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The CD44 cell surface molecule has been shown to be the principal cell surface receptor for hyaluronan (or hyaluronic acid), a glycosaminoglycan component of marrow extracellular matrix. However, its affinity for hyaluronan is not constitutive, since it depends on the cell type, the stage of differentiation and on activation by external stimuli including certain anti-CD44 antibodies and phorbol esters. Except for a few lymphoid cell lines, hematopoietic cells do not spontaneously bind hyaluronan and initial studies reported that, contrary to lymphocytes, myeloid cells could not be activated to bind hyaluronan. Because CD44 plays an important role in the initial phases of hematopoiesis, as shown by experiments using blocking anti-CD44 monoclonal antibodies, its capacity to mediate adhesion of primitive myeloid cells has been investigated. It was found that CD44 could mediate spontaneous adhesion to hyaluronan of immature myeloid cell lines KG1, KG1a, and TF1, which serve as a model for hematopoietic progenitors. However, despite expressing high amounts of CD44, no more than 15% of bone marrow progenitors could adhere to hyaluronan. Recent experiments have shown that a very important feature of CD44 is its capacity to be rapidly activated by certain antibodies and cytokines (GM-CSF and KL) from a low affinity to a high affinity state for hyaluronan. These data shed light on striking similarities in the functional regulation of CD44 and of the two integrin receptors VLA-4 (a4b1), and VLA-5 (a5b1), which are also expressed on hematopoietic progenitors. The relevance of these data to the regulation of normal hematopoiesis and mobilization of CD34+ progenitors in the view of cell grafting is analyzed. In addition, we show that in idiopathic myelofibrosis, the amount of hyaluronan is markedly increased in the extracellular matrix from the myeloproliferative spleen. Considering that the production of cytokines is enhanced in this disease, we discuss whether CD44-hyaluronan interaction may have a role in the pathophysiology of this myeloproliferative syndrome.

Cytokines are known to be important regulators of normal hemopoiesis, acting in concert with components of the bone marrow microenvironment. Interactions with this microenvironment are known to regulate the proliferation, differentiation, and homing of hemopoietic progenitor (CD34+) cells. Adhesive interactions with the extracellular matrix retain CD34+ cells in close proximity to cytokines, but may also provide important costimulatory signals. Thus, the functional states of adhesion receptors are critical properties of CD34+ cells, but the physiological mechanisms responsible for regulating functional properties of cell adhesion receptors on primitive hemopoietic cells are still unknown. We confirm that the integrins very late antigen (VLA)-4 and VLA-5 are expressed on the CD34+ cell lines MO7e, TF1, and on normal bone marrow CD34+ progenitor cells, but in a low affinity state, conferring on them a weak adhesive phenotype on fibronectin (Fn). Herein, we show that the cytokines interleukin (IL)-3, granulocyte-macrophage CSF (GM-CSF), and KIT ligand (KL) are physiological activators of VLA-4 and VLA-5 expressed by MO7e, TF1, and normal bone marrow CD34+ progenitor cells. Cytokine-stimulated adhesion on Fn is dose dependent and transient, reaching a maximum between 15 and 30 min and returning to basal levels after 2 h. This cytokine-dependent activation is specific for VLA-4 and VLA-5, since activation of other beta 1 integrins was not observed. The addition of second messenger antagonists staurosporine and W7 abolished all cytokine-stimulated adhesion to Fn. In contrast, genistein inhibited KL-stimulated adhesion, but failed to inhibit GM-CSF- and IL-3-stimulated adhesion. Our data suggest that cytokines GM-CSF and IL-3 specifically stimulate beta 1 integrin function via an "inside-out" mechanism involving protein kinase activity, while KL stimulates integrin activity through a similar, but initially distinct, pathway via the KIT tyrosine-kinase. Thus, in addition to promoting the survival, proliferation, and development of hemopoietic progenitors, cytokines also regulate adhesive interactions between progenitor cells and the bone marrow microenvironment by modifying the functional states of specific integrins. These data are of importance in understanding the fundamental processes of beta 1 integrin activation and cellular response to mitogenic cytokines as well as on the clinical setting where cytokines induce therapeutic mobilization of hematopoietic progenitors.

Abstract 2402 The MYB oncogene is widely expressed in acute leukaemias and is important for the continued proliferation of leukemia cells, raising the possibility that MYB may be a therapeutic target. However realization of this potential requires (i) a significant therapeutic window for MYB inhibition, given its essential role in normal haemopoiesis; and (ii) an approach for developing an effective therapeutic. We previously showed that the interaction of Myb with the coactivator CBP/p300 is essential for Myb's intrinsic transforming activity (1). Here we use haemopoietic cells from the Booreana mouse strain, which carries a mutation in Myb that prevents interaction with CBP/p300 (see Fig. 1 and ref. 2), to examine the requirement for this interaction in myeloid transformation and leukaemogenesis. Using this strain and a strain (plt6) carrying a “complementary” mutation in p300 (3), we show that the Myb-p300 interaction is essential for in vitro transformation by the myeloid leukaemia oncogenes AML1-ETO, AML1-ETO9a, MLL-ENL, and MLL-AF9. We further show that unlike cells from wild-type (WT) mice, Booreana cells fail to induce leukaemia upon transplantation into irradiated recipients following transduction with an AML1-ETO9a retrovirus (Fig. 2). These data, as well as reinforcing the notion that MYB is an essential cofactor for myeloid leukaemogenesis, highlight disruption of the Myb-p300 interaction as a potential therapeutic strategy for AML. Furthermore, our results suggest that such a strategy would have a useable therapeutic index since Booreana mice, unlike Myb null mice, are viable. We have started to explore the use of small peptides to provide proof-of-principle for this approach. We have also begun to explore the molecular basis of the requirement for MYB, and the Myb-p300 interaction, in AML using gene expression profiling. This has highlighted several Myb target genes – identified by our previous ChIP-Seq analysis (4) - that have been previously implicated in myeloid leukaemogenesis, as being differentially expressed between WT and Booreana cells transduced with AML1-ETO9a. One striking example is the apparent Myb-p300 dependent activation of Ets2 and Gfi1, accompanied by repression of Gata2 and Gfi1b; this pattern has previously been reported in MLL-AF9 and MOZ-TIF-induced murine AML (5). Disclosures: No relevant conflicts of interest to declare.

Abstract 216 Up to 5% allogeneic healthy donors and up to 40–60% of chemotherapy-treated patients in autologous setting, fail to reach minimal threshold of 2×106 blood CD34+cells/kg in response to G-CSF, precluding transplantation. Plerixafor, a small inhibitor of the chemokine receptor CXCR4, used for 4 days in combination with G-CSF enables this minimal threshold to be reached in up to 60% patients who previously failed to mobilise in response to G-CSF alone. However, the remaining 40% of patients who failed to mobilise in response to G-CSF alone, still fail to mobilize adequately with G-CSF + Plerixafor. In an attempt to further boost HSC mobilization in response to combinations of G-CSF and Plerixafor, we have investigated the role of the hypoxia-sensing pathway in HSC mobilization. HIF-1α (Hypoxia-inducible factor-1α) controls HSC proliferation and self-renewal in poorly perfused hypoxic bone marrow (BM) niches where very quiescent HSC with highest self-renewal potential reside. When O2 concentration is above 2% in the cell microenvironment, HIF-1α protein is rapidly hydroxylated on Pro residues by prolyl hydroxylases PHD1-3. This recruits the E3 ubiquitin ligase VHL, which targets HIF-1α to rapid proteasomal degradation. When O2concentration is below 2% (hypoxia), PHDs are inactive; HIF-1α protein is stabilized, associates with its β subunit ARNT, translocates to the nucleus and activates of transcription and hypoxia-responsive genes. In this study, we have investigated the effect of pharmacological stabilization of HIF-1α protein on HSC mobilization in mice using the HIF-PHD inhibitor FG-4497. We report that FG-4497 treatment stabilizes HIF-1α protein in mouse BM. We find that FG-4497 synergizes with G-CSF and Plerixafor to enhance HSC mobilization. C57/Bl6 mice were in 4 treatment groups: (G) 250μg/kg/day G-CSF alone for 2 days; (GF) G-CSF for 2 days + 20mg/kg/day FG-4497 for 3 days; (GP) G-CSF for 2 days together with16mg/kg Plerixafor 1 hour prior harvest; (GPF) G-CSF together with Plerixafor and FG-4497 with same dosing as above. Mobilization of colony-forming cells (CFC), phenotypic Lin-CD41-Sca1+Kit+CD48-CD150+ HSC, and functional HSC in long-term competitive transplantation assays were measured. Mice in the GF group (G-CSF + FG-4497) mobilized CFC to the blood 4-fold and phenotypic HSC 3-fold more than mice mobilized with G-CSF alone (p To determine which cell types drive HSC mobilization in a HIF-1α-dependant manner, we crossed HIF1αflox/floxmice with osxCre (HIF-1α gene deletion in osteoprogenitors), LysMCre (deletion in myeloid cells), or with SclCreER mice (tamoxifen-induced deletion in HSC). While studies in LysMCre and SclCreER mice are ongoing, we find that deletion of HIF-1α gene in osteoprogenitors (osxCre mice) decreased 2.5-fold the number of CFU/mL blood following 2 and 3 days treatment with G-CSF. This suggests that HIF-1α in osteoprogenitors and their osteoblastic progenies is necessary for optimal mobilization in response to G-CSF. In conclusion, our data highlight the importance of HIF-1α in HSC mobilization and provide a novel therapeutic strategy for increasing HSC mobilization above levels obtained with combinations of G-CSF and Plerixafor. Thus PHD inhibitors could be useful agents in patients who still fail to mobilize in response to G-CSF and plerixafor. Disclosures: Walkinshaw: Fibrogen Inc.: Employment, Equity Ownership.

