An Unexpected Role for the Complement C5b-C9 Membrane Attack Complex (MAC) In Trafficking of Hematopoietic Stem/Progenitor Cells - a Novel Unexpected Link Between Innate Immunity and Hematopoiesis

Blood ◽  
2010 ◽  
Vol 116 (21) ◽  
pp. 555-555
Author(s):  
Chihwa Kim ◽  
Wu Wan ◽  
Rui Liu ◽  
Magdalena Kucia ◽  
Janina Ratajczak ◽  
...  
Keyword(s):  
Innate Immunity ◽  
Progenitor Cells ◽  
Peripheral Blood ◽  
Conflicts Of Interest ◽  
Proteolytic Enzymes ◽  
Biological Fluids ◽  
Membrane Attack Complex ◽  
Target Cells ◽  
Complement Cascade ◽  
Hematopoietic Stem

Abstract Abstract 555 We previously reported that complement cascade (CC) is activated in bone marrow (BM) during mobilization of hematopoietic stem/progenitor cells (HSPCs; Blood 2003;101:3784; Blood 2004;103:2071; and Blood 2005;105:40) and that C5 cleavage fragments direct egress of HSPCs from BM into peripheral blood (PB) (Leukemia 2009;23:2052 and Leukemia 2010;24:976). Accordingly, C5 cleavage fragments (C5a and desArgC5a) stimulate myeloid cells in BM to secrete proteolytic enzymes and chemoattract granulocytes into peripheral blood (PB). Therefore, granulocytes form a first wave of cells that permeabilize the BM-PB endothelial barrier and prime it for subsequent egress of HSPCs. We have also observed that activation of the distal part of the complement cascade (CC), which leads to formation of C5b-C9 (also known as the membrane attack complex [MAC]), is crucial for egress/mobilization of HSPCs. It is known that proteins that form MAC can be inserted into cell membranes, resulting in cell lysis, or may remain in biological fluids as soluble MAC (sMAC) and in this “non-lytic” form may interact with target cells. We have already reported that sMAC releases bioactive lipid - sphingosine-1 phosphate (S1P) from erythrocytes, which is a major chemoattractant in mobilized peripheral blood (mPB) for HSPCs (Leukemia 2010;24:976). Since the level of sMAC increases in PB during mobilization as well as following conditioning for transplantation, we became interested in whether this protein complex affects the biology of normal HSPCs. First, we observed that, while sMAC does not affect proliferation and viability of clonogenic progenitors, it activates phosphorylation of MAPKp42/44 and AKT in both human CD34+ and murine SKL cells. Furthermore, sMAC primes and enhances chemotactic responsiveness of HSPCs to S1P and SDF-1 gradients and increases adhesiveness of these cells to BM stroma and endothelium. This effect is probably lipid raft mediated, because exposure of cells to methylo-b-cyclodextrin before chemotaxis abrogates this phenomenon. We also found that HSPCs, as well as PB mononuclear cells exposed to sMAC, secrete increased levels of PGE2 and metalloproteinases, which indicates that an increase in sMAC level in PB after conditioning for transplantation may enhance the homing properties of HSPCs. Thus, our results in toto provide novel evidence that sMAC is an underappreciated and potent regulator of HSPC trafficking and plays an important role, both direct and indirect (via released from cells S1P), in mobilization and homing of HSPCs after transplantation. In support of this notion, we found that mice displaying defects in CC activation and sMAC generation display a defect in homing of HSPCs. Thus, our data provide yet more evidence that innate immunity and the complement cascade regulate trafficking of HSPCs by (1) releasing active C3 and C5 cleavage fragments that increase the level of bioactive lipids chemoattractants in PB and BM and by (2) modulating the migratory properties of HSPCs with sMAC. We propose modulation of CC as a novel strategy for controlling both mobilization and homing of HSPCs. Disclosures: No relevant conflicts of interest to declare.

Novel Observation That Mice Lacking the Fifth Complement Cascade Protein Component (C5) Are Very Poor Stem Cell Mobilizers Explained by Defective Egress of Granulocytes: A Novel Role for Bone Marrow Granulocytes to Act as “ice Breaker” Cells in Facilitating Egress of Hematopoietic Stem/Progenitor Cells

Blood ◽  
2008 ◽  
Vol 112 (11) ◽  
pp. 67-67
Author(s):  
Wan Wu ◽  
Hakmoo Lee ◽  
Marcin Wysoczynski ◽  
Magdalena Kucia ◽  
Janina Ratajczak ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Progenitor Cells ◽  
Peripheral Blood ◽  
White Blood Cells ◽  
Membrane Attack Complex ◽  
Optimal Dose ◽  
Histochemical Staining ◽  
Complement Cascade ◽  
Hematopoietic Stem ◽  
Small Vessels

