM). To assess the effect of plasma S1P versus plasma SDF-1 as chemoattractants mediating egress of HSPCs from BM, we employed plasma derived from control and PHZ-treated mice, W146, a receptor-specific inhibitor for the S1P receptor type 1 (S1P1), and the CXCR4 antagonist AMD3100 in Transwell migration assays. We observed that chemotaxis of BM-purified HSPCs was inhibited by blocking the S1P–S1P1 but not the SDF-1–CXCR4 axes, which demonstrates that the S1P level in plasma is a crucial chemoattractant for HSPCs present under normal steady-state conditions and in PHZ-treated mouse plasma. This observation also clearly shows that the S1P gradient, even under steady-state conditions, is already high enough to promote egress of HSPCs from BM into PB and supports our previous observations that, while the SDF-1–CXCR4 axis plays an important role in retention of HSPCs in BM niches, the SDF-1 plasma level is too low to induce egress of HSPCs (Leukemia 2010;24:976). In our in vivo mobilization studies, we observed that, in contrast to AMD3100 administration, PHZ-induced hemolysis alone had a negligible effect on mobilization of HSPCs, with a peak at 6 h after infusion of this hemolysis-inducing agent (Figure 1). However, when we combined PHZ with AMD3100, mice mobilized twice as many HSPCs as with administration of AMD3100 alone. The degree of mobilization of HSPCs correlated with the free Hb level in plasma and with activation of the complement cascade. Conclusions At the steady-state conditions S1P level in PB is already a strong chemotactic factros for BM-residing HSPCs. More importantly, to explain the differential mobilization of PNH-affected HSPCs versus normal HSPCs (Leukemia 2012;26:1722), we show here for the first time that hemolysis alone, even if it doubles the S1P level in PB, requires attenuation of CXCR4–SDF-1-mediated retention in BM niches. Thus, PNH-affected HSPCs, due to defective lipid raft formation, have impaired CXCR4-mediated retention in BM niches and are preferentially mobilized into PB. Finally, our data explain why, compared with PNH, HSPCs are mobilized to a much lesser degree in other hemolytic syndromes (e.g., sickle cell anemia). Disclosures: No relevant conflicts of interest to declare.

The sphingosine 1-phosphate receptor agonist FTY720 supports CXCR4-dependent migration and bone marrow homing of human CD34+ progenitor cells

Blood ◽  
2004 ◽  
Vol 103 (12) ◽  
pp. 4478-4486 ◽  
Author(s):  
Takafumi Kimura ◽  
Andreas M. Boehmler ◽  
Gabriele Seitz ◽  
Selim Kuçi ◽  
Tina Wiesner ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Progenitor Cells ◽  
Actin Polymerization ◽  
Hematopoietic Stem ◽  
Human Cd34 ◽  
Sphingosine 1 Phosphate ◽  
Stem And Progenitor Cells ◽  
Stromal Cell Derived Factor

Abstract The novel immunosuppressant FTY720 activates sphingosine 1-phosphate receptors (S1PRs) that affect responsiveness of lymphocytes to chemokines such as stromal cell-derived factor 1 (SDF-1), resulting in increased lymphocyte homing to secondary lymphoid organs. Since SDF-1 and its receptor CXCR4 are also involved in bone marrow (BM) homing of hematopoietic stem and progenitor cells (HPCs), we analyzed expression of S1PRs and the influence of FTY720 on SDF-1/CXCR4-mediated effects in human HPCs. By reverse transcriptase-polymerase chain reaction (RT-PCR), S1PRs were expressed in mobilized CD34+ HPCs, particularly in primitive CD34+/CD38- cells. Incubation of HPCs with FTY720 resulted in prolonged SDF-1-induced calcium mobilization and actin polymerization, and substantially increased SDF-1-dependent in vitro transendothelial migration, without affecting VLA-4, VLA-5, and CXCR4 expression. In nonobese diabetic-severe combined immunodeficient (NOD/SCID) mice, the number of CD34+/CD38- cells that homed to the BM after 18 hours was significantly raised by pretreatment of animals and cells with FTY720, tending to result in improved engraftment. In addition, in vitro growth of HPCs (week-5 cobblestone area-forming cells [CAFCs]) was 2.4-fold increased. We conclude that activation of S1PRs by FTY720 increases CXCR4 function in HPCs both in vitro and in vivo, supporting homing and proliferation of HPCs. In the hematopoietic microenvironment, S1PRs are involved in migration and maintenance of HPCs by modulating the effects of SDF-1. (Blood. 2004;103:4478-4486)

Novel Mechanistic Insight Into Mobilization of Hematopoietic Stem/Progenitor Cells (HSPCs): Complement Cascade and Membrane Attack Complex Activated in Bone Marrow Sinusoids During Mobilization Release From Erythrocytes Sphingosine-1 Phosphate – An Underappreciated Chemoattractant Executing Egress of HSPCs.