Abstract 1889 We have previously shown that G-CSF administration impairs HSC niches in the mobilized bone marrow (BM). G-CSF causes rapid suppression (within 2 days) of endosteal osteoblasts and bone formation with concomitant down-regulation of Kit ligand, CXCL12 and angiopoietin-1. This effect is mediated by the depletion of specific populations of BM macrophages1. Considering the very rapid impairment of HSC niches in response to G-CSF, we hypothesized that 1) the most primitive HSC should also mobilize at this very early stage within the first 48 hours of G-CSF treatment, and 2) that down-regulation of HSC niche function should also alter the number or function of HSC remaining in the mobilized BM. To test this, 125μg/kg rhuG-CSF was injected twice daily to C57BL/6 mice; blood and BM harvested at days 2 and 5 of G-CSF treatment to be transplanted into congenic recipients in a long-term competitive repopulation assay (LT-CR). Transplantation of 25μL blood showed a gradual increase in the number of LT-CR cells mobilized in response to G-CSF as measured by donor chimerism in myeloid and B lineages at 16 weeks post-transplant. Expectedly repopulating units (RU) per mL blood progressively increased from 0.2 ± 0.0 (n=6) in steady-state to 2.9 ± 1.6 (n=9) and 82.6 ± 40.4 (n=9) at days 2 and 5 of G-CSF treatment respectively. At 16 weeks post-transplant, BM from primary recipients were transplanted into secondary recipients. Surprisingly, secondary recipients of blood samples collected after 2 and 5 days of G-CSF treatment had equivalent levels of donor chimerism (37.2% ± 6.6% for 2 days G-CSF and 47.1% ± 7.8% for 5 days G-CSF, n = 8 per group). Therefore, although the absolute number of RU mobilized at day 2 of G-CSF was 28-fold lower than at day 5 of G-CSF administration, more primitive serially reconstituting HSC were mobilized at equivalent levels at days 2 and 5 of G-CSF treatment. This supports our hypothesis that most potent serially reconstituting HSC are mobilized as early as day 2 of G-CSF treatment consistent with the disappearance of osteoblasts1. To test the potential of HSC remaining in the BM, BM cells from G-CSF mobilized mice were transplanted in competition with BM cells from congenic mice in steady-state. Donor chimerism at 16 weeks post-transplant showed that competitive repopulation of BM cells was severely impaired at day 5 of G-CSF treatment with the number of RU per 200,000 BM cells decreasing from 4.1 ± 1.4 in steady-state and 5.2 ± 1.6 at day 2 of G-CSF treatment, to only 0.14 ± 0.05 at day 5 of G-CSF treatment. To test whether this 29-fold decrease in competitive repopulation was due to increased HSC proliferation, we measured BrdU incorporation for the last 2.5 days prior to BM harvest as well as cell cycle analysis with Ki67 and Hoechst33342. The proportion of quiescent Lin- Sca1+ Kit+ CD48- phenotypic HSC in G0 phase decreased from 62.8 ±4.0% in steady-state to 43.5±8.2% at day 2 of G-CSF, but surged back to 80.5±1.9% and 75.1±3.5% at days 3.5 and 5 of G-CSF treatment. The proportion of HSC in G1 and S/G2/M phases followed the opposite pattern, up at day 2, down at days 3.5 and 5. This was confirmed by BrdU incorporation for 2.5 days with the number of BrdU+ cells among Lin- Sca1+ KIT+ CD48- cells rising from 35.1±4.0% in steady-state, to 51.2±4.5% at day 2 of G-CSF and going down to 18.1±1.9% at day 3.5 and 23.3±5.5% at day 5 of G-CSF. Therefore, G-CSF recruits phenotypic HSC into cell cycle within the first 2 days of administration, but HSC return to quiescence despite continuing G-CSF. Therefore decreased repopulation potential at day5 of G-CSF is not due to increased cycling. Finally, we noted that the number of Lin-Sca1+KIT+CD48-CD150+ HSC and Lin-Sca1+KIT+CD48-CD150- multipotent progenitors were reduced 2.4- and 2.8-fold respectively (p Disclosures: No relevant conflicts of interest to declare.

Abstract 2334 The endosteal region of the bone marrow (BM) is hypoxic in steady-state and most quiescent hematopoietic stem cells (HSC) reside in hypoxic, poorly perfused niches. Mobilizing doses of G-CSF renders most of the BM space hypoxic. Most cellular effects of hypoxia are mediated by O2-labile hypoxia-inducible transcription factors (HIF). At O2 concentration above 2%, HIF-α is rapidly hydroxylated on Pro residues by the prolyl hydroxylases PHD1, PHD2, and PHD3. HIF-α prolyl hydroxylation recruits the E3 ubiquitin ligase VHL, which targets HIF-α to the proteasome. In hypoxia (O2 < 2%), HIF-α proteins are stable, associate with the β subunit ARNT, and translocate to the nucleus to activate transcription. It has emerged that HIF-1α regulates HSC proliferation and is critical to maintain long-term HSC self-renewal in vivo. In this study, we investigated the effect of pharmacological stabilization of HIF-1α protein on HSC cycling and mobilization in mice using two different HIF prolyl hydroxylase (PHD) inhibitors, dimethyloxalyl glycine (DMOG) and FG-4497. We first assessed whether DMOG and FG-4497 stabilized HIF-1α protein in BM leukocytes in vivo by western-blot. Following a single injection of 400mg/kg DMOG, HIF-1α protein was stabilized for up to 6 hrs in BM leukocytes. With 20mg/kg FG-4497, HIF-1α protein persisted over 12 hours. HIF-1α protein was below detection in the BM from saline injected animals. C57BL/6 mice were injected daily with 400mg/kg DMOG to measure effect on HSC cycling and BrdU incorporation by flow cytometry. Using Hoecht33342 and FITC-conjugated anti-Ki67 antibody, a 18 day DMOG treatment increased the proportion of Lin-negative Kit+ Sca1+ CD48- (L-K+S+48-) HSC in phase G0 from 61±11% to 84±6% (p Since HIF-PHD inhibitors slow HSC cycling in vivo, we tested whether they could protect HSC from sublethal irradiation. Mice were treated with 400mg/kg DMOG or saline for 22 days and then irradiated with 9.0Gy. Both cohorts were leukopenic betweens days 7 and 14 post-irradiation but DMOG treated mice had significantly higher blood leukocytes at days 22 and 30, and higher platelet numbers day 22 and all subsequent time-points suggesting that DMOG enhances HSC survival with higher blood recovery. Finally mice were injected with DMOG for 4 days and pegylated rhuG-CSF 3 and 1.5 days before harvest. DMOG doubled mobilization in response to G-CSF with 1,620±530 CFC / mL blood in response G-CSF alone, and 3,250±830 CFC / mL in the G-CSF + DMOG group (p In conclusion, these data highlight the importance of the hypoxia pathway and HIF in the regulation of HPSC cycling and trafficking in vivo. Furthermore HIF-PHD inhibitors may provide therapeutic opportunities to protect HSC from deleterious effects of irradiation as well as increasing mobilization efficiency for transplantation. Disclosures: Walkinshaw: Fibrogen Inc.: Employment, Equity Ownership.