Abstract We reported that complement cascade (CC) becomes activated in bone marrow (BM) during mobilization of hematopoietic stem/progenitor cells (HSPCs) by immunoglobulin (Ig)-dependent pathway and/or by alternative Ig-independent pathway and, as result of this, several potent bioactive CC anaphylatoxins (C3a, desArgC3a, C5a and desArgC5a) are released (Blood2003;101,3784; Blood2004;103,2071; Blood2005;105,40). Bioactive CC anaphylatoxins (C5a and desArgC5a) are also potent chemoattractants of granulocytes that bind to G-protein-coupled, seven trans-membrane span C5a receptors (C5aR and C5L2) on these cells. To learn more on the role of C5 cleavage fragments in HSPC mobilization, we studied mobilization in C5−/− and C5aR−/− mice as well as their normal wildtype littermates. Mobilization was induced by granulocyte colony-stimulating factor (G-CSF; high 250 μg/kg/6 days and low dose 50 μg/kg/6 days) or zymosan (20 mg/1kg/1 hour), which activate classical and alternative pathways of CC, respectively. We evaluated mobilization efficiency by counting the number of SKL cells, colony-forming unit granulocyte-macrophages (CFU-GMs), and white blood cells circulating in peripheral blood. In parallel, we employed transmission electron microscopy (TEM) to study the morphology and integrity of BM vessels in the BM-blood barrier. Activation of CC was measured by ELISA for C3 cleavage fragments and by histochemical staining for membrane attack-complex (MAC) depositions in BM tissue. We found by ELISA and histochemistry that CC activation correlates with the level of HSPC mobilization in wildtype mice and that mobilization of HSPCs was always preceded by the release of granulocytes from BM. Thus, granulocytes are the first wave of cells that increase in number during mobilization in peripheral blood. Mobilization studies in C5−/− revealed that these animals are very poor mobilizers. TEM studies demonstrated that hematopoietic cells together with granulocytes accumulated around small vessels in the BM of C5−/− animals, but they did not migrate or cross the BM-endothelial barrier. Since C5 cleavage fragments C5a and desArgC5a are potent chemoatrractants for granulocytes but not HSPCs, we hypothesize that a lack of both these anaphylatoxins in C5−/− animals prevents egress of granulocytes from BM, which always precedes egress of HSPCs. Furthermore, in C5aR−/−, mice mobilization was normal after administration of a high optimal dose of G-CSF. However, mobilization was significantly lower after a suboptimal dose of G-CSF or administration of zymosan. This indicates that another alternative receptor for C5a and desArgC5a (C5L2) may compensate for C5aR deficiency and that it plays a role in the egress of granulocytes from the BM as well. Thus, this study demonstrates that cells from the granulocytic lineage are actively involved in mobilization in a C5a,-desArgC5a-C5aR manner not only by secreting proteases that create a proteoytic environment in BM, but also as a kind of “ice-breaker” type cells necessary for disintegration of the endothelial-BM barrier to enable HSPCs to egress from the BM microenvironment. In cases of granulocytopenia or if granulocytes are not mobilized as seen in C5−/− mutants, mobilization of HSPCs is very poor. Thus, modulation of CC activation in the BM and stimulation of granulocyte egress from the BM into circulation may help to develop more efficient strategies for HSPC mobilization.

Mobilization Studies in Complement-Deficient Mice Reveal That AMD3100 Mobilization Depends On Complement Cascade Activation and Suggest Involvement of “Membrane Attack Complex - MAC” in Egress of Hematopoietic Stem/Progenitor Cells.

Blood ◽  
2009 ◽  
Vol 114 (22) ◽  
pp. 367-367
Author(s):  
Marcin Wysoczynski ◽  
HakMo Lee ◽  
Rui Liu ◽  
Wan Wu ◽  
Janina Ratajczak, ◽  
...  
Keyword(s):  
Progenitor Cells ◽  
Conflicts Of Interest ◽  
Membrane Attack Complex ◽  
Classical Pathway ◽  
Compensatory Mechanism ◽  
Complement Cascade ◽  
Cxcr4 Antagonist ◽  
Hematopoietic Stem ◽  
Deficient Mice