Blood ◽  
2009 ◽  
Vol 114 (22) ◽  
pp. 31-31 ◽  
Author(s):  
HakMo Lee ◽  
Marcin Wysoczynski ◽  
Wan Wu ◽  
Rui Liu ◽  
Magdalena Kucia ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Progenitor Cells ◽  
Conflicts Of Interest ◽  
Membrane Attack Complex ◽  
Heat Inactivation ◽  
Cationic Peptides ◽  
Complement Cascade ◽  
Cxcr4 Antagonist ◽  
Hematopoietic Stem ◽  
Sphingosine 1 Phosphate

Abstract Abstract 31 We reported that complement cascade (CC) is activated in bone marrow (BM) during mobilization of hematopoietic stem/progenitor cells (HSPCs) and that CC clevage fragments direct egress of HSPCs from BM into peripheral blood (PB) (Blood 2003;101,3784; Blood 2004;103,2071; Blood 2005;105,40). We also reported that C5 cleavage fragments play a crucial role in the mobilization process by: i) inducing proteolytic activity in the BM environment; ii) directing BM egress of granulocytes that “pave a road” for HSPCs; and iii) inducing secretion of cationic peptides from activated granulocytes that prime HSPC egress (Leukemia 2009; in press). In this study, we sought to determine which major chemottractant is present in PB that is responsible for egress of HSPCs and whether activation of CC plays some role in its level/expression. We noticed that plasma derived from normal and mobilized PB strongly chemoattracts murine and human HSPCs. This chemotactic effect was not dependent on plasma SDF-1 levels because: i) it occurs unaffectedly in the presence of CXCR4 antagonist AMD3100; ii) it was still robust to heat-inactivated sera; and iii) ELISA studies revealed negligible concentrations of SDF-1, which did not correlate with good or poor mobilizer status. However, to our surprise, we noticed that plasma isolated from G-CSF-mobilized mice and patients contains traces of free hemoglobin, which suggests some level of hemolysis occurs in mobilized PB. As such, we performed chemotactic assays in the presence of different concentrations of lysed erythrocytes and noticed that such diluted lysates are potent chemoattractants for HSPCs. The chemotactic activities of these lysates were resistant to heat inactivation similarly as patient sera. Based on this, we focused on S1P, a thermo-resistant lipid that, as reported, chemoattracts HSPCs and whose major reservoirs are erythrocytes (FASEB J 2007:21;1202). In fact we found by ELISA that S1P level increases during mobilization in PB and that SP1 is the most potent chemoattractant for BM-residing HSPCs, much stronger than SDF-1 - if both compounds are compared in physiologically relevant concentrations. Furthermore, activation of S1P receptors on BM-derived HSPCs augmented responsiveness to SDF-1 gradient up to 50%. However, these chemotactic effects of S1P were not visible for previously mobilized PB or umbilical cord blood HSPCs, which we explain by a fact that these mobilized cells are already desensitized to S1P gradient. Therefore, we propose the following scenario. First, a mobilizing agent (e.g., G-CSF) induces activation of CC in BM that subsequently contributes to the release of protelolytic enzymes from granulocytes that perturb SDF-1-CXCR4/VLA-4-VCAM1 interactions and stimulate egress of activated granulocytes from BM that “pave a road” for egress of HSPCs. Simultaneously, the final product of CC activation (C5b-C9), the membrane attack complex (MAC), induces in BM sinusoids the release of S1P from erythrocytes. S1P accumulating in BM sinusoids and cationic peptides released from activated granulocytes, but not changes in plasma SDF-1 levels, are crucial executors of HSPCs egress from BM into PB. Thus, our results provide novel evidence that CC activation/membrane attack complex (MAC)-induced elevated plasma S1P level is essential for egress/mobilization of HSPCs. Disclosures: No relevant conflicts of interest to declare.

A Stro-1+ human universal stromal feeder layer to expand/maintain human bone marrow hematopoietic stem/progenitor cells in a serum-free culture system

2006 ◽  
Vol 34 (10) ◽  
pp. 1353-1359 ◽  
Author(s):  
Raquel Gonçalves ◽  
Cláudia Lobato da Silva ◽  
Joaquim M.S. Cabral ◽  
Esmail D. Zanjani ◽  
Graça Almeida-Porada
Keyword(s):  
Bone Marrow ◽  
Progenitor Cells ◽  
Culture System ◽  
Human Bone ◽  
Human Bone Marrow ◽  
Feeder Layer ◽  
Hematopoietic Stem ◽  
Serum Free
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