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J P Lévesque

Publications and source records attributed to J P Lévesque.

14 recordsLinked to original sources
PubMed2001

Vascular cell adhesion molecule-1 (CD106) is cleaved by neutrophil proteases in the bone marrow following hematopoietic progenitor cell mobilization by granulocyte colony-stimulating factor.

Mobilized progenitor cells currently represent the most commonly used source of hematopoietic progenitor cells (HPCs) to effect hematopoietic reconstitution following myeloablative chemotherapies. Despite their widespread use, the molecular mechanisms responsible for the enforced egress of HPCs from the bone marrow (BM) into the circulation in response to mobilizing agents such as cytokines remain to be determined. Results of this study indicate that expression of vascular cell adhesion molecule-1 (VCAM-1) is strongly reduced in vivo in the BM during HPC mobilization by granulocyte colony-stimulating factor (G-CSF) and stem cell factor. Two serine proteases, namely, neutrophil elastase and cathepsin G, were identified, which cleave VCAM-1 and are released by neutrophils accumulating in the BM during the course of immobilization induced by G-CSF. The proposal is made that an essential step contributing to the mobilization of HPCs is the proteolytic cleavage of VCAM-1 expressed by BM stromal cells, an event triggered by the degranulation of neutrophils accumulating in the BM in response to the administration of G-CSF.

Animals↗
PubMed1999

Cytoskeleton and integrin-mediated adhesion signaling in human CD34+ hemopoietic progenitor cells.

Significant progress has been made recently in the understanding of cell adhesion signaling. Many components of focal adhesion complexes have been identified in fibroblasts and endothelial cells, showing considerable overlap and complementarity between growth signaling mediated by growth factor receptors and adhesive signaling mediated by cell adhesion receptors such as integrins. These studies showed that the cytoskeleton is essential for the correct intracellular localization of large signaling complexes that regulate the cellular machinery. Although adhesive interactions are essential to maintain steady-state hemopoiesis, the study of the function and role of adhesive interactions in hemopoietic progenitor and stem cells is less advanced. As in fibroblasts, functional overlap between hemopoietic growth factor receptors and cell adhesion receptors has been demonstrated, with the cytoskeleton likely playing a critical role in integrating information provided by soluble factors and cell adhesion molecules constituting the hemopoietic microenvironment. The intention of this article is to give a critical review of the current knowledge about the cytoskeleton and integrin-mediated signaling in hemopoietic progenitor cells.

Animals↗
PubMed1999

PSGL-1-mediated adhesion of human hematopoietic progenitors to P-selectin results in suppression of hematopoiesis.

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.

ADP-ribosyl Cyclase↗
PubMed1998

Osteoclast-mediated bone resorption is stimulated during short-term administration of granulocyte colony-stimulating factor but is not responsible for hematopoietic progenitor cell mobilization.

The cellular and molecular mechanisms responsible for hematopoietic progenitor cell (HPC) mobilization from bone marrow (BM) into peripheral blood after administration of cytokines such as granulocyte colony-stimulating factor (G-CSF) are still unknown. In this study we show that high concentrations of soluble calcium induce the detachment of BM CD34(+) HPC adherent on fibronectin, a major component of BM extracellular matrix. Because G-CSF has been shown to induce osteoporosis in patients with congenital neutropenia and in G-CSF-overexpressing transgenic mice, we hypothesized that short-term G-CSF administration may be sufficient to induce bone resorption, resulting in the release of soluble calcium in the endosteum leading in turn to the inhibition of attachment to fibronectin and the egress of HPC from the BM. We show herein that in humans, serum osteocalcin concentration, a specific marker of bone formation, is strongly reduced after 3 days of G-CSF administration. Furthermore, in patients mobilized with G-CSF either alone or in association with stem cell factor or interleukin-3, the reduction of serum osteocalcin is significantly correlated with the number of HPC mobilized in peripheral blood. Urine levels of deoxypyridinoline (DPyr), a specific marker of bone resorption, gradually elevated during the time course of G-CSF administration until day 7 after cessation of G-CSF, showing a simultaneous stimulation of bone degradation during G-CSF-induced HPC mobilization. In an in vivo murine model, we found that the number of osteoclasts was dramatically increased paralleling the elevation of DPyr after G-CSF administration. When pamidronate, an inhibitor of osteoclast-mediated bone resorption, was administered together with G-CSF in mice, the G-CSF-induced increase of DPyr levels was completely abolished whereas the numbers of colony-forming cells mobilized in peripheral blood were not decreased, but unexpectedly increased relative to the numbers elicited by G-CSF alone. Collectively, our data therefore show that short-term administration of G-CSF induces bone degradation by a simultaneous inhibition of bone formation and an enhanced osteoclast-mediated bone resorption. This increased bone resorption is inhibited by pamidronate without reducing G-CSF-induced HPC mobilization, suggesting that the activation of bone resorption after G-CSF administration is not the direct cause of HPC mobilization as initially hypothesized, but a parallel event.

