Potential role for hyaluronan and the hyaluronan receptor RHAMM in mobilization and trafficking of hematopoietic progenitor cells

Although the mechanism(s) underlying mobilization of hematopoietic progenitor cells (HPCs) is unknown, detachment from the bone marrow (BM) microenvironment and motility are likely to play a role. This work analyzes the motile behavior of HPCs and the receptors involved. CD34(+)45(lo/med)Scatterlo/med HPCs from granulocyte colony-stimulating factor (G-CSF)-mobilized blood and mobilized BM were compared with steady-state BM for their ability to bind hyaluronan (HA), their expression of the HA receptors RHAMM and CD44, and their motogenic behavior. Although RHAMM and CD44 are expressed by mobilized blood HPCs, function blocking monoclonal antibodies (MoAbs) identified RHAMM as a major HA binding receptor, with a less consistent participation by CD44. Permeabilization of mobilized blood HPCs showed a pool of intracellular (ic) RHAMM and a smaller pool of icCD44. In contrast, steady-state BM HPCs have significantly larger pools of icRHAMM and icCD44. Also, in contrast to mobilized blood HPCs, for steady-state BM HPCs, MoAbs to RHAMM and CD44 act as agonists to upregulate HA binding. The comparison between mobilized and steady-state BM HPCs suggests that G-CSF mobilization is associated with depletion of intracellular stores of HA receptors and modulates HA receptor usage. To confirm that mobilization alters the HA receptor distribution and usage by HPCs, samples of BM were collected at the peak of G-CSF mobilization in parallel with mobilized blood samples. HA receptor distribution of mobilized BM HPCs was closely matched with mobilized blood HPCs and different from steady-state BM HPCs. Mobilized BM HPCs had lower pools of icHA receptors, similar to those of mobilized blood HPCs. Treatment of mobilized BM HPCs with anti-RHAMM MoAb decreased HA binding, in contrast to steady-state BM HPCs. Thus, G-CSF mobilization may stimulate an autocrine stimulatory loop for HPCs in which HA interacts with basal levels of RHAMM and/or CD44 to stimulate receptor recycling. Consistent with this, treatment of HPCs with azide, nystatin, or cytochalasin B increased HA binding, implicating an energy-dependent process involving lipid rafts and the cytoskeleton. Of the sorted HPCs, 66% were adherent and 27% were motile on fibronectin plus HA. HPC adherence was inhibited by MoAbs to beta1 integrin and CD44, but not to RHAMM, whereas HPC motility was inhibited by MoAb to RHAMM and beta1 integrin, but not to CD44. This finding suggests that RHAMM and CD44 play reciprocal roles in adhesion and motility by HPCs. The G-CSF-associated alterations in RHAMM distribution and the RHAMM-dependent motility of HPCs suggest a potential role for HA and RHAMM in trafficking of HPCs and the possible use of HA as a mobilizing agent in vivo.     

E-selectin and very late activation antigen-4 mediate adhesion of hematopoietic progenitor cells to bone marrow endothelium

 Adhesion of CD34⁺ hematopoietic progenitor cells (HPCs) to sinusoidal endothelium probably plays a key role in homing of transplanted CD34⁺ HPCs to the bone marrow (BM). We have investigated the role of various adhesion molecules in the interaction of purified CD34⁺ HPCs derived from BM or peripheral blood (PB) and a human BM-derived endothelial cell line. Adhesion of CD34⁺ HPCs to endothelial cells was measured with the use of a double-color flow microfluorimetric adhesion assay. In this assay, adhesion is measured under stirring conditions, simulating blood flow in sinusoidal marrow vessels. Adhesion of PB CD34⁺ cells to human BM endothelial cells (HBMECs) was observed only after interleukin (IL)-1β prestimulation of the endothelial cells. This adhesion was strongly increased after addition of phorbol-myristate acetate (PMA). Adhesion of PB CD34⁺ cells to IL-1β-prestimulated HBMECs was inhibited by blocking monoclonal antibodies (mAbs) against E-selectin and by neuraminidase treatment of the PB CD34⁺ cells. mAbs against very late activation antigen (VLA)-4 inhibited adhesion only when the E-selectin-mediated interaction was prevented. No clear inhibiting effect was found with blocking mAbs against β₂-integrins. Stimulation with the β₁-integrin-activating mAb, 8A2, induced adhesion of CD34⁺ cells to endothelial cells. In conclusion, stimulation of both endothelial cells and CD34⁺ HPCs is necessary for adhesion of CD34⁺ HPCs to endothelial cells. We furthermore demonstrated that E-selectin and VLA-4 mediated this adhesion. Received: 26 April 1999 / Accepted: 8 February 2000     