Using optimal culture conditions in which the transforming growth factor beta 1 (TGF-beta 1) inhibitory loop has been interrupted by antisense TGF-beta 1 oligonucleotides or anti-TGF-beta serum, we have compared the proliferative capacities and the abilities of the CD34+ CD38- cell populations from bone marrow and umbilical cord blood to generate early progenitors in long-term cultures. The CD34+ CD38- fraction of umbilical cord blood accounts for 4% of the CD34+ fraction compared to only 1% in bone marrow, indicating that umbilical cord blood may be relatively enriched in stem cells. We estimate that the CD34+ CD38- cells from a typical umbilical cord blood sample produce equivalent numbers of colony-forming units (CFU)-granulocyte/erythrocyte/macrophage/megakaryocyte, twice as many CFU-granulocyte/macrophage (GM) and 3 times as many burst-forming units-erythroid as the same population from an average bone marrow sample used in adult transplantation. In addition, the colonies resulting from the umbilical cord blood samples were significantly larger than those from bone marrow, indicating a greater growth potential. However, the content of later progenitors, which may be important for short-term reconstitution, was less in umbilical cord blood-derived than in bone marrow-derived cell preparations, as estimated by a 4-fold lower production of CFU-GM in long-term cultures of CD34+ CD38+ cells. This deficit is partially compensated by the higher growth capacity of the resulting CFU-GM. These studies suggest that umbilical cord blood is a suitable source of cells for adult transplantation.

Hematopoietic stem cells (HSC) reside in specialized niches in the bone marrow (BM), that regulate their survival, proliferation and differentiation. Two types of HSC niches have been reported: endosteal niches in close contact with osteoblasts, and endothelial niches near vascular sinuses. Whether these niches have distinct functions in controlling HSC fate remains unknown. One difference between these two niches is the constitutive expression of E-selectin and P-selectin by BM endothelial cells. E- and P-selectin are two cell adhesion molecules that modulate hematopoietic progenitor cell (HPC) survival, proliferation and differentiation in vitro. We now show that deletion of E-selectin, but not P-selectin, delays HSC turn-over in the BM in vivo. Mice lacking either E-selectin (E−/ −), P-selectin (P−/ −) or both (PE−/ −) were given bromo-deoxyuridine (BrdU) in their drinking water for up to 14 days. Lineage-negative c-KIT+ Sca-1+ CD34− (LKS34) cells were sorted from the BM and stained for BrdU incorporation into genomic DNA. Although it took only 3.6 days for 50% of LKS34 cells from wild-type (WT) and P−/ − mice to incorporate BrdU, 9 days were required for 50% BrdU incorporation in LKS34 cells from E−/ − and PE−/ − double KO mice. Thus, HSC cycling time is 2.5 times slower in the absence of E-selectin. To confirm these findings, LKS cells were stained with rhodamine123, a vital dye that is retained by metabolically active cells but effluxed from quiescent HSC. A higher proportion of LKS cells from E−/ − mice were rhodamine dull (34±2%) than WT LKS (23±1%; p=0.037) confirming that a greater proportion of HSC from E−/ − mice are quiescent. To further support these findings, we determined the effect of E-selectin deletion on HSC recovery following cytotoxic stress with a single dose of 5-fluorouracil (5FU 150mg/kg). As KIT is strongly down-regulated in the BM of 5FU-treated mice, we examined frequency and BrdU incorporation in Lin− Sca1+ CD41− CD48− CD150+ long-term reconstituting HSC. We found HSC recovery to be enhanced in E−/ − mice with a 5-fold increase in HSC numbers per femur compared to WT mice at day 7 post-5FU. Despite the more rapid recovery of E−/ − HSC, BrdU incorporation remained significantly lower in E−/ − HSC on days 3 and 7 post-5FU suggesting the decreased HSC turn-over in the absence of E-selectin protects them from the cytotoxic effect of 5FU. To determine whether this effect was mediated by the two described E-selectin receptors PSGL-1 and/or CD44, BrdU incorporation experiments were repeated with mice lacking both the PSGL-1 and CD44 genes. LKS cell turnover in these mice was identical to that of WT suggesting that the effect is mediated by a distinct unknown receptor(s) on HSC. The fact that a novel E-selectin receptor on HSC/HPC is involved was confirmed both using flow cytometry with selectin-IgM chimeras as well as cell adhesion assays using plastic-adsorbed selectin-IgG chimeras. In both assays, 90-95% of LKS cells from CD44−/ − PSGL-1−/ − double KO mice bound E-selectin whereas adhesion to P-selectin was completely lost. Taken together our findings suggest that E-selectin, whose constitutive expression is restricted to BM endothelial cells, plays an important role in the regulation HSC turnover in vivo, endothelial niches, where E-selectin is expressed, support more rapid HSC turn-over within the BM, and this effect is mediated by unknown E-selectin receptors distinct from PSGL-1 or CD44.

Although the molecular mechanisms responsible for hematopoietic stem cell (HSC) mobilization in response to G-CSF or chemotherapy have not been fully elucidated, various murine models demonstrate that both the expansion of the neutrophil pool within the bone marrow (BM) and the down-regulation of the chemokine CXCL12/SDF-1 play a critical role. The main function of neutrophils is to destroy pathogens and necrotic tissues during inflammation. Inflamed and damaged tissues are often very hypoxic due to local disruption of blood supply. Although most cells are stressed in hypoxic conditions, neutrophils and macrophages are attracted and activated by hypoxia, a property dependent on the stabilization of hypoxia-inducible transcription factor-1α (HIF-1α) which is absolutely required for neutrophil and macrophage function. Mice with a conditional deletion of the HIF-1α gene specifically in myeloid cells cannot mount an inflammatory response with neutrophils and macrophage displaying impaired motility, invasiveness and bacterial killing. We therefore hypothesized that hypoxia may play a major role in HSC mobilization. Considering that granulocyte/monocyte progenitors consume 30 times more O2 than their fully differentiated progeny and that mobilized BM is enriched in those progenitors, we calculated that mobilized BM should consume twice more O2 than steady-state BM (3.40μmole O2/femur/hour vs 1.64μmole O2/femur/hour in the mouse). According to the Krogh’s model that predicts O2 distribution in solid tissues, this increased O2 consumption should lead to O2 exhaustion resulting in hypoxia within mobilized BM. We demonstrated this in vivo by injecting the hypoxia-specific probe pimonidazole hydrochloride into mobilized and non-mobilized BALB/c mice 3 hours prior to sacrifice. Pimonidazole covalently binds to tissue proteins when O2 tension is below 10mmHg (O2 tension is 95mmHg in arterial blood, 40mmHg in venous and capillary blood and 150mmHg in the atmosphere at sea level). Staining of femoral BM with a monoclonal antibody (mAb) specific for pimonidazole showed that the hypoxic area (≤10mmHg or