Abstract Abstract 367 We reported that complement cascade (CC) becomes activated in bone marrow (BM) during mobilization of hematopoietic stem/progenitor cells (HSPCs) by immunoglobulin (Ig)-dependent pathway and/or by alternative Ig-independent pathway as seen during G-CSF- or Zymosan mobilization, respectively. As a result, several potent bioactive CC anaphylatoxins (C3 and C5 cleavage fragments) are released that regulate egress of HSPCs (Blood 2003;101,3784; Blood 2004;103,2071; Blood 2005;105,40, Leukemia 2009; in press.). This explains why: i) NOD/SCID and RAG-/- animals that do not activate the Ig-dependent CC classical pathway; ii) C2fB-/- and C3-/- mice that do not activate the classical and alternative CC pathways; and iii) C5-/- mice that do not activate the distal pathway of CC are all poor G-CSF- and/or Zymosan mobilizers. In this study, we evaluated the role of CC in mobilization induced by CXCR4 antagonist AMD3100. We noticed that all CC activation-deficient mice mentioned above, except C5-/- mice, mobilize normally in response to AMD3100 administration. Accordingly, the number of mobilized CD34- SKL cells, leucocytes, and CFU-GM clonogeneic progenitors in mutant mice was similar to wt littermates. More important we observed that AMD3100 mobilization of HSPCs was preceded by a massive egress of leucocytes from BM and that AMD3100 was able to stimulate in these cells i) phosphorylation of MAPKp42/44 and ii) secretion of MMP-9. At the same time, ELISA data to detect CC activation revealed that serum levels of CC cleavage fragments, which were low in the initial phase of AMD3100 mobilization during granulocyte egress, become elevated later during HSPC egress. Thus, our data show that despite a fact that G-CSF and AMD3100 mobilize HSPCs by involving different mechanisms, activation of CC is a common phenomenon occurring during mobilization induced by both compounds. This further supports a pivotal role of CC activation in the egress of HSPCs from BM; however, both compounds activate CC differently. While G-CSF administration initiates CC activation at its proximal C1q-C3 level, AMD3100 induces CC activation at the distal C5 level, pointing to a crucial role of C5 cleavage in executing mobilization. To support this, all mice employed in our studies that display defects in activation of proximal stages of CC (NOD/SCID, RAG, C2fB-/-, and C3-/-) are normal AMD3100 mobilizers. However, C5 is cleavage required for mobilization occurs in the plasma of these animals latter on - directly by proteases released from AMD3100-stimulated granulocytes that egress from the BM as a first wave of mobilized cells. This compensatory mechanism cannot occur from obvious reasons in C5-/- mice. We conclude that AMD3100-directed mobilization similarly as G-CSF-induced one depends on activation of CC; however, AMD3100 in contrast to G-CSF activates CC at distal stages – directly by proteases released from mobilized/activated granulocytes. Cleavage of C5 and release of C5a and desArgC5a create a sinusoid-permissive environment in BM for HSPCs egress. This suggests involvement of both C5 cleavage fragments as well as a potential role of downstream elements of CC activation - membrane attack complex - MAC (C5b-C9) in stem cell mobilization. Therefore, some poor AMD3100 patient responders could possess a defect in activation of the distal steps of CC. Disclosures: No relevant conflicts of interest to declare.

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.

The Tetraspanin CD9 Regulates Engraftment and Mobilization of Human CD34+ Hematopoietic Stem/Progenitor Cells and Modulates VLA-4 Activity

Blood ◽  
2015 ◽  
Vol 126 (23) ◽  
pp. 1169-1169
Author(s):  
Kam Tong Leung ◽  
Karen Li ◽  
Yorky Tsin Sik Wong ◽  
Kathy Yuen Yee Chan ◽  
Xiao-Bing Zhang ◽  
...  
Keyword(s):  
Stem Cell ◽  
Progenitor Cells ◽  
Peripheral Blood ◽  
Scid Mouse ◽  
Conflicts Of Interest ◽  
Chimeric Mice ◽  
Hematopoietic Stem ◽  
Cell Engraftment ◽  
Cd34 Cells