CD1d expression on and regulation of murine hematopoietic stem and progenitor cells

In the present study, surface CD1d, which is involved in immune cell interactions, was assessed for effects on hematopoiesis. Mouse BM hematopoietic stem cells (HSCs) and hematopoietic progenitor cells (HPCs) express CD1d. The numbers and cycling status of HPCs in the BM and spleen of different strains of cd1d(-/-) mice were enhanced significantly, suggesting that CD1d is a negative regulator of HPCs. In support of this, CD1d was required for the SCF and Flt3 ligand synergistic enhancement of CSF induction of HPC colony formation and for HPC response to myelosuppressive chemokines. Colony formation by immature subsets of HPCs was greatly enhanced when normal, but not cd1d(-/-), BM cells were pretreated with CD1d Abs in vitro. These effects required the full CD1d cytoplasmic tail. In contrast, long-term, but not short-term, repopulating HSC engraftment was impaired significantly, an effect that was minimally influenced by the presence of a truncated CD1d cytoplasmic tail. Pretreatment of normal BM cells with CD1d Abs greatly enhanced their engraftment of HSCs. The results of the present study implicate CD1d in a previously unrecognized regulatory role of normal and stressed hematopoiesis.     

Enhanced engraftment of hematopoietic stem/progenitor cells by the transient inhibition of an adaptor protein, Lnk

Hematopoietic stem cells (HSCs) are the key elements responsible for maintaining blood-cell production throughout life and for lymphohematopoietic reconstitution following bone marrow (BM) transplantation. Enhancement of the engrafting potential and expansion capabilities of HSCs as well as hematopoietic progenitor cells (HPCs) has been a long-time desire as a means of reducing the risks and difficulties that accompany BM transplantation. The ability of HSCs/HPCs to reconstitute the hematopoietic system of irradiated hosts is negatively regulated by an intracellular adaptor protein, Lnk. Here we have identified the functional domains of Lnk and developed a dominant-negative (DN) Lnk mutant that inhibits the functions of Lnk endogenously expressed in the HSCs/HPCs and thereby potentiates the HSCs/HPCs for engraftment. Importantly, even transient expression of DN-Lnk in HSCs/HPCs facilitated their engraftment under nonmyeloablative conditions and fully reconstituted the lymphoid compartments of immunodeficient host animals. HPCs expressing DN-Lnk were efficiently trapped by immobilized vascular cell adhesion molecule-1 (VCAM-1) in a transwell migration assay, suggesting involvement of Lnk in the regulation of cell mobility or cellular interaction in microenvironments. Transient inhibition of Lnk or Lnk-mediated pathways could be a potent approach to augment engraftment of HSCs/HPCs without obvious side effects.     

Chemokine stromal cell-derived factor-1alpha modulates VLA-4 integrin-dependent adhesion to fibronectin and VCAM-1 on bone marrow hematopoietic progenitor cells