Mobilization of hematopoietic stem cells (HSC) involves the disruption of 1) the adhesive interaction between VCAM-1 and α4-integrins and 2) the chemotactic interaction between CXCL12 and CXCR4, interactions which are both required for the retention of HSC within the bone marrow (BM). Experiments in mice deficient in neutrophil proteases have shown that while the disruption of the VCAM-1/α4 integrin interaction is entirely due to the proteolytic cleavage of VCAM-1 by proteases released from neutrophils accumulating in mobilized BM, the down-regulation of CXCL12 involves protease-independent mechanisms. We have recently shown that osteoblasts are the main source of CXCL12 in the BM and that both the number of osteoblasts lining the endosteum and bone formation are dramatically reduced during G-CSF administration as reflected by bone pain often experienced by mobilized donors. Consequently, the decreased level of CXCL12 levels in mobilized BM could be due to inhibition of osteoblasts, an essential component of the hematopoietic niche. Quantitative real-time RT-PCR were performed on mouse BM cells to follow osteocalcin mRNA levels. Osteocalcin mRNA dropped 2–3 logs during mobilization induced by either G-CSF or cyclophosphamide showing that the inhibition of osteoblast function is not restricted to G-CSF-induced mobilization. Morphometric analyses of tibia sections showed a quasi disappearance of osteoblasts and osteoid as early as day 2 of G-CSF injection. In humans, we observed a significant reduction of osteocalcin protein concentration in the plasma during mobilization induced by either G-CSF alone, G-CSF+ KIT ligand or IL3+GM-CSF, showing that in both humans and mice this effect is not restricted to G-CSF. In cultures of purified human osteoblasts, neither G-CSF, KIT ligand, IL3 nor GM-CSF inhibited osteocalcin production demonstrating that inhibition of osteoblast function is not a direct effect of these cytokines. In parallel experiments, addition of differentiated BM CD34− leukocytes to osteoblast cultures, resulted in a dose-dependant inhibition of osteocalcin production showing that the effect is mediated by mature leukocytes. Since IL6 and soluble IL6 receptor (sIL6R) are important mediators of bone formation, we tested these two cytokines on purified osteoblasts and found that the combination of IL6+sIL6R was a potent inhibitor of osteocalcin production while these cytokines had no effect when used alone. Furthermore, we find cocultures of osteoblasts and BM leukocytes results in a 30-fold increase in IL6 production compared to monocultures of osteoblasts or BM leukocytes. Finally, in humans, plasma concentration of sIL6R is significantly increased during HSC mobilization and this increase is significantly correlated with the number of circulating CFU-GM. Taken together, these data indicate that the inhibition of osteoblast function during HSC mobilization is an indirect effect involving mature BM leukocytes, IL6 and sIL6R.

There is accumulating evidence that interaction of stromal cell derived factor-1 (SDF-1/CXCL12) with its cognate receptor, CXCR4, generates signals that regulate hematopoietic progenitor cell (HPC) trafficking in the bone marrow. During G-CSF induced HPC mobilization, SDF-1 protein expression in the bone marrow decreases, thereby attenuating CXCR4 signaling. We recently reported that G-CSF treatment induced a decrease in bone marrow SDF-1 mRNA that closely mirrored the fall in SDF-1 protein, suggesting that G-CSF targets one or more SDF-1 producing cell population in the bone marrow. However, the identity of cell populations in the bone marrow that express SDF-1 is controversial. In the present study, we address this issue by sorting cells into mature hematopoietic, hematopoietic progenitor, endothelial, and osteoblast cell populations. Real time RT-PCR analyses showed that osteoblasts and to a lesser degree endothelial cells are the major sources of SDF-1 production in the bone marrow. Surprisingly, on a per cell basis, SDF-1 expression per osteoblast was only modestly (less than two-fold) reduced in mice treated with G-CSF. These data raised the possibility that, rather than affecting SDF-1 expression per osteoblast, G-CSF regulated the number of osteoblasts in the bone marrow. To explore this possibility, osteoblast number in the bone marrow was measured by histomorphometry. Indeed, after 5 days of G-CSF treatment, a significant reduction in the number of endosteal osteoblasts was observed [number of osteoblasts per mm bone perimeter ± SEM: 74.8 ± 13.5 (untreated) versus 33.3 ± 3.8 (G-CSF)]. Moreover, expression of osteocalcin (a specific marker of mature osteoblasts) in the bone marrow was sharply reduced during G-CSF treatment: a 47 ± 12 fold reduction in osteocalcin mRNA (relative to b-actin mRNA) was observed in the bone marrow of G-CSF-treated mice compared with untreated mice. Finally, calcein double-labeling experiments showed that the mineral apposition rate was significantly reduced in G-CSF-treated mice. However, RT-PCR analyses showed that the G-CSF receptor is not expressed on osteoblasts. Accordingly, G-CSF had no direct effect on osteoblast activity in vitro. Collectively, these data show that G-CSF potently suppresses osteoblast number/activity in the bone marrow through an indirect mechanism. Since osteoblasts are thought to play a key role in establishing and maintaining the stem cell niche in the bone marrow, these data raise the possibility that G-CSF, by regulating osteoblast function (including SDF-1 expression), may have profound effects on the stem cell niche that ultimately contribute to HPC mobilization.

The use of mobilised blood to effect haematological rescue now exceeds that of bone marrow. Despite the now broad application of blood stem cells (BSC) in a variety of clinical settings little is known of the precise mechanisms responsible for their release into the peripheral circulation. Under steady state conditions, primitive haemopoietic progenitor cells (HPC) are restricted to the bone marrow and circulate at low to undetectable levels. Although the basis for this specific retention of HPC within the BM remains to be defined, adhesive interactions between HPC and their surrounding stromal cell and ECM environment are hypothesised to play a major role. Mobilisation occurs in response to a variety of perturbations including physical exercise, following myelosupressive chemotherapy and following administration of a number of different cytokines. The kinetics of release may vary from as little as a few minutes to several weeks. Such observations suggest that a single unifying mechanism is therefore unlikely to be responsible for mobilisation in all cases and in all likelihood the predominant mechanism(s) will vary according to the mobilising agent employed. Nevertheless, mobilising agents are proposed to act to a greater or lesser extent by perturbing the function of the various cell adhesion molecules (CAM) or ligands which restrict HPC to the BM. In accord with this notion, studies by other have shown that antibody induced perturbation of VLA-4 function results in mobilisation of HPC in primates. How therefore do cytokines, the most commonly mobilising agents, result in HPC release? The most frequently employed agent to induce HPC release is granulocyte colony-stimulating factor (G-CSF). We have previously demonstrated that in vitro exposure of human CD34+ cells (HPC) to G-CSF, in common with several cytokines including IL-3, GM-CSF and SCF, results in transient increases in the affinity of the β 1 integrins VLA-4 and VLA-5 for their ligands fibronectin (FN) and VCAM-I. Thus in addition to the well documented mitogenic effects of cytokines these molecules also regulate the adhesive properties of HPC. Data will therefore be presented which examine the regulation of integrin function on human HPC and its relevance to the phenomenon of mobilisation. In addition we will also review recent observations in man and in a murine model which suggest an unanticipated link between bone turnover and BSC release. Collectively these studies provide some novel insights into the phenomenon of mobilisation but also suggest that the actual mechanisms involved are more complex than is currently appreciated.

In a defined medium in which transferrin (3 micrograms/ml) was the only source of exogenous proteins, Raji cells of the human pre-B lymphoblastoid cell line died within 48 h after forming polykaryons. The simple addition of purified C3 at a concentration equal to or higher than 3 micrograms/ml allowed Raji cells to divide. This preliminary report provides a defined system for studying the mitogenic effect of human C3 or C3 fragments upon proliferation of human B-cells lines.

By using human bone marrow cells enriched for early progenitors by selective immunoadsorption and plated at low cell density (10(3) to 10(4) cells/mL/9.6 cm2) in semisolid methylcellulose culture, we have analyzed the cooperative effects of human colony-stimulating factor 1 (CSF-1), granulocyte-macrophage-CSF (GM-CSF), interleukin-1 alpha (IL-1 alpha), and gibbon as well as human recombinant IL-3 on the formation of monocytic colonies. CSF-1 alone stimulated mature monocytic colony formation by human CFU-M. However, in the presence of IL-3 and erythropoietin, CSF-1 stimulated maximal immature monocytic colony formation at low concentrations and inhibited the formation of granulomonocytic, erythrocytic, and mixed colonies. Cultures with CSF-1 and IL-3 contained more immature monocytic colonies than did cultures with CSF-1 alone. IL-1 alpha alone had little effect. However, IL-1 alpha in combination with optimal concentrations of either CSF-1, GM- CSF, or IL-3 increased the number of colonies containing immature or mature monocytic colonies.