Abstract Migration, homing and engraftment of hematopoietic stem/progenitor cells depend critically on the SDF-1/CXCR4 axis. We previously identified the tetraspanin CD9 as a downstream signal of this axis, and it regulates short-term homing of cord blood (CB) CD34+ cells (Leung et al, Blood, 2011). However, its roles in stem cell engraftment, mobilization and the underlying mechanisms have not been described. Here, we provided evidence that CD9 blockade profoundly reduced long-term bone marrow (BM; 70.9% inhibition; P = .0089) and splenic engraftment (87.8% inhibition; P = .0179) of CB CD34+ cells (n = 6) in the NOD/SCID mouse xenotransplantation model, without biasing specific lineage commitment. Interestingly, significant increase in the CD34+CD9+ subsets were observed in the BM (9.6-fold; P < .0001) and spleens (9.8-fold; P = .0014) of engrafted animals (n = 3-4), indicating that CD9 expression on CD34+ cells is up-regulated during engraftment in the SDF-1-rich hematopoietic niches. Analysis of paired BM and peripheral blood (PB) samples from healthy donors revealed higher CD9 expressions in BM-resident CD34+ cells (46.0% CD9+ cells in BM vs 26.5% in PB; n = 13, P = .0035). Consistently, CD34+ cells in granulocyte colony-stimulating factor (G-CSF)-mobilized peripheral blood (MPB) expressed lower levels of CD9 (32.3% CD9+ cells; n = 25), when compared with those in BM (47.7% CD9+ cells; n = 16, P = .0030). In vitro exposure of MPB CD34+ cells to SDF-1 significantly enhanced CD9 expression (1.5-fold increase; n = 4, P = .0060). Treatment of NOD/SCID chimeric mice with G-CSF decreased the CD34+CD9+ subsets in the BM from 79.2% to 62.4% (n = 8, P = .0179). These data indicate that CD9 expression is down-regulated during egress or mobilization of CD34+ cells. To investigate the possible mechanisms, we performed a VCAM-1 (counter receptor of the VLA-4 integrin) binding assay on BM CD34+ cells. Our results demonstrated that CD34+CD9+ cells preferentially bound to soluble VCAM-1 (17.2%-51.4% VCAM-1-bound cells in CD9+ cells vs 12.8%-25.9% in CD9- cells; n = 10, P ≤ .0003), suggesting that CD9+ cells possess higher VLA-4 activity. Concomitant with decreased CD9 expression, MPB CD34+ cells exhibited lower VCAM-1 binding ability (2.8%-4.0% VCAM-1-bound cells; n = 3), when compared to BM CD34+ cells (15.5%-37.7%; n = 10, P < .0130). In vivo treatment of NOD/SCID chimeric mice with G-CSF reduced VCAM-1 binding of CD34+ cells in the BM by 49.0% (n = 5, P = .0010). Importantly, overexpression of CD9 in CB CD34+ cells promoted VCAM-1 binding by 39.5% (n = 3, P = .0391), thus providing evidence that CD9 regulates VLA-4 activity. Preliminary results also indicated that enforcing CD9 expression in CB CD34+ cells could enhance their homing and engraftment in the NOD/SCID mouse model. Our findings collectively established that CD9 expression and associated integrin VLA-4 activity are dynamically regulated in the BM microenvironment, which may represent important events in governing stem cell engraftment and mobilization. Strategies to modify CD9 expression could be developed to enhance engraftment or mobilization of CD34+ cells. Disclosures No relevant conflicts of interest to declare.

scholarly journals Evidence That T Cells Specific for Non-Hematopoietic Cells Trigger the Development of Acquired Aplastic Anemia

Blood ◽  
2015 ◽  
Vol 126 (23) ◽  
pp. 1168-1168
Author(s):  
Tatsuya Imi ◽  
Hiroyuki Maruyama ◽  
Takamasa Katagiri ◽  
Yoshitaka Zaimoku ◽  
Kana Maruyama ◽  
...  
Keyword(s):  
T Cells ◽  
Aplastic Anemia ◽  
Progenitor Cells ◽  
Response Rate ◽  
Conflicts Of Interest ◽  
Uniparental Disomy ◽  
Target Cells ◽  
Principal Mechanism ◽  
Hematopoietic Stem ◽  
Number Of Patients