Stromal cell-derived factor-1alpha (SDF-1alpha) is a potent chemoattractant for hematopoietic progenitor cells (HPC), suggesting that it could play an important role during their migration within or to the bone marrow (BM). The integrin VLA-4 mediates HPC adhesion to BM stroma by interacting with CS-1/fibronectin and VCAM-1. It is required during hematopoiesis and homing of HPC to the BM. As HPC migration in response to SDF-1alpha might require dynamic regulation of integrin function, we investigated if SDF-1alpha could modulate VLA-4 function on BM CD34(hi) cells.CD34(hi) BM cells and hematopoietic cell lines were tested for the effect of SDF-1alpha on VLA-4-dependent adhesion to CS-1/fibronectin and VCAM-1, as well as to BM stroma. CD34(hi) BM cells that adhered to VLA-4 ligands after SDF-1alpha treatment were characterized in colony-forming and long-term culture-initiating cell (LTC-IC) assays.SDF-1alpha rapidly (1 minute) and transiently upregulated the adhesion of CD34(hi) BM cells and hematopoietic cell lines to both CS-1/fibronectin and VCAM-1, and to BM stromal cells. The upregulation of VLA-4-dependent cell adhesion by SDF-1alpha targeted primitive LTC-IC as well as committed CD34(hi) cells. SDF-1alpha-triggered enhancement in VLA-4 function was inhibited by pertussis toxin (PTx) and cytochalasin D, indicating the involvement of G(i) protein downstream signaling and an intact cytoskeleton. Instead, activation of p44/42 MAP kinases by SDF-1alpha did not functionally correlate with enhancement of VLA-4-dependent cell adhesion.Modulation of VLA-4-mediated CD34(hi) BM cell adhesion by SDF-1alpha could play a key role in their migration within and to the BM and therefore influence their proliferation and differentiation.     

Distinct roles of integrins alpha6 and alpha4 in homing of fetal liver hematopoietic stem and progenitor cells

Homing of hematopoietic stem cells (HSCs) into the bone marrow (BM) is a prerequisite for establishment of hematopoiesis during development and following transplantation. However, the molecular interactions that control homing of HSCs, in particular, of fetal HSCs, are not well understood. Herein, we studied the role of the alpha6 and alpha4 integrin receptors for homing and engraftment of fetal liver (FL) HSCs and hematopoietic progenitor cells (HPCs) to adult BM by using integrin alpha6 gene-deleted mice and function-blocking antibodies. Both integrins were ubiquitously expressed in FL Lin(-)Sca-1(+)Kit(+) (LSK) cells. Deletion of integrin alpha6 receptor or inhibition by a function-blocking antibody inhibited FL LSK cell adhesion to its extracellular ligands, laminins-411 and -511 in vitro, and significantly reduced homing of HPCs to BM. In contrast, the anti-integrin alpha6 antibody did not inhibit BM homing of HSCs. In agreement with this, integrin alpha6 gene-deleted FL HSCs did not display any homing or engraftment defect compared with wild-type littermates. In contrast, inhibition of integrin alpha4 receptor by a function-blocking antibody virtually abrogated homing of both FL HSCs and HPCs to BM, indicating distinct functions for integrin alpha6 and alpha4 receptors during homing of fetal HSCs and HPCs.     

Cell surface peptidase CD26/DPPIV mediates G-CSF mobilization of mouse progenitor cells

CXC ligand 12 (CXCL12; also known as stromal cell-derived factor 1alpha/SDF-1alpha) chemoattracts hematopoietic stem and progenitor cells (HSCs/HPCs) and is thought to play a crucial role in the mobilization of HSCs/HPCs from the bone marrow. CD26 (dipeptidylpeptidase IV [DPPIV]) is a membrane-bound extracellular peptidase that cleaves dipeptides from the N-terminus of polypeptide chains. CD26 has the ability to cleave CXCL12 at its position-2 proline. We found by flow cytometry that CD26 is expressed on a subpopulation of normal Sca-1+c-kit+lin- hematopoietic cells isolated from mouse bone marrow, as well as Sca-1+c-kit-lin- cells, and that these cells possess CD26 peptidase activity. To test the functional role of CD26 in CXCL12-mediated normal HSC/HPC migration, chemotaxis assays were performed. The CD26 truncated CXCL12(3-68) showed an inability to induce the migration of sorted Sca-1+c-kit+lin- or Sca-1+c-kit-lin- mouse marrow cells compared with the normal CXCL12. In addition, CXCL12(3-68) acts as an antagonist, resulting in the reduction of migratory response to normal CXCL12. Treatment of Sca-1+c-kit+lin- mouse marrow cells, and myeloid progenitors within this population, or Sca-1+c-kit-lin- cells with a specific CD26 inhibitor, enhanced the migratory response of these cells to CXCL12. Finally, to test for potential in vivo relevance of these in vitro observations, mice were treated with CD26 inhibitors during granulocyte colony-stimulating factor (G-CSF)-induced mobilization. This treatment resulted in a reduction in the number of progenitor cells in the periphery as compared with the G-CSF regimen alone. This suggests that a mechanism of action of G-CSF mobilization involves CD26.     