Cellular interactions are critical for the regulation of hematopoiesis. The sialomucin PSGL-1/CD162 mediates the attachment of mature leukocytes to P-selectin. We now show that PSGL-1 also functions as the sole receptor for P-selectin on primitive human CD34+ hematopoietic progenitor cells (HPC). More importantly, ligation of PSGL-1 by immobilized or soluble ligand or anti-PSGL-1 antibody results in a profound suppression of HPC proliferation stimulated by potent combinations of early acting hematopoietic growth factors. These data demonstrate an unanticipated but extremely marked growth-inhibitory effect of P-selectin on hematopoiesis and provide direct evidence that PSGL-1, in addition to its well-documented role as an adhesion molecule on mature leukocytes, is a potent negative regulator of human hematopoietic progenitors.

We have previously established that the mitogenic effect of fibrinogen on hemopoietic cell lines Raji and JM is mediated via a specific receptor (Levesque, J.-P. et al.: Proc. Natl. Acad. Sci. USA 83:6494-6498, 1986). In this study, we have further characterized the fibrinogen domain involved in the binding to the mitogenic receptor. This binding was not inhibited either by a monoclonal antibody against the C-terminal sequence of the fibrinogen gamma chains or by synthetic peptides containing the Arg-Gly-Asp sequence. Such inhibition is specific of the platelet fibrinogen receptor, the glycoprotein IIb-IIIa complex. Fragments containing the fibrinogen D domain were the only plasmin degradation products of fibrinogen which were mitogenic. These fragments acted via direct binding on the mitogenic receptor with a Kd of 2.24 X 10(-6) M. This value was similar to the KI value of unlabeled fragments D (2.47 X 10(-6) M). Our results suggest the presence of two different functional types of fibrinogen receptors: the glycoprotein IIb-IIIa receptor responsible both for platelet aggregation and leukocyte adhesion and killing, and the mitogenic receptor involved in proliferation control of hemopoietic cells.

A gradient has been developed for separating free ligand from ligand bound to cells growing in suspension. This method can be used with all kinds of ligand but it is particularly useful for those ligands having the tiresome tendency to adhere to the cells non-specifically or to polymerize by themselves. This is the case of fibronectin, fibrinogen, immunoglobulins and many other plasma proteins. The gradient consists of two layers: an upper aqueous phase and a lower hydrophobic organic phase. The aqueous phase, a sucrose buffer, allows the cells to become well dispersed before they enter the hydrophobic phase which excludes the free ligand efficiently. This reduces non-specific binding and allows the accurate measurement of specific binding which could not be obtained with a gradient made of a single phase. Depending upon the size and the density of the cells, and the nature of the ligand, the assay method can be modified by changing the density or the nature of the hydrophilic and hydrophobic phases.

The possibility that the mitogenic effect of fibrinogen, a major plasma protein (3 mg/ml), is mediated by specific membrane receptors was studied. Specific binding analysis showed that fibrinogen receptors are present only on hemopoietic cell lines that respond to its mitogenic effect. The mitogenic fibrinogen receptor is not recognized by antibodies specific for the platelet fibrinogen receptor or is not competitively blocked by synthetic peptides containing the Arg-Gly-Asp sequence, which is common to fibronectin, fibrinogen, vitronectin, and other cell-attachment proteins. The lymphoma-derived pre-B-cells (Raji) have 149,000 receptors, whereas the lymphoma-derived T cells (JM), which are 3 times smaller, have 54,000 receptors. These receptors have a Kd of 2 X 10(-7) M. They are inducible by stimuli specific for the cell lineage: activators of the breakdown of phosphatidylinositol phosphates, such as platelet activating factor for Raji cells, and adenylate cyclase agonists and cAMP analogues for JM cells. The stimuli have no mitogenic effect in the absence of fibrinogen; they do not change the Kd. Each stimulus increases the number of fibrinogen receptors in a dose-dependent manner, which correlates strongly (r = -0.98, n = 5) with an increased growth rate of cells in the presence of fibrinogen. This correlation concludes that the mitogenic effect of fibrinogen is controlled via receptor modulation.

The effect of ligand interactions with the C3d/C3dg complement receptor (CR2) on proliferation of human B lymphoblastoid cells was investigated by using cell cultures performed at low density (1 to 1.5 x 10(3) cells/ml) in a serum-free defined medium to which only transferrin had been added. This medium does not allow proliferation of Raji cells which die within 48 hr with formation of polykaryons. Addition of purified human C3 to the cultures resulted in a dose-dependent proliferation of the cells. A steady growth of Raji cells with a doubling time of 36 hr was observed in cultures containing 10 micrograms/ml of C3. A growth rate similar to that observed in the presence of native C3 was found in the presence of equimolar concentrations of purified C3dg but not of C3c. F(ab')2 anti-C3d but not F(ab')2 anti-C3c antibodies inhibited the mitogenic effect of C3. Preincubation of Raji cells with monoclonal antibody OKB7 which directly inhibits the binding of C3dg to CR2, totally suppressed C3-induced growth of the cells. C3 did not enhance growth of the T lymphoma-derived cell line JM and monocytic cell line U937 which do not express CR2. These results provide direct evidence that the interaction between CR2 and C3 fragments stimulates proliferation of human cells of the B lineage. Because CR2 also acts as a receptor for Epstein-Barr virus on B cells, our results may pertain to the B cell mitogenic properties of the virus.

Leupeptin, a thiol- and serine-proteinase inhibitor of low molecular weight, quickly enters viable cells. This property has been used to protect cells during thawing against intracellular proteolytic activities released by injured lysosomes. The bone marrow nucleated cells were frozen without rate-controlled freezing devices. Concentrations ranging from 0.1 to 1 microM of leupeptin allow to recover 87% of the most immature multipotent bone marrow progenitors which can develop in vitro into large multilineage colonies, instead of 58% recovery without leupeptin. The protective effect of leupeptin is particularly useful to freeze cells difficult to cryopreserve or when freezing-control equipment are not available.

Abstract 564 The behaviour of a hematopoietic stem cell (HSC) is regulated by its immediate micro-environment or niche. We have identified a novel function for the adhesion molecule E-selectin which is constitutively expressed on bone marrow (BM) vasculature. Using mice knocked-out for E- (E-/-) or P-selectin (P-/-) genes, we investigated whether selectin absence alters HSC behaviour in vivo. We found HSC cycling in the absence of E-selectin to be significantly delayed 2.5-fold in BrdU incorporation assays compared to either P-/- or WT (mice were administered BrdU for 3d then BrdU incorporation in BM Lineage-KIT+Sca1+(LKS+)CD34- or LKS+CD48-CD150+cells measured). To confirm these findings, LKS+ cells were stained with rhodamine123, a vital dye retained by metabolically active cells but not quiescent HSC. More LKS+ cells from E-/- mice were rhodamine dull (34±2%) than WT (23±1%; p=0.037) confirming that a greater proportion of HSC from E-/- mice are quiescent. We then determined whether administration of E-selectin antagonists alone could similarly delay HSC turnover. Mice were administered the glycomimetic E-selectin antagonist GMI-1070, for set periods of time before harvest. We found HSC turnover to be significantly delayed following GMI-1070 administration (1.4 fold less BrdU incorporation, p=0.011) with a concomitant 1.4-fold increase in the number of Rho123 dull LSK+ quiescent HSC per femur (p=0.020). Non-cycling, quiescent HSC are known to be more resistant to chemotherapy and irradiation. Indeed 7 days following 5-FU administration, we found that E-/- mice had faster BM HSC recovery / less HSC damage compared to WT mice, both by phenotype analysis and in a competitive long-term reconstituting assay. Following 5-FU administration the number of reconstituting units/femur in WT mice decreased 5.1-fold but only decreased 2.3-fold in similarly treated E-/- mice. Interestingly, when mice were pre-treated with GMI-1070 before 5-FU, there was significantly enhanced blood neutrophil recovery compared to mice administered 5-FU alone (blood neutrophils were 710±205 ×103/mL with GMI-1070, compared to 234±141 ×103/mL without, at day 9 post-5-FU, p=0.0001). Similarly when mice were severely irradiated and test bleeds performed weekly, a more rapid haematopoietic recovery was observed in E-/- compared to WT mice. In summary, we have identified a novel function for the adhesion molecule E-selectin. HSC turnover is dramatically reduced in E-/- mice an effect that can be replicated by transient administration of E-selectin antagonist mimetics. Furthermore blood leukocyte and HSC numbers recover faster following cytotoxic or irradiation injury in the absence or blockage of E-selectin-mediated cell adhesion. Thus E-selectin may well be a crucial component of the proliferative HSC niche regulating HSC turnover. Blockage of E-selectin adhesive interaction by GMI-1070, a novel E-selectin antagonist that has completed phase I clinical trails, may represent a promising treatment for the protection of HSC during chemotherapy. Disclosures: Winkler: Glycomimetics Inc: Research Funding. Smith:GlycoMimetics, Inc: Employment. Patton:GlycoMimetics, Inc: Employment. Magnani:GlycoMimetics, Inc.: Employment. Levesque:Glycomimetics Inc.: Research Funding.