Adult↗
PubMed1998

Stem cell factor as a single agent induces selective proliferation of the Philadelphia chromosome positive fraction of chronic myeloid leukemia CD34(+) cells.

The interaction between p145(c-KIT) and p210(bcr-abl) in transduced cell lines, and the selective outgrowth of normal progenitors during long-term culture of chronic myeloid leukemia (CML) cells on stroma deficient in stem-cell factor (SCF) suggests that the response of CML cells to SCF may be abnormal. We examined the proliferative effect of SCF(100 ng/mL), provided as the sole stimulus, on individual CD34(+) cells from five normal donors and five chronic-phase CML patients. Forty-eight percent of isolated single CML CD34(+) cells proliferated after 6 days of culture to a mean of 18 cells, whereas only 8% of normal CD34(+) cells proliferated (mean number of cells generated was 4). SCF, as a single agent, supported the survival and expansion of colony-forming unit-granulocyte-macrophage (CFU-GM) from CML CD34(+)CD38(+) cells and the more primitive CML CD34(+)CD38(-) cells. These CFU-GM colonies were all bcr-abl positive, showing the specificity of SCF stimulation for the leukemic cell population. Coculture of CML and normal CD34(+) cells showed exclusive growth of Ph+ cells, suggesting that growth in SCF alone is not dependent on secretion of cytokines by CML cells. SCF augmentation of beta1-integrin-mediated adhesion of CML CD34(+) cells to fibronectin was not increased when compared with the effect on normal CD34(+) cells, suggesting that the proliferative and adhesive responses resulting from SCF stimulation are uncoupled. The increased proliferation may contribute to the accumulation of leukemic progenitors, which is a feature of CML.

Adult↗
PubMed1998

Mitogenic effect of fibrinogen on hematopoietic cells: involvement of two distinct specific receptors, MFR and ICAM-1.

In addition to its well-known functions in blood clotting and cell adhesion, fibrinogen has been reported to be a mitogen for lymphoid cell lines and for human hematopoietic progenitors. Two specific receptors, the mitogenic fibrinogen receptor (MFR) and intercellular adhesion molecule-1 (ICAM-1/CD54), have been identified as possible candidates for the mediation of the mitogenic effect of fibrinogen. However, it has been questioned whether the MFR and ICAM-1 are truly distinct molecules. Using an antiserum specific for the MFR, we demonstrate that the MFR is a cell surface molecule clearly distinct from ICAM-1. Both receptors can be expressed separately or coexpressed on different cell types. Moreover, they are regulated differently: ICAM-1 is calcium-dependent whereas the MFR is not and the MFR is down-regulated by fibrinogen whereas ICAM-1 is not. The inhibition by an anti-MFR serum of the mitogenic effect of fibrinogen confirms the mitogenic function of the MFR.

Antibodies↗
PubMed1998

Dual control by divalent cations and mitogenic cytokines of alpha 4 beta 1 and alpha 5 beta 1 integrin avidity expressed by human hemopoietic cells.