Implication of CD44 in adhesion-mediated overproduction of TGF-β and IL-1 in monocytes from patients with bone marrow fibrosis

Myelofibrosis (MF) is characterized by bone marrow (BM) fibrosis and excessive deposits of extracellular matrix (ECM) proteins which include fibronectin (FN), collagen type I and hyaluronic acid (HA). We have previously reported that adhesion to polystyrene overactivates MF monocytes. We now confirm their activation by increased CD25 expression and tyrosine phosphorylation. We hypothesize that ECM protein-adhesion molecule interactions induce overproduction of fibrogenic cytokines in MF monocytes leading to BM fibrosis. In this study we found that FN, collagen type I and HA induce 2–3-fold more TGF-β and 6–9-fold more interleukin (IL)-1 in MF monocytes than normal controls (NC). Since CD44 can function as the natural ligand for these proteins, its role was studied. We found that CD44 mediated most of the TGF-β and IL-1 produced. Immunoprecipitation of CD44 revealed three proteins at approximately 110 kD in MF monocytes and one in NC. Our results indicate that adhesion is important in overproduction of TGF-β and IL-1, and that their production is at least partly mediated by adhesion molecule–ECM protein interactions. These results implicate at least one adhesion molecule, CD44, in the pathophysiology of BM fibrosis.     

Expression and affinity of homing-related molecules on steady-state adult and neonate human PB CD34+ cells and their SRC activity

Although the vast majority of hematopoietic progenitor cells (HPCs) reside within the bone marrow (BM), a small number of HPCs also continuously circulate in the peripheral blood (PB). The examination of the fate of blood-borne HPCs in parabiotic mice, which are surgically conjoined and share a common circulation, recently revealed that steady-state PB HPCs play a physiological role in, at least, the functional re-engraftment of unconditioned BM. To assess the possibility that human HPCs have a similar function, in this study we examined the expression level and affinity of the homing-related molecules, as well as the SCID mouse reconstituting cell (SRC) activity of human PB CD34+ cells, and compared adults with neonates. There was no remarkable difference between adults and neonates in the expression of E- and/or P-selectin ligands by PB CD34+ cells or in these cells' affinity to VCAM-1. In contrast, the expression level of CXCR4 on PB CD34+ cells was much lower in adults than in neonates. Adult cells also showed a much lower SRC activity than neonates. These results suggest that human PB HPCs may contribute to steady-state hematopoiesis in the BM of neonates to some extent, but not so much in adults.     

Hematopoietic capability of CD34+ cord blood cells: a comparison with CD34+ adult bone marrow cells

The characteristics of hematopoietic progenitor and stem cell (HPC/HSC) populations in mammals vary according to their ontogenic stage. In humans, HPC/HSCs from umbilical cord blood (CB) are increasingly used as an alternative to HPC/HSCs from adult bone marrow (BM) for the treatment of various hematologic disorders. How the hematopoietic activity of progenitor and stem cells in CB differs from that in adult BM remains unclear, however. We compared CD34+ cells, a hematopoietic cell population, in CB with those in adult BM using phenotypic subpopulations analyzed by flow cytometry, the colony-forming activity in methylcellulose clonal cultures, and the repopulating ability of these cells in NOD/Shi-scid (NOD/SCID) mice. Although the proportion of CD34+ cells was higher in adult BM than in CB mononuclear cells, the more immature subpopulations, CD34+ CD33- and CD34+ CD38- cells, were present in higher proportions in CD34+ CB cells. Clonal culture assay showed that more multipotential progenitors were present in CD34+ CB cells. When transplanted into NOD/SCID mice. CD34+ adult BM cells could not reconstitute human hematopoiesis in recipient BM, but CD34+ CB cells achieved a high level of engraftment, indicating that CD34+ CB cells possess a greater repopulating ability. These results demonstrated that human hematopoiesis changes with development from fetus to adult. Furthermore, CD34+ CB cells contained a greater number of primitive hematopoietic cells, including HSCs, than did adult BM, suggesting the usefulness of CD34+ CB cells not only as a graft for therapeutic HSC transplantation but also as a target cell population for ex vivo expansion of transplantable HSCs and for gene transfer in gene therapy.     