Inflammation is a natural part of wound healing but it can also cause secondary (bystander) damage and/or negatively interfere with endogenous repair mechanisms if non-resolving. Regulation of inflammation is traditionally looked at from the perspective of danger signals, cytokines and chemokines, and their respective receptors. A neuronal contribution to the regulation of inflammation is, however, increasingly appreciated, and this has important implications for the bodily response under conditions where the nervous system itself may be damaged. In this review article, we provide an up-to-date overview of the current literature on neural innervation of primary and secondary lymphoid organs, focusing in particular on the bone marrow and spleen, its significance in relation to immune function and, lastly, also briefly discussing how a major neurotraumatic event like spinal cord injury (SCI) may impact on this.

Bone forming osteoblasts an are essential component of bone marrow (BM) niches necessary for the maintenance of hematopoietic stem cells (HSC) and B-cell progenitors. Previous studies from our and other groups have shown that the mobilizing agent granulocyte colony-stimulating factor (G-CSF) reduces the number and function of osteoblasts lining the bone at the endosteum. By histomorphometry, we now demonstrate that like G-CSF, the chemotherapy agent cyclophosphamide (CY) also induces a rapid and dramatic reduction in osteoblast number and osteoid surface at days 6–8 when hematopoietic stem and progenitor cells (HSPC) are mobilized into the blood. Both G-CSF- and CY-induced HSPC mobilizations coincide with a 90% reduction in bone mineralization and formation as measured by incorporation of calcein, a Ca2+-chelating fluorophore, in the mineralizing bone. As osteoblasts form specific niches for HSC and primitive B lymphoid progenitors, we measured expression of hematopoietic and lymphopoietic cytokines at the endosteum to gain insight on how these endosteal niches are affected during HSPC mobilization. As expected, osteoblast and osteoprogenitor specific mRNA were markedly reduced during mobilization induced by G-CSF or CY with significant reductions in Runx2 (5- fold), osterix (12–32-fold), PTHR1 (20-fold) and osteocalcin (50–100-fold) mRNA. To determine whether this reduction in osteoblast number/function affected the expression of cytokines essential for the maintenance of HSC and B progenitor at the endosteum, we measured mRNA encoding CXCL12, IL-7, BAFF, KIT ligand and angiopoietin-1 (Ang-1) in total endosteal cells as well as in endothelial cells (Lin−CD45−CD31+), mesenchymal osteoprogenitors (Lin−CD45−CD31−Sca-1+CD51+) and osteoblasts (Lin−CD45− CD31−Sca- 1−CD51+) sorted from the endosteum. In saline-treated mice, IL-7 and CXCL12 were mostly expressed by osteoblasts, BAFF mostly by osteoprogenitors, Ang-1 by both osteoblasts and osteoprogenitors, whereas KIT ligand was expressed by both osteoblasts and endothelial cells. G-CSF caused a 3-fold reduction in the expression of IL-7, BAFF, and KIT ligand, a 4-fold reduction in Ang-1, and a 5–10-fold reduction in CXCL12 mRNA at the endosteum, demonstrating an impairment of endosteal niches during G-CSF administration. Despite maintenance of lineage-negative (Lin−) Sca-1+ KIT+ cell number in the BM, Lin− Sca-1+ KIT+ CD41− CD48− CD150+ phenotypic HSC were decreased 3.4- fold in mobilized BM. By measuring in vivo Hoechst33342 incorporation following a 10 minute perfusion, we found that these phenotypic HSC relocated from niches distal from BM blood sinuses (low Ho33342 incorporation in vivo) to more perivascular locations (high Ho33342 incorporation). Finally, the numbers of B progenitors were dramatically reduced in mobilized BM with enhanced B cell apoptosis. Mobilization of B progenitors into blood and spleen did not compensate for their reduction in the BM, resulting in a net loss in all B cell progenitors in whole mice. From our findings, we conclude that reduced osteoblast number/function during G-CSF-induced HSC mobilization not only leads to the impairment of HSC endosteal niches but also an arrest in medullar B lymphopoiesis.

Background: Despite the introduction of 21st-century surgical and neoadjuvant treatment modalities, survival of patients with osteosarcoma (OS) has not improved in two decades. Advances will depend in part on the development of clinically relevant and reliable animal models. This report describes the engineering and validation of a humanized tissue-engineered bone organ (hTEBO) for preclinical research on primary bone tumors in order to minimize false-positive and false-negative results due to interspecies differences in current xenograft models. Methods: Pelvic bone and marrow fragments were harvested from patients during reaming of the acetabulum during hip arthroplasty. HTEBOs were engineered by embedding fragments in a fibrin matrix containing bone morphogenetic protein-7 (BMP-7) and implanted into NOD-scid mice. After 10 weeks of subcutaneous growth, one group of hTEBOs was harvested to analyze the degree of humanization. A second group was injected with human luciferase-labeled OS (Luc-SAOS-2) cells. Tumor growth was followed in vivo with bioluminescence imaging. After 5 weeks, the OS tumors were harvested and analyzed. They were also compared with tumors created via intratibial injection. Results: After 10 weeks of in vivo growth, a new bone organ containing human bone matrix as well as viable and functional human hematopoietic cells developed. Five weeks after injection of Luc-SAOS-2 cells into this humanized bone microenvironment, spontaneous metastatic spread to the lung was evident. Relevant prognostic markers such as vascular endothelial growth factor (VEGF) and periostin were found to be positive in OS tumors grown within the humanized microenvironment but not in tumors created in murine tibial bones. Hypoxia-inducible transcription factor-2α (HIF-2α) was detected only in the humanized OS. Conclusions: We report an in vivo model that contains human bone matrix and marrow components in one organ. BMP-7 made it possible to maintain viable mesenchymal and hematopoietic stem cells and created a bone microenvironment mimicking human physiology. Clinical Relevance: This novel platform enables preclinical research on primary bone tumors in order to test new treatment options.