Abstract A T-cell attack against hematopoietic stem cells (HSCs) is believed to be the principal mechanism underlying the development of acquired aplastic anemia (AA). The presence of leukocytes that lack HLA class I alleles as a result of the copy number-neutral loss of heterozygosity of the HLA haplotype due to uniparental disomy in the short arm of chromosome 6 (6pLOH) is compelling evidence for the involvement of cytotoxic T lymphocytes (CTLs) in the HSC destruction; this is based on the high response rate to immunosuppressive therapy (IST) in 6pLOH(+) patients (Katagiri, et al. Blood, 2011). However, target cells of the putative CTLs have not yet been characterized because of the small number of patients analyzed via flow cytometry (FCM) in our previous study. FCM can be substituted for SNP arrays by detecting HLA-A allele-lacking leukocytes (HLA-LLs) caused by 6pLOH. To gain insight into the CTL target responsible for the development of AA, we examined a total of 223 patients (213 idiopathic and 10 hepatitis-associated AA; 93 severe and 130 non-severe AA) for the presence of HLA-LLs and determined the lineage combinations of the aberrant leukocyte population. Of the 223 patients, 145 (65.0%) were heterozygous for the HLA-A allele and could be assessed for the presence of HLA-LLs by FCM. Eighteen (25.4%; 10 with severe AA and 8 with non-severe AA) of the 71 pre-treatment patients, and 26 (35.1%; 13 with severe AA and 13 with non-severe AA) of the 74 post-treatment patients were found to be positive for HLA-LLs. The lineage combinations of HLA-LLs in the 44 HLA-LL(+) patients were granulocytes (Gs), monocytes (Ms), B cells (Bs), and T cells (Ts, GMBT) in 13, GMB in 16 and GM in 11 patients. Surprisingly, HLA-LLs were found in Bs alone in three patients, and in one patient, the lineage combination pattern was TB (Figure). The presence of 6pLOH was confirmed via deep sequencing of isolated Bs from one of the three Bs alone patients. These lineage combination patterns were not observed to change for 1-40 months in 22 of 23 patients whose blood samples were available for follow-up analyses. In one patient with the GMB pattern, HLA-LLs decreased from 11.7% before treatment to 0% after 12 months of ATG therapy. The response rate to IST in GMBT patients (3/4, 75%) and in patients with GMB or GM (9/10, 90%) were similarly higher than in patients without HLA-LLs (22/39, 56%) or in patients who were homozygous for the HLA-A allele (23/36, 64%). Two of the three Bs alone patients showed complete responses at the time of sampling three years after ATG therapy and 20 years after CsA therapy, and another patient had a secondary myelodysplastic syndrome two years after response to ATG. The TB alone patient developed AA 20 years earlier, but had not been treated until recently because there was no need for blood transfusions, and is now improving in response to eltrombapag. This study revealed that the targets of putative CTLs in more than half of AA patients are hematopoietic progenitor cells with limited differentiation but long-lasting capacity, and in some patients, they are lymphoid progenitor cells that do not contribute to hematopoiesis. This suggests that CTL attack against non-HSCs including lymphoid precursors could trigger BM failure. Consistent with our previous report, the bystander effects caused by the immune response to non-HSCs such as myelosuppressive cytokines may play a major role in the development of AA. Disclosures No relevant conflicts of interest to declare.

The Truncated Form of Hoxa1 Regulates Proliferation and Differentiation of Hematopoietic Stem and Progenitor Cells.

Blood ◽  
2009 ◽  
Vol 114 (22) ◽  
pp. 3654-3654
Author(s):  
Jean Hendy ◽  
Stewart A Fabb ◽  
Meaghan Wall ◽  
Paul J Simmons ◽  
Lorraine J Gudas ◽  
...  
Keyword(s):  
Progenitor Cells ◽  
Peripheral Blood ◽  
Hox Genes ◽  
Cultured Cells ◽  
Conflicts Of Interest ◽  
Proliferative Potential ◽  
Hematopoietic Stem ◽  
Post Transplant ◽  
Proliferation And Differentiation