Role of CXCR4 chemokine receptor blockade using AMD3100 for mobilization of autologous hematopoietic progenitor cells

G-CSF mobilization of hematopoietic progenitor cells (HPCs) is mediated through enzyme release from maturing myeloid cells, leading to digestion of adhesion molecules, trophic chemokines and their receptors, and the extracellular matrix. HPCs traffic to and are retained in the marrow through the trophic effects of the chemokine SDF-1alpha/CXCL12 binding to its receptor, CXCR4. AMD3100 reversibly inhibits SDF-1alpha/CXCR4 binding, and AMD3100 administration mobilizes CD34+ cells into the circulation. AMD3100 has been tested in several clinical trials which demonstrate that it improves the number of CD34+ cells mobilized including patients failing to mobilize with G-CSF alone. Engraftment of AMD3100-mobilized cells is prompt and durable. Toxicities are mild and infrequent. Lymphoma and myeloma cells do not appear to be mobilized. AMD3100 appears to be a promising agent for HPC mobilization.     

Chemokine stromal cell-derived factor-1alpha modulates VLA-4 integrin-mediated multiple myeloma cell adhesion to CS-1/fibronectin and VCAM-1

The chemokine stromal cell-derived factor-1alpha (SDF-1alpha) and its G-protein-linked receptor CXCR4 are involved in hematopoietic progenitor cell and lymphocyte migration. The integrin VLA-4 is a cell adhesion receptor for CS-1/fibronectin and VCAM-1 and constitutes one of the main adhesion receptors mediating myeloma cell adhesion to bone marrow (BM) stroma in multiple myeloma (MM). It is shown here that MM CD38(hi)CD45RA(-) BM cells and myeloma-derived cell lines expressed CXCR4 and displayed a moderate chemotactic response to SDF-1alpha. Because cell migration in response to SDF-1alpha might require a dynamic regulation of integrin function, it was investigated whether SDF-1alpha can modulate VLA-4 function on myeloma cells. SDF-1alpha rapidly and transiently up-regulated VLA-4-mediated myeloma cell adhesion to both CS-1/fibronectin and VCAM-1, which was inhibited by pertussis toxin and cytochalasin D, indicating the involvement of G(i) protein downstream signaling and an intact cytoskeleton. Modulation of VLA-4-dependent myeloma cell adhesion by SDF-1alpha could contribute to the trafficking and localization of these cells in the BM microenvironment.     

A novel anti-inflammatory function of human galectin-1: inhibition of hematopoietic progenitor cell mobilization