Cytotoxic chemotherapies used to treat acute leukemia and high-grade lymphoma target rapidly-dividing cells including the malignant cells but also normal HSC needed to replenish the blood and immune system, as well as mucosal cells lining the respiratory and gastrointestinal tracts. Consequences include therapy-induced mucositis (providing a portal of entry for bacteria) and neutropenia leading to increased morbidity and potentially life-threatening infections. In acute myeloid leukemia and high-grade lymphoma patients, treatment-related mortality can reach 20% (Elmaagacli, BMT, 2007). We now report a novel therapy (therapeutic blockade of E-selectin) that may enhance survival after repeated rounds of high-dose chemotherapy. In mice we find the mechanism contributing to this enhanced survival to be a combination of accelerated neutrophil recovery and a dramatic reduction in therapy-related weight loss and intestinal mucositis when E-selectin is therapeutically blocked or genetically-deleted. Our studies were performed using two treatment regimens (chemotherapy or radiation). For chemotherapy, cohorts of mice received repeated rounds of the anti-metabolite, 5-fluorouracil (5-FU) at a dose of 150mg/kg every 10 days without prophylactic antibiotic support. Using this regime, 80% of E-selectin gene-deleted mice (E-/-) survived >6 weeks. In sharp contrast all wild-type mice died (p Deletion of the E-selectin gene also prevented therapy-induced weight loss (from 16% average weight loss in wild-type mice, to only 2% in E-/- mice after 2 rounds 5-FU, p Therapy-induced mucositis is greatly exacerbated by a wave of secondary inflammation following initial direct damage. We found administration of the selective E-selectin antagonist GMI-1271 (20 mg/kg bi-daily) for 5 days following each round of chemotherapy enhanced neutrophil recovery and similarly protected mice from weight loss and mucositis following chemotherapy. Importantly the administration of GMI-1271 effectively blocked secondary migration of inflammatory F4/80+ Ly-6C+ macrophages to intestines of mice following chemotherapy or irradiation. In conclusion, our results suggest that inflammatory macrophages play a major role in chemotherapy-induced mucositis and are recruited to damaged intestine following E-selectin up-regulation. Importantly there is no current highly-effective treatment to prevent mucositis. Therapeutic blockade of E-selectin by administration of GMI-1271 at the same time as chemotherapy represents a potential effective and novel therapy to block recruitment of inflammatory macrophages to damaged mucosa whilst also enhancing neutrophil recovery, thus protecting patients from two chemotherapy-induced side-effects. Therapeutic blockade of E-selectin should provide an opportunity for improved completion rates of toxic chemotherapy or radiotherapy regimes and reduce therapy-related morbidity and mortality. Disclosures: Magnani: GlycoMimetics, Inc.: Employment, Equity Ownership.

Normal human hemopoietic cells such as early bone marrow progenitors, or lymphoma-derived cell lines such as Raji or JM cells, possess a low-affinity receptor specific for fibrinogen. This receptor triggers a mitogenic effect. It differs from the glycoprotein IIb-IIIa which is involved in fibrinogen-induced platelet aggregation. We demonstrate here that this mitogenic fibrinogen receptor (MFR) can be internalized or reexpressed, depending on culture conditions. Internalization was temperature-dependent. At 37 degrees C in the presence of cycloheximide or actinomycin D, the half-life of cell surface MFRs was 2 h, independent of receptor occupancy. Binding of fibrinogen to the MFR resulted in a down-regulation which was fibrinogen dose-dependent. This occurred in serum-supplemented medium but not in defined medium supplemented with fatty acids. Reexpression of MFRs could be induced in 28 to 42 h by serum removal. The down-regulation of mitogenic receptors in plasma or serum could explain why normal cells do not proliferate in the peripheral blood.

The cytokines granulocyte colony-stimulating factor (G-CSF) and Flt3 ligand (Flt3-L) mobilize hematopoietic stem and progenitor cells into the peripheral blood of primates, humans, and mice. We recently reported that G-CSF administration causes a transient blockade of medullar erythropoiesis by suppressing erythroblastic island (EI) macrophages in the bone marrow. In the study described here, we investigated the effect of mobilizing doses of Flt3-L on erythropoiesis in mice in vivo. Similar to G-CSF, Flt3-L caused whitening of the bone marrow with significant reduction in the numbers of EI macrophages and erythroblasts. This was compensated by an increase in the numbers of EI macrophages and erythroblasts in the spleen. However, unlike G-CSF, Flt3-L had an indirect effect on EI macrophages, as it was not detected at the surface of EI macrophages or erythroid progenitors.

The vascular adhesion molecule E-selectin is a key component of the bone marrow (BM) vascular niche, awakening otherwise dormant hematopoietic stem cells (HSC) (Winkler et al., Nat Med 2012). Vascular niches also mediate malignant cell survival. We find E-selectin becomes upregulated on BM vasculature during leukemia and that adhesion of BM AML blasts to E-selectin promotes survival signaling. This is unique to E-selectin - not observed with adhesion to P-selectin, PECAM1, or integrin ligands ICAM1, VCAM1. In vivo we find absence (in SELE-/- mice) or therapeutic blockade of E-selectin by administration of E-selectin antagonist GMI-1271 to mice, induces 9-fold greater chemosensitivity in 11q23-rearranged AML Initiating Cells (LIC) to cytarabine therapy in vivo and doubles survival over that achieved by chemotherapy alone. Together these data support the Phase I/II clinical trial of GMI-1271 in combination with intensive chemotherapy to improve efficacy of therapy for AML (NCT02306291), and raise the further questions: 1) what are the AML LIC ligands interacting with E-selectin at the vascular niche and 2) what are the pathways initiated by E-selectin adhesion that mediate chemoresistance. Using murine models of AML generated by retroviral transduction of the 11q23 fusion oncogene MLL-AF9 into HSC, we find leukemic blasts rapidly upregulate E-selectin binding potential upon oncogenic transformation. To investigate if this is due to aberrant glycosylation facilitating the generation of de novo AML cell surface receptors, we generated AML from HSC knocked-out for the two canonical E-selectin receptors PSGL1 and CD44. Surprisingly we found although HSC from CD44-/-PSGL1-/- mice still bound to E-selectin (indicating HSC express additional E-selectin ligands), AML cells generated from these same HSCs no longer did. Thus the functional E-selectin ligands utilized by murine HSC and AML blasts differ. When transplanted into recipient mice CD44-/-PSGL1-/- AML engrafted with similar disease progression to matching wildtype AML but demonstrated ~100-fold greater sensitivity to high-dose cytarabine chemotherapy. When GMI-1271 was co-administered together with chemotherapy no further boost in chemosensitivity was observed in CD44-/-PSGL1-/- AMLs. This complements in vitro data showing E-selectin adhesion-mediated chemoresistance is lost when CD44 and/orPSGL1are absent in AML cells. Together suggesting that CD44 and/or PSGL1 are the key receptors involved in E-selectin-mediated pro-survival signaling. To understand the pro-survival signaling pathways involved, E-selectin antagonist GMI1271 or saline control were administered 5 days to mice with AML and BM KIT+ leukemic blasts sorted. RNA sequencing revealed only a small number (~200) of mRNA transcripts differentially regulated following therapeutic E-selectin blockade. Of note these included down-regulation in several components of the PI3K/AKT/NF-kB signaling pathway frequently involved in cell survival. Indeed AKT Ser473 and NF-kB p65 Ser536 were found to be rapidly phosphorylated within 15 minutes of E-selectin adhesion in AML blasts in vitro. This phosphorylation was specific to E-selectin adhesion and not observed with other vascular adhesion molecules tested suggesting that direct E-selectin-mediated AKT activation in AML cells at the vascular niche may be the potential mechanism driving vascular mediated chemoresistance. To confirm whether therapeutic blockade of E-selectin alone dampens intracellular AKT signaling in AML blasts, cohorts of mice with wildtype or matching CD44-/-PSGL1-/- AML were administered GMI-1271 for 5 days then BM AML blasts collected for signaling studies. Both the administration of E-selectin antagonist or absence of CD44 and/or PSGL-1 dampened AKTSer473 phosphorylation in BM AML blasts in vivo, correlating with their heightened sensitivity to chemotherapy . In summary, these findings suggest E-selectin ligands on specific CD44 and/or PSGL1 glycoforms are the predominant determinants through which the induction/amplification of chemoresistant pathways in AML blasts occur following vascular adhesion and further that these pathways are coupled to an early phosphorylation event of AKT - and finally explain potential mechanism how GMI-1271 administration is able to attenuate vascular-mediated signaling and restore susceptibility of AML to chemotherapy. Disclosures Magnani: GlycoMimetics, Inc.: Employment, Equity Ownership, Membership on an entity's Board of Directors or advisory committees.