Abstract Abstract 3654 Poster Board III-590 Homeobox (Hox) genes have been shown to play critical roles in the regulation of hematopoiesis. Unlike most other Hox genes, Hoxa1 has two alternatively spliced transcripts: full-length Hoxa1 (Hoxa1-993) and truncated Hoxa1 (Hoxa1-399), which is similar to Hoxa1-993 but lacks the homeobox domain. Roles for either of these Hox transcripts in hematopoiesis have not yet been described, and the function of Hoxa1-399 in organogenesis remains largely unknown. We found that Hoxa1-993 is expressed most strongly in hematopoietic stem cells (HSCs), less in progenitor cells and is absent in mature cells. Hoxa1-399, which is spliced within the exon of Hoxa1-993 and hence can be generated by Hoxa1-993-expressing cells, is more prominently expressed in progenitor cells than HSCs. Wildtype (WT) Hoxa1-overexpressing bone marrow (BM) cells (WT-Hoxa1-993/399 cells, abbreviated here as WT-Hoxa1) proliferated for up to 15 weeks in ex vivo culture, increasing in cell number by an average of 240-fold weekly. These cultured cells predominantly expressed Hoxa1-993, but also expressed low levels of Hoxa1-399 due to splicing occurring within the exon, and were polyclonal with various integration sites. In contrast, control- and Hoxa1-399-overexpressing BM cells proliferated for a maximum of 3 weeks, with average weekly increases of 50-fold and 7-fold respectively. Colony-forming cells (CFCs) generated by WT-Hoxa1 GFP+ BM cells had approximately 8-fold increased numbers of cells compared to control and Hoxa1-399 GFP+ BM cells (cells/CFC: WT-Hoxa1: 1.3 × 106 ± 1 × 105; control: 1.6 × 105 ± 3.9 × 104; Hoxa1-399: 3.3 × 105 ± 3.1 × 104 n=5, P<0.05 WT-Hoxa1 vs control and Hoxa1-399). There was no difference in the number of day 12 colony-forming unit-spleen (CFU-S) formed from 2500 control or WT-Hoxa1 GFP+ BM cells (11.2±0.5 and 12.6±1.3 respectively). In contrast, 2500 Hoxa1-399 GFP+ BM cells produced significantly fewer CFU-S (8.9±0.6) compared to both control and WT-Hoxa1 GFP+ BM cells (n=4, P<0.02). To assess HSC potential, lethally irradiated CD45.2+ recipients (n=6/group) were injected with 5×106 congenic CD45.1+ BM immediately post-transduction without selection (all groups had similar transduction efficiencies). All recipients had >80% donor cells (CD45.1+, GFP+/−) in their peripheral blood (PB) post-transplant. As early as 5 weeks post-transplant the average %GFP+ cells in recipients were similar for control- (28.7±4.3%) and WT-Hoxa1-overexpressing BM cells (26.4±2.0%) and multi-lineage repopulating potential in both populations persisted for 6 months post-transplant. Strikingly, BM cells overexpressing Hoxa1-399 had markedly reduced repopulating ability as early as 5 weeks post-transplant (4.1±0.6% GFP+, P<0.05 Hoxa1-399 vs. control or WT-Hoxa1), which declined further during 6 months of transplant. To further explore the role of Hoxa1-993 and Hoxa1-399 in hematopoiesis, we created a mutant Hoxa1 that expressed Hoxa1-993 but was no longer capable of generating Hoxa1-399 (muHoxa1-993). MuHoxa1-993-overexpressing BM cells had extensive proliferative potential in culture and produced approximately 2-fold more cells per CFC than WT-Hoxa1 (cells/CFC: muHoxa1-993: 2.1 × 106 ± 2 × 105; P<0.05 vs WT-Hoxa1, Hoxa1-399 and control). MuHoxa1-993-overexpressing HSCs had long-term multi-lineage repopulating potential but generated significantly increased numbers of CD4+ T lymphocytes accompanied by reduced numbers of B220+ B lymphocytes (P<0.005). Most strikingly, mice transplanted with muHoxa1-993-overexpressing BM cells exhibited thrombocytopenia as early as 5 weeks post-transplant (platelet counts (x 106) per ml of PB: control: 640 ± 79; WT-Hoxa1: 735 ± 97; muHoxa1-993: 231 ± 36; P<0.005 muHoxa1-993 vs control and WT-Hoxa1). This thrombocytopenia persisted long-term and inversely correlated with the %GFP+ muHoxa1-993-overexpressing cells detectable in the peripheral blood of the transplanted mice. Preliminary data suggest the thrombocytopenia occurs because of an impairment in megakaryocyte maturation. These data therefore suggest that both Hoxa1-993 and Hoxa1-399 have regulatory roles in hematopoiesis. Furthermore, a balance in the expression of the two Hoxa1 transcripts is essential for normal proliferation and differentiation of HSCs and progenitor cells. Disclosures: No relevant conflicts of interest to declare.

Loss of Foxo3a In FA Mice Leads to Hematopoietic Stem Cell Exhaustion That Can Be Attenuated by Natural Anti-Oxidant Quercetin.

Blood ◽  
2010 ◽  
Vol 116 (21) ◽  
pp. 1166-1166
Author(s):  
Jie Li ◽  
Jared Sipple ◽  
Qishen Pang
Keyword(s):  
Reactive Oxygen Species ◽  
Progenitor Cells ◽  
Peripheral Blood ◽  
Conflicts Of Interest ◽  
Genetic Disorder ◽  
Specific Interaction ◽  
Reactive Oxygen ◽  
Oxygen Species ◽  
Hematopoietic Stem ◽  
Anti Oxidant

Abstract Abstract 1166 Fanconi anemia (FA) is a genetic disorder characterized by genomic instability, bone marrow (BM) failure and predisposition to cancer. However, FA mouse models do not show spontaneous genetic instability. Previous study shows that FOXO3a is associated with the FA pathway through oxidative stress-specific interaction with FANCD2. To address the consequence of loss of FOXO3a function in FA hematopoiesis, we generated Foxo3a-/-Fancd2-/- and Foxo3a-/-Fancc-/- double-knockout (DKO) mice by crossing Foxo3a+/− with Fancd2+/− or Fancc+/−; mice. Reactive oxygen species are increased in low-density BM (LDBM) cells isolated from DKO mice compared to those from single KO (SKO) or wt mice. Analysis of hematologic parameters shows significantly increased number of nucleate cells and high ratio of eosinophils in peripheral blood of DKO mice. CFU assay shows more progenitor cells in peripheral blood isolated from DKO mice. Moreover, BM progenitor cells from DKO mice exhibit lower adhesion but higher migration activity, compared to those from wt or SKO mice. Consistent with this, Cdc42 pull-down assay shows lower Cdc42 activity in DKO LDBM cells than in wt or SKO cells, indicating that decreased Cdc42 may contribute to the observed aberrant adhesion and migration activities. DKO mice show significant decrease in primitive progenitor (Lin-Sca-1+c-kit+; LSK) cells, increase in BrdU+ and G1-phase LSK cells, and impaired repopulating capacity after competitive BM transplantation, which can be attenuated by the anti-oxidant Quercetin. Taken together, loss of Foxo3a in FA mice results in FA-like syndrome, which may be resulted from increased reactive oxygen species accumulation. Disclosures: No relevant conflicts of interest to declare.

