Adhesion Of Progenitor Cells Research Articles

Adhesion and Migration Are Differentially Regulated in Hematopoietic Progenitor Cells by Cytokines and Extracellular Matrix

AbstractThe conditions that control the migratory status of hematopoietic progenitor cells on extracellular matrix (ECM) and that decide whether a cell migrates or adheres are incompletely understood. We analyzed the migratory behavior of murine hematopoietic progenitor cells factor-dependent-cell-paterson (FDCP)-mix and purified lin−Sca1+ bone marrow cells on ECM. We found that migration on fibronectin (Fn) or laminin (Lam) becomes dependent on β1-integrins if a surface restraint force is introduced by tilting the ECM-coated culture vessels. Under these conditions, migration specifically occured on Fn and Lam, and was not detected on collagen IV-, hyaluronate-, or bovine serum albumin- coated surfaces. Migration depended on the continuous presence of hematopoietic cytokines interleukin-3 (IL-3), granulocyte colony-stimulating factor (G-CSF ), macrophage-CSF (M-CSF), granulocyte-macrophage-CSF (GM-CSF ), or stem cell factor (SCF), whereas other cytokines, such as IL-8, macrophage inflammatory protein-1α, macrophage-chemotactic and activating factor, and erythropoietin resulted in very little or no migratory response. IL-3 induced migration was synergistically enhanced by other CSFs, but was completely inhibited by addition of transforming growth factor-β1. In contrast to firm local adhesion of previously cytokine depleted progenitors that was rapidly inducible within 1 hour after exposure to cytokines, preincubation on Fn matrix for 4 to 6 hours was required before cytokines could induce migration. A sudden increase of cytokine concentration reversibly inhibited migration and induced a fully adhesive state; this effect could be prolonged by consecutive stimulation with heterologous cytokines. Whereas cytokines activated resting progenitor cells to migrate on ECM, cell migration speed was regulated by Fn concentration. These results indicate that β1-integrin–mediated progenitor cell adhesion and migration are differentially regulated by external stimuli and suggest that this regulation corresponds to different activation states of β1-integrins in hematopoietic progenitor cells.

Influence of Interleukin-3 and Other Growth Factors on α4β1 Integrin-Mediated Adhesion and Migration of Human Hematopoietic Progenitor Cells

AbstractThe mechanisms by which hematopoietic progenitor cells are normally anchored in stromal niches and yet can be mobilized by specific growth factors are poorly understood. It is likely, however, that integrins and their extracellular matrix (ECM) ligands play a key role in this process, and recent evidence suggests that integrin function is modulated by signals originating from activated growth factor receptors. We have now examined this further by studying the role of growth factors on α4β1 integrin-mediated adhesion of human CD34+ hematopoietic progenitor cells to specific recombinant fibronectin fragments coated onto tissue culture dishes. Cells were prepared from cord blood and peripheral blood harvests. During a 30-minute adhesion assay a mean of 74% of CD34 cells attached to the so-called H120 fragment of fibronectin, which contains the strongest α4β1 integrin-binding sequence. The level of cell adhesion was significantly reduced by low concentrations of interleukin-3 (IL-3) (2.5 to 10 ng/mL), whereas stem cell factor (SCF ) and granulocyte colony-stimulating factor (G-CSF ) at these concentrations did not affect adherence of the cells. Migratory behavior of CD34 cells was examined using fibronectin fragments adsorbed onto a Transwell filter. The H120 fragment supported much higher levels of cell migration than the H0 fragment of fibronectin, which contains a weak α4β1 integrin binding sequence. Over a 16-hour assay, migration of peripheral blood progenitor cells was increased slightly by SCF and by G-CSF. However, a marked stimulation was observed with IL-3, which significantly increased migration. Similar effects were noted with cord blood cells, although a small proportion of cells were able to migrate in the absence of growth factors. These results indicate that there is a highly selective and functional link between the α4β1 integrin and IL-3/IL-3–receptor that could affect the position of stem and progenitor cells in the marrow stroma and influence their growth and development.

Fibronectin Improves Transduction of Reconstituting Hematopoietic Stem Cells by Retroviral Vectors: Evidence of Direct Viral Binding to Chymotryptic Carboxy-Terminal Fragments

Efficient transduction of reconstituting hematopoletic stem cells (HSC) is currently only possible by cocultivation of target cells directly on producer cell lines, a method not applicable to human gene therapy protocols. Our laboratory has previously shown adhesion of primitive hematopoletic stem and progenitor cells to the carboxy-terminal 30/35-kD fragment of the extracellular matrix molecule fibronectin (FN 30/35) (Nature 352:438, 1991) and increased transduction of human hematopoietic progenitor cells via retroviral vectors while adherent to this fragment (J Clin Invest 93:1451, 1994). Here we report that (1) transduction of reconstituting murine HSC assayed 12 months after infection with retrovirus supernatant on FN 30/35 is as effective as cocultivation directly on producer cells; (2) recombinant retrovirus particles directly adhere to FN 30/35 in a quantitative and dose-dependent fashion; and (3) increased transduction efficiency on FN 30/ 35 does not appear to be associated with increased cell proliferation or activation of protein phosphorylation typically induced by integrin-fibronectin interactions. Therefore, we speculate that supernatant infection of HSC on FN 30/35 leads to colocalization of retrovirus particles and target cells on FN 30/35 molecule with a large increase in local virus titer presented to the cell. These findings have direct and important implications for the modification of current human gene therapy protocols.