Peritoneal macrophages from mice injected with aclacinomycin (ACM) (4 mg/kg, i.p.) showed increased functional activity, as assessed by increased antitumoral activity in vitro and in vivo and zymosan-triggered chemoluminescence. They also showed ultrastructural signs of activation (increased number of cytoplasmic organelles), and atypical alterations (giant vacuoles and giant lysosomes containing heterogenous myelinoid bodies, lipofuscine-like substance, cytoplasmic debris, and a fine granular material). As these atypical alterations could be due to the generation of superoxide following ACM injection, superoxide dismutase (SOD) was injected 1 h prior to ACM administration. Neither the morphological characteristics of activation, nor the enhanced metabolic and antitumoral activities induced by ACM were affected by SOD pretreatment, but the atypical alterations were inhibited in a dose-dependent manner. Heat-inactivated SOD did not prevent their appearance. The atypical alterations were not found in peritoneal macrophages from talc or lipopolysaccharide-injected mice, but they were present in Adriamycin-treated mice and were also prevented by SOD pretreatment, indicating that the alterations are due to anthracycline treatment. Finally, [125I]SOD was phagocytized by peritoneal macrophages in vitro and in vivo and not by L1210 tumoral cells, explaining why the atypical alterations induced by ACM were no longer seen after SOD pretreatment. The unchanged direct oncostatic activity of ACM following SOD pretreatment suggests that this combination may have some wider perhaps clinical, potential.

Abstract 4005 The CXCR4 antagonist AMD3100 is progressively replacing cyclophosphamide as adjuvant to G-CSF to mobilize hematopoietic stem cells (HSC) for autologous transplants in patients who failed prior mobilization with G-CSF alone. We and others have recently demonstrated that G-CSF-induced mobilization and the associated response of HSC niches and bone formation depend on bone marrow (BM) macrophages1–3. Moreover medullar B lymphopoiesis is dependent on bone-forming osteoblasts in vivo. We therefore compared the effects of these three mobilizing agents (6 day course of G-CSF, versus a single injection of cyclophosphamide, versus 6 day course of AMD3100) on endosteal osteoblasts, bone formation, BM macrophages, expression of HSC-supportive cytokines and B lymphopoiesis in the mouse. G-CSF administration significantly reduced the number of endosteal osteoblasts and niche-supporting macrophages. G-CSF also inhibited expression of chemokines and cytokines such as CXCL12, Kit-ligand, angiopoietin-1 and IL-7 in the endosteal region resulting in a profound inhibition of medullar B lymphopoiesis with suppression of pre-pro-B, pro-B, pre-B cells and mature sIgM+ B cells as long as G-CSF was administered and HSC mobilization persisted. Cyclophosphamide also suppressed osteoblasts, niche-supportive macrophages and inhibited endosteal expression of CXCL12 and angiopoietin-1 but did not inhibit KIT-ligand expression. Of note, the down-regulation of CXL12 was more pronounced in response to cyclophosphamide than G-CSF. This could explain the higher mobilizing effect of cyclophosphamide. In contrast to G-CSF, medullar B lymphopoiesis rebounded in synchrony with endosteal IL-7 expression during cyclophosphamide-induced mobilization despite persistent suppression of osteoblasts and bone formation. Despite a marked rebound in the number of immature B cell progenitors in cyclophosphamide-mobilized BM, B cell progenitors could not mature into sIgM+ B cells which remained very depressed in BM, spleen and blood even 10 days after a single cyclophosphamide injection. Finally, while AMD3100 was effective at mobilizing HSC, it did not suppress osteoblasts, cytokine expression at the endosteum or B lymphopoiesis. In conclusion, although G-CSF, cyclophosphamide and AMD3100 efficiently mobilize HSC into the blood, their effects on HSC niches, bone formation and B lymphopoiesis are distinct. Furthermore the contrasted effects of G-CSF, cyclophosphamide and AMD3100 on medullar B lymphopoiesis indicates that grafts mobilized with these three agents may have different immune properties. (1) Winkler IG, Sims NA, Pettit AR, et al. Bone marrow macrophages maintain hematopoietic stem cell (HSC) niches and their depletion mobilizes HSCs. Blood. 2010;116(23):4815–4828. (2) Christopher MJ, Rao M, Liu F, Woloszynek JR, Link DC. Expression of the G-CSF receptor in monocytic cells is sufficient to mediate hematopoietic progenitor mobilization by G-CSF in mice. J Exp Med 2011;208(2):251–260. (3) Chow A, Lucas D, Hidalgo A, et al. Bone marrow CD169+ macrophages promote the retention of hematopoietic stem and progenitor cells in the mesenchymal stem cell niche. J Exp Med 2011;208(2):261–271. Disclosures: No relevant conflicts of interest to declare.

Matrix metalloproteinase (MMP) activity is regulated by tissue inhibitor of metalloproteinases (TIMPs). We found that while TIMP-1 and -2 expressions were unaffected, and TIMP-4 was not expressed, TIMP-3 mRNA expression decreased 10-fold within the bone marrow (BM) during G-CSF induced mobilization. In addition, through reverse zymography, the level of biologically active TIMP-3 protein was also shown to decrease during mobilization. Down-regulation of TIMP-3 may contribute to the accumulation of active MMPs within the BM, allowing for the release of hematopoieticstem/progenitor cells (HSPC) from the BM matrix. By qRT-PCR we have shown 10-fold greater TIMP-3 expression in endosteal mRNA compared to central BM mRNA in mouse femur (p=0.008). To assess which bone associated cell populations expressed the majority of TIMP-3, pooled bones were crushed, collagenase treated and FACS sorted. Mesenchymal progenitors (CD45-Lin-Sca1+) expressed the highest level of TIMP-3 followed by endothelial cells (CD45-Lin-CD31+) and mature osteoblasts (CD45-Lin-Sca1-CD51+). Erythroid progenitors (CD45+Ter119+Kit+), megakaryocyte progenitors (CD45+CD41+Kit+) and megakaryocytes (CD45+CD41+Kit−) from BM were also found to express TIMP-3, but at a level at least 10-fold lower than those of non-hematopoietic stromal cells. All other BM hematopoietic cell types tested were negative for TIMP-3 expression. Immunohistofluorescence on bone sections validated TIMP-3 expression in megakaryocytes, endothelial cells and osteoblasts. Expression of TIMP-3 in mouse platelets was confirmed by reverse zymography. To investigate TIMP-3 function we over-expressed huTIMP-3 in mice via retroviral transduction with MND-X-IRES-eGFP (MXIE) retroviral vector. BM cells retrovirally transduced with MXIE-huTIMP-3 or empty MXIE control was transplanted into lethally irradiated congenic mice. Engraftment and transduction levels were determined by GFP expression. At 3-months post-transplant there were no significant differences in body weight, total blood, spleen or BM cell counts between the two groups. qRT-PCR data showed that over-expressing huTIMP-3 did not alter the expression level of endogenous mTIMP-3. Flow cytometry analysis showed that in mice transduced with MXIE-huTIMP-3, the frequency of GFP+ B cells (CD11b-B220+) was reduced by 50% in the blood from 23.88±12.00% to 11.94±7.85% (p=0.0315) and by 64% in the BM from 25.06±13.78% to 9.02±7.67% (p=0.0188) when compared to MXIE controls. Conversely, the frequency of GFP+ huTIMP-3 expressing myeloid cells (CD11b+) was significantly increased in the blood from 55.69±17.13% to77.91±6.31% (p=0.0005), BM from 58.67±16.32% to 77.32±12.02% (p=0.0244) and spleen from 14.07±3.75% to 28.82±6.85% (p=0.0002). Unexpectedly, the frequency of untransduced GFP- myeloid and B cells were similar between the two groups. Although huTIMP-3 over-expression did not significantly alter the number of GFP+ HSPC (Linage-Sca1+Kit+, LSK) per femur (MXIE 0.03±0.03%, MXIE-huTIMP-3 0.01±0.01%, p=0.1139), LSK turnover in huTIMP-3 over-expressing cells was increased in vivo from 4.36±2.83% to 13.31±5.61% (p=0.0159) as determined by BrdU incorporation following 3 days of BrdU administration. Similarly, a trend was also observed in vitro after 12days of culture, LSK sorted from MXIE-huTIMP-3 mice proliferate faster than MXIE controls from 2.55^6cells/ml±1.05 to 9.6^6cells/ml±0.54 (p=0.1). In summary, huTIMP-3 over-expression in mice increased HSPC proliferation in vivo and in vitro. And whilst the huTIMP-3 over-expression in mice was not at a sufficient level to observe a global effect on total BM haematopoiesis, our data suggests that forced huTIMP-3 over-expression in vivo skews differentiation towards myelopoiesis at the detriment of lymphopoiesis.