Cell-Intrinsic and Cell-Extrinsic Involvement Of C/EBPβ In The Regulation Of Hematopoietic Stem Cells

Blood ◽  
2013 ◽  
Vol 122 (21) ◽  
pp. 1202-1202
Author(s):  
Akihiro Tamura ◽  
Hideyo Hirai ◽  
Yoshihiro Hayashi ◽  
Asumi Yokota ◽  
Atsushi Sato ◽  
...  
Keyword(s):  
Stem Cells ◽  
Steady State ◽  
Hematopoietic Stem Cells ◽  
Progenitor Cells ◽  
Peripheral Blood ◽  
Leucine Zipper ◽  
Conflicts Of Interest ◽  
Chronic Phase ◽  
Hematopoietic Stem ◽  
The Difference

Abstract Our previous findings have revealed the requirement of CCAAT Enhancer Binding Protein β (C/EBPβ), a leucine zipper transcription factor, in emergency granulopoiesis (Hirai et al. Nat Immunol, 2006). During emergency situations such as infection, C/EBPβ is involved in the sufficient supply of granulocytes through amplification of hematopoietic stem/progenitor cells (Satake et al. J Immunol, 2012). In addition, we have shown that C/EBPβ is upregulated by downstream signaling of BCR-ABL and promotes myeloid expansion and leukemic stem cells exhaustion in chronic phase chronic myeloid leukemia (Hayashi et al. Leukemia, 2013). These observations suggested that C/EBPβ plays important roles in normal hematopoietic stem cells (HSCs). Here we investigated the cell-intrinsic and -extrinsic function of C/EBPβ in the regulation of HSCs by analyzing C/EBPβ knockout (KO) mice. At steady state, no obvious defects have been reported in hematopoiesis of C/EBPβ KO mice. Accordingly, the frequencies of long-term and short-term HSCs and various kinds of progenitor cells in bone marrows (BM) of C/EBPβ KO mice were identical to those in BM of wild type (WT) mice. To examine the functional consequences of C/EBPβ deletion, competitive repopulation assay was performed. In brief, 5x105 BM cells from WT or C/EBPβ KO mice (CD45.2+) and the same number of competitor CD45.1+ BM cells were transplanted into lethally irradiated CD45.1+ mice and the chimerisms of CD45.2+ cells in the peripheral blood of the recipient mice were monitored monthly. The chimerisms of C/EBPβ KO cells were significantly lower than that of WT cell at 1 month after transplantation and the differences were maintained thereafter (Figure A). In order to elucidate the reason for the difference, homing ability of C/EBPβ KO cells were assessed. Lineage depleted CD45.2+ WT or C/EBPβ KO BM cells together with the equal number of lineage negative CD45.1+ BM cells were transplanted into lethally irradiated CD45.1+ mice and the frequencies of CD45.2+ cells were analyzed 16 hours after transplantation. The frequencies of CD45.2+ WT and C/EBPβ KO donor cells in the recipient BMs were identical and the data indicated that the differences in the chimerisms after primary BM transplantation were due to the difference in the initial expansion of transplanted cells after equivalent levels of homing. To see the roles of C/EBPβ in hematopoiesis under stressed conditions, CD45.1+ mice were transplanted with CD45.2+ WT or C/EBPβ KO BM cells with equal numbers of CD45.1+ BM cells and these mice were administered with 150mg/kg 5-fluorouracil (5-FU) once a month and the chimerisms of peripheral blood were monitored every time before the next 5-FU administration. In consistent with the results mentioned above, the frequencies of CD45.2+ C/EBPβ KO cells were significantly lower than those of CD45.2+ WT cells 1 month after transplantation. After repetitive administration of 5-FU, however, the chimerisms of CD45.2+ C/EBPβ KO cells gradually caught up with those of CD45.2+ WT cells, suggesting that C/EBPβ is involved in the exhaustion of HSCs under stressed conditions (Figure B). To explore the functions of C/EBPβ in hematopoietic microenvironments, 1x106 CD45.1+ BM cells from WT mice were transplanted into irradiated (5Gy or 7Gy) WT or C/EBPβ KO mice (CD45.2+). All the WT recipient mice survived after 5Gy or 7Gy irradiation (4/4 and 4/4, respectively). In contrast, only 2/4 and 1/4 C/EBPβ KO recipient mice survived after 5Gy or 7Gy irradiation, respectively. We are currently trying to identify the cells expressing C/EBPβ in BM microenvironments and investigating the mechanisms for the higher sensitivity of C/EBPβ KO mice to irradiation. In summary, these data suggested that C/EBPβ is required for initial expansion of hematopoietic stem/progenitor cells at the expense of HSCs under stressed conditions, while it is dispensable for maintenance of HSCs at steady state. We are now investigating the cellular and molecular targets of C/EBPβ in HSC regulation and would like to elucidate the cell-intrinsic and cell-extrinsic mechanisms in regulation of the homeostasis of hematopoietic system by C/EBPβ. Disclosures: No relevant conflicts of interest to declare.