Beta-1 integrins have essential functions in hemopoietic and immune systems by controlling phenomenons such as cell homing and cell activation. The function alpha 4 beta 1 and alpha 5 beta 1 integrins is regulated by divalent cations and, as demonstrated more recently, by mitogenic cytokines which activate them by "inside-out" mechanisms. Using the adhesive interaction of a cytokine-dependent human hemopoietic cell line to immobilized fibronectin, we have analyzed the requirements in divalent cations Mn2+, Mg2+ and Ca2+ for alpha 4 beta 1 and alpha 5 beta 1 activation by "inside-out" mechanisms triggered by cytokines such as granulocyte-macrophage colony stimulating factor or KIT ligand, or by external conformational constraints with the function-activating anti-beta 1 integrin monoclonal antibody 8A2. The intrinsic difference between these two modes of beta 1 integrin activation was revealed by their different requirements in divalent cations. We found that in the absence of any divalent cations, alpha 4 beta 1 and alpha 5 beta 1 were non-functional even after further stimulation by cytokines or 8A2. However, whilst either Ca2+, Mg2+ or Mn2+ were able to restore adhesive functions of alpha 4 beta 1 and alpha 5 beta 1 when activated by 8A2, only Mg2+ and Mn2+ were able to support activation of alpha 4 beta 1 and alpha 5 beta 1 by cytokines. Furthermore, high concentrations of Ca2+ exceeding 20 mM dramatically inhibited cell adhesion to fibronectin induced by Mn2+ and cytokines but not by 8A2. On the contrary, in the presence of both Ca2+ and Mg2+, Mn2+ had an additive effect on the activation of alpha 4 beta 1 and alpha 5 beta 1 by mitogenic cytokines. The presence of the absence of these divalent cations did not inhibit early tyrosine phosphorylation induced by the binding of KIT ligand to its tyrosine-kinase receptor KIT. Therefore, we propose that in hemopoietic cells, Ca2+, Mg2+ and Mn2+ may modulate in vivo alpha 4 beta 1 and alpha 5 beta 1 regulation by mitogenic cytokines, a phenomenon involved in the regulation of hemopoietic progenitor cell homing within the bone marrow.

Anti-Allergic Agents↗
PubMed1996

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.

Adult↗
PubMed1995

Early CD34high cells can be separated into KIThigh cells in which transforming growth factor-beta (TGF-beta) downmodulates c-kit and KITlow cells in which anti-TGF-beta upmodulates c-kit.

We have previously shown that early human CD34high hematopoietic progenitors are maintained quiescent in part through autocrine transforming growth factor-beta 1 (TGF-beta 1). We also demonstrated that, in the presence of interleukin-3, interleukin-6, granulocyte colony-stimulating factor, and erythropoietin, TGF-beta 1 antisense oligonucleotides or anti-TGF-beta serum have an additive effect with KIT ligand (Steel factor [SF]), which suggests that they control different pathways of regulation in these conditions. This finding also suggests that autocrine TGF-beta 1 might suppress c-kit expression in primitive human hematopoietic progenitors. We have now distinguished two subpopulations of CD34high cells. One subpopulation expresses a c-kit mRNA that can be downmodulated by exogenous TGF-beta 1 within 6 hours. Another subpopulation of early CD34high cells expresses a low or undetectable level of c-kit mRNA, but its expression can be upmodulated within 6 hours by anti-TGF-beta. These effects disappear 48 hours after induction and cannot be maintained longer than 72 hours, even if TGF-beta 1 or anti-TGF-beta serum are added every day. Similar kinetics, although delayed, are observed with KIT protein expression. On the contrary, no specific effect of TGF-beta 1 was observed on c-fms, GAPDH, and transferrin receptor gene expression in these early progenitors. These results clarify the complex interaction between TGF-beta 1 and SF in normal early hematopoietic progenitors. SF does not switch off the TGF-beta 1 inhibitory pathway. Autocrine TGF-beta 1 appears to maintain these cells in a quiescent state, suppressing cell division by downmodulating the receptor of SF, a key cytokine costimulator of early progenitors.

Antibodies, Monoclonal↗
PubMed1995

Cytokines increase human hemopoietic cell adhesiveness by activation of very late antigen (VLA)-4 and VLA-5 integrins.

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.

Adult↗
PubMed1991

Mitogenic properties of major extracellular proteins.

The major plasma and extracellular matrix proteins are multifunctional molecules. Some, such as fibrinogen or C3, have one domain that binds adhesion receptors and another that specifically binds and activates a separate, mitogenic receptor. In this review, Jean-Pierre Lévesque, Antoinette Hatzfeld and Jacques Hatzfeld describe adhesion and mitogenic receptors that bind to distinct domains of the same extracellular matrix protein and discuss the possibility of common ancestral genes for cell adhesion molecules, extracellular matrix proteins, integrins, immunoglobulins, growth factors and their receptors.

Complement C3↗
PubMed1991

DNA transfection in COS cells: a low-cost serum-free method compared to lipofection.

We describe a defined medium that allows efficient DNA transfections in COS cells and transient expression of the corresponding recombinant protein in serum-free conditions. With a modified DEAE-dextran/chloroquine method, we obtained 80% more transfected cells expressing the recombinant human interleukin-2 receptor than with transfection with cationic liposomes, one of the most efficient techniques to date. The absence of serum in the culture medium should reduce subsequent purification steps for production of recombinant mammalian proteins. Moreover, it should allow investigations dealing with the role of serum or other exogenous factors on mRNA stability or post-translation events during protein synthesis.

Cell Line↗