OBJECTIVE: The immunosuppressive and anti-inflammatory activity of mammalian galectin-1 (Gal-1) has been well established in experimental in vivo animal models and in vitro studies. Since the proliferation and migration of leukocytes represent a necessary and important step in response to the inflammatory insult, we have investigated whether Gal-1 affects the mobilization of hematopoietic progenitor cells (HPC) induced by cyclophosphamide (CY) and granulocyte colony-stimulating factor (G-CSF). METHODS: Bone marrow HPCs were mobilized with CY/G-CSF or CY/G-CSF plus human recombinant Gal-1 in BDF1 mice. Bone marrow (BM) and blood cells were taken at different time points and analyzed for their in vivo repopulating ability in lethally irradiated syngeneic animals. The number of myeloid progenitor cells in BM and blood samples was determined by colony-forming cell assay. Expression of surface markers (Sca-1, CD3epsilon, CD45R/B220, Ter-119, GR-1, and CD11b) on nucleated marrow cells was measured by flow cytometry. The lymphocytes, granulocytes, and monocytes in blood samples were counted after Giemsa staining. RESULTS: Gal-1 dramatically inhibited CY/G-CSF-induced HPC migration to the periphery as well as decreased peripheral neutrophilia and monocytosis in a dose- and time-dependent manner. In contrast, Gal-1 itself stimulated HPC expansion and accumulation within the BM. The presence of the lectin for inhibition of HPC mobilization was essential during the second half of the treatment. Moreover, Gal-1 inhbited transendothelial migration of BM-derived HPCs in response to SDF-1 in vitro. CONCLUSION: Gal-1 blocked BM progenitor cell migration induced by CY/G-CSF treatment, indicating a novel anti-inflammatory function of the lectin. We suggest that the inhibition of HPC mobilization occurs mainly via obstructing the transendothelial migration of BM-derived cells including primitive hematopoietic and committed myeloid progenitor cells and mature granulocytes and monocytes.     

Primitive hematopoietic cells in murine bone marrow express the CD34 antigen

The CD34 antigen is expressed on most, if not all, human hematopoietic stem cells (HSCs) and hematopoietic progenitor cells, and its use for the enrichment of HSCs with repopulating potential is well established. However, despite homology between human and murine CD34, its expression on subsets of primitive murine hematopoietic cells has not been examined in full detail. To address this issue, we used a novel monoclonal antibody against murine CD34 (RAM34) to fractionate bone marrow (BM) cells that were then assayed in vitro and in vivo with respect to differing functional properties. A total of 4% to 17% of murine BM cells expressed CD34 at intermediate to high levels, representing a marked improvement over the resolution obtained with previously described polyclonal anti-CD34 antibodies. Sixty percent of CD34+ BM cells lacked lineage (Lin) markers expressed on mature lymphoid or myeloid cells. Eighty-five percent of Sca-1+Thy-1(10)Lin- /10 cells that are highly enriched in HSCs expressed intermediate, but not high, levels of CD34 antigen. The remainder of these phenotypically defined stem cells were CD34-. In vitro colony-forming cells, day-8 and -12 spleen colony-forming units (CFU-S), primitive progenitors able to differentiate into B lymphocytes in vitro or into T lymphocytes in SCID mice, and stem cells with radioprotective and competitive long-term repopulating activity were all markedly enriched in the CD34+ fraction after single-parameter cell sorting. In contrast, CD34-BM cells were depleted of such activities at the cell doses tested and were capable of only short-term B-cell production in vitro. The results indicate that a significant proportion of murine HSCs and multilineage progenitor cells express detectable levels of CD34, and that the RAM34 monoclonal antibody is a useful tool to subset primitive murine hematopoietic cells. These findings should facilitate more direct comparisons of the biology of CD34+ murine and human stem and progenitor cells.     

Cytokine regulation of proliferation and cell adhesion are correlated events in human CD34+ hemopoietic progenitors

Adhesive interactions with the extracellular matrix of the bone marrow (BM) stroma are of critical importance in the regulation of hematopoiesis. In part, these interactions are presumed to play an important role in retaining CD34+ hematopoietic progenitor cells (HPCs) within the BM environment, in close proximity with BM stromal cells and the cytokines they produce. Evidence of a more direct role for cell adhesion in the regulation of hematopoiesis is provided by recent data showing that adhesive interactions can also provide important costimulatory signals. We have previously shown that normal CD34+ HPCs express high levels of fibronectin (Fn) receptors very late antigen-4 (VLA-4) and VLA-5 in a low-affinity state, which do not allow HPCs to strongly adhere on immobilized Fn, and that cytokines such as interleukin-3, granulocyte-monocyte colony-stimulating factor, and stem cell factor transiently activate these receptors, providing HPCs with an adhesive phenotype on Fn. Thus, knowledge of the functional states of adhesion receptors is critical to our understanding of the physiological mechanisms responsible for the regulation of normal hematopoiesis. Herein, we show that combinations of cytokines that synergize to stimulate the proliferation of CD34+ HPCs result in additive stimulation of the adhesion of these cells to Fn. Thus, the activation level of Fn receptors expressed by normal CD34+ HPCs is highly correlated with their proliferative state, suggesting a functional link between these two events. Therefore, we propose a 2- step model with an initial activation of VLA-4 and VLA-5 generated by cytokine receptors that is followed by a secondary signal resulting from Fn binding to VLA-4 and VLA-5, which may cooperate with those generated by cytokine receptors.     