A Novel Observation That Heme Oxygenase-1 (HO-1) Deficient Mice Are Easy Mobilizers and That HO-1 Plays An Important Role in Maintaining Expression of SDF-1 in Bone Marrow (BM) Stroma and Promotes Retention of Hematopoietic Stem/Progenitor Cells (HSPCs) in the Bone Marrow Microenvironment

Blood ◽  
2011 ◽  
Vol 118 (21) ◽  
pp. 315-315
Author(s):  
Marcin Wysoczynski ◽  
Janina Ratajczak ◽  
Gregg Rokosh ◽  
Roberto Bolli ◽  
Mariusz Z Ratajczak
Keyword(s):  
Bone Marrow ◽  
Progenitor Cells ◽  
Heme Oxygenase ◽  
Peripheral Blood ◽  
Conflicts Of Interest ◽  
Mrna Level ◽  
Heme Oxygenase 1 ◽  
Hematopoietic Stem ◽  
Normal Peripheral Blood ◽  
Cell Counts

Abstract Abstract 315 Background. Heme oxygenase (HO) is an enzyme that catalyzes the degradation of heme. Two distinct HO isoforms have been identified: HO-2, which is constitutively expressed, and HO-1, which is stress-responsive and plays an important function in various physiological and pathophysiological states associated with cellular stress. HO-1 plays a role in ischemic/reperfusion injury, atherosclerosis, and cancer. It has also been reported that HO-1 regulates expression of a-chemokine stromal derived factor-1 (SDF-1) in myocardium (J Mol Cell Cardiol.2008;45:44–55). Aim of study. Since SDF-1 plays a crucial role in retention and survival of hematopoietic stem cell/progenitor cells (HSPCs) in BM, we become interested in whether deficiency of HO-1 affects normal hematopoiesis and retention of HSPCs in BM. Experimental approach. To address this issue, we employed several complementary strategies to investigate HO-1−/−, HO+/–, and wild type (wt) mouse littermates for i) the expression level of SDF-1 in BM, ii) the number of clonogenic progenitors from major hematopoietic lineages in BM, iii) peripheral blood (PB) cell counts, iv) chemotactic responsiveness of HSPCs to an SDF-1 gradient, iv) adhesiveness of clonogenic progenitors, v) the number of circulating HSPCs in PB, and vi) the degree of mobilization in response to granulocyte-colony stimulating factor (G-CSF) or AMD3100 assessed by enumerating the number of CD34–SKL cells and clonogeneic progenitors (CFU-GM) circulating in PB. Results: Our data indicate that under normal, steady-state conditions, HO-1−/− and HO+/– mice have normal peripheral blood cell counts and numbers of circulating CFU-GM. Interestingly, lack of HO-1 leads to an increase in the number of erythroid (BFU-E) and megakaryocytic (CFU-GM) progenitors in BM. Next, BMMNCs from HO-1−/−have normal expression of the SDF-1-binding receptor, CXCR4, but a 5-times lower level of CXCR7, which is another SDF-1-binding receptor. Of note, we observed that the mRNA level for SDF-1 in BM-derived fibroblasts was ∼4 times lower. This corresponded with the observation in vitro that HSPCs from HO-1−/−animals responded more robustly to an SDF-1 gradient, and HO-1−/−animals mobilized a higher number of CD34–SKL cells and CFU-GM progenitors into peripheral blood in response to G-CSF and AMD3100. Conclusions: Our data demonstrate for the first time that heme oxygenase plays an important and underappreciated role in BM retention of HSPCs and may affect their trafficking. Since small non-toxic molecular inhibitors of HO-1 have been developed for clinical use (e.g., metaloporhirins), blockage of HO-1 could be a novel strategy for mobilizing HSPCs. Our recent in vivo mobilization studies lend support to this hypothesis. Disclosures: No relevant conflicts of interest to declare.

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