Pluripotent and myeloid-committed CD34+ subsets in hematopoietic stem cell allografts

The present study compared the contents of pluripotent and lineage-committed hematopoietic progenitor cells (HPCs) in various types of allografts. Bone marrow (BM) allografts and single leukapheresis products (LPs) collected from G-CSF-mobilized donors contained similar amounts of pluripotent HPCs (CD34(+)CD38(-)) and total CD34(+) cells. However, the content of late-myeloid HPCs (CD34(+)CD33(+)CD15(+)) were significantly higher in BM grafts compared to LPs (P>0.02), whereas the contents of early-myeloid HPCs (CD34(+)CD33(+)CD15-) were 2.5-fold higher in LPs (P<0.03). In comparison to grafts from adult donors, cord blood (CB) grafts contained 26-65-fold lower amounts of early-myeloid HPCs (P<0.001), but only 8-12-fold lower contents of pluripotent HPCs (P<0.04). Additional findings demonstrated that among all tested parameters the numbers of early-myeloid HPCs were the most accurate measure of the total colony-forming cell (CFC) numbers in allografts. Hence, the earlier engraftment observed after transplantation of LPs compared to BM grafts might be explained by the higher content of early-myeloid HPCs/CFCs in LPs. Moreover, the slow engraftment following CB transplantation might not be affected essentially by the low number of myeloid HPCs, but rather by pluripotent HPCs. Finally, this study reports a new gating strategy for the enumeration of pluripotent CD34(+)CD38(-) subsets.     

Total body irradiation causes profound changes in endothelial traffic molecules for hematopoietic progenitor cell recruitment to bone marrow

Nonirradiated bone marrow (BM) venules and sinusoids in murine skull support hematopoietic progenitor cell (HPC) rolling through constitutively expressed endothelial (P- and E-) selectins and VCAM-1. Using intravital microscopy, we tested whether host conditioning with total body irradiation (TBI) changes the molecular mechanisms by which murine HPCs from fetal livers (FL) interact with BM endothelial cells. Although a high dose of TBI did not affect the overall frequency of HPC rolling in BM microvessels, the underlying molecular mechanisms differed from those in nonirradiated BM. TBI induced VCAM-1 up-regulation in BM microvessels, whereas P-selectin expression was reduced and the low baseline level of E-selectin remained unchanged. Only the administration of anti-VCAM-1, but not anti-P- or -E-selectin monoclonal antibodies, decreased FL HPC rolling. Rolling was frequently followed by firm arrest (sticking), even in nonirradiated BM microvessels in which sticking was entirely pertussis toxin-insensitive-that is, Galpha(i)-coupled signaling events (eg, through chemokines) were apparently not required. TBI increased the frequency of sticking FL HPC. This irradiation-induced additional sticking was reversed when FL HPCs were pretreated with pertussis toxin, suggesting that TBI induced elevated expression of a Galpha(i)-protein-coupled chemotactic signal in the BM. This chemoattractant was probably distinct from SDF-1alpha because, unlike adult HPCs, FL HPCs (day 11 of gestation) responded poorly to SDF-1alpha in vitro. These results demonstrate that TBI induces profound changes in the expression of endothelial traffic molecules in the BM, and they indicate that FL HPCs can home to the BM in the absence of SDF-1alpha and other Galpha(i)-protein-coupled signals.