Fibronectin improves transduction of reconstituting hematopoietic stem cells by retroviral vectors: evidence of direct viral binding to chymotryptic carboxy-terminal fragments

Efficient transduction of reconstituting hematopoletic stem cells (HSC) is currently only possible by cocultivation of target cells directly on producer cell lines, a method not applicable to human gene therapy protocols. Our laboratory has previously shown adhesion of primitive hematopoletic stem and progenitor cells to the carboxy-terminal 30/35- kD fragment of the extracellular matrix molecule fibronectin (FN 30/35) (Nature 352:438, 1991) and increased transduction of human hematopoietic progenitor cells via retroviral vectors while adherent to this fragment (J Clin Invest 93:1451, 1994). Here we report that (1) transduction of reconstituting murine HSC assayed 12 months after infection with retrovirus supernatant on FN 30/35 is as effective as cocultivation directly on producer cells; (2) recombinant retrovirus particles directly adhere to FN 30/35 in a quantitative and dose- dependent fashion; and (3) increased transduction efficiency on FN 30/ 35 does not appear to be associated with increased cell proliferation or activation of protein phosphorylation typically induced by integrin- fibronectin interactions. Therefore, we speculate that supernatant infection of HSC on FN 30/35 leads to colocalization of retrovirus particles and target cells on FN 30/35 molecule with a large increase in local virus titer presented to the cell. These findings have direct and important implications for the modification of current human gene therapy protocols.

Identification of novel K562 membrane proteins that adhere to bone marrow fibroblasts

Adhesion of myeloid leukemia cells to the bone marrow (BM) microenvironment is mediated in part by Beta 1 and Beta 2 integrins. Cells of the erythroleukemia line K562, derived from a patient with chronic myeloid leukemia, bind to BM fibroblasts (BMFs) but the adhesion cannot be accounted for by integrins or other known adhesion proteins including CD44 or members of the Ig or selectin families. Membrane fragments from K562 cells were radioiodinated and allowed to adhere to BMF monolayers. Adherent proteins were solubilized together with the fibroblasts, analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and visualized by autoradiography. Four adherent proteins were consistently observed. Two of these, with reduced molecular weights of 52 kD and 35 to 37 kD, were prominent. Addition of soluble thrombospondin and heparin but not fibronectin inhibited binding of K562 membrane proteins to adherent BMFs and immobilized thrombospondin- and heparin-bound K562 proteins. The 52-kD protein has a multimeric structure nonreduced and has characteristics of a glycoprotein. Its adhesion to fibroblasts is divalent cation and temperature sensitive. The 35- to 37-kD protein, whose function is divalent cation but not temperature sensitive, is phosphoinositol-linked and has characteristics identical to an adherent 35- to 37-kD protein identified on murine progenitor cells. Membrane preparations from two cases of acute myeloid leukemia showed an adherent 35- to 37-kD protein and in one case an adherent 52-kD protein without other adherent bands. A K562 subclone with reduced adherence to BMFs showed reduced amounts of adherent 52-kD and 35- to 37-kD proteins. These proteins may be responsible for the adhesion of malignant and normal hematopoietic progenitor cells to the BM microenvironment.

Identification of novel K562 membrane proteins that adhere to bone marrow fibroblasts

Abstract Adhesion of myeloid leukemia cells to the bone marrow (BM) microenvironment is mediated in part by Beta 1 and Beta 2 integrins. Cells of the erythroleukemia line K562, derived from a patient with chronic myeloid leukemia, bind to BM fibroblasts (BMFs) but the adhesion cannot be accounted for by integrins or other known adhesion proteins including CD44 or members of the Ig or selectin families. Membrane fragments from K562 cells were radioiodinated and allowed to adhere to BMF monolayers. Adherent proteins were solubilized together with the fibroblasts, analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and visualized by autoradiography. Four adherent proteins were consistently observed. Two of these, with reduced molecular weights of 52 kD and 35 to 37 kD, were prominent. Addition of soluble thrombospondin and heparin but not fibronectin inhibited binding of K562 membrane proteins to adherent BMFs and immobilized thrombospondin- and heparin-bound K562 proteins. The 52-kD protein has a multimeric structure nonreduced and has characteristics of a glycoprotein. Its adhesion to fibroblasts is divalent cation and temperature sensitive. The 35- to 37-kD protein, whose function is divalent cation but not temperature sensitive, is phosphoinositol- linked and has characteristics identical to an adherent 35- to 37-kD protein identified on murine progenitor cells. Membrane preparations from two cases of acute myeloid leukemia showed an adherent 35- to 37- kD protein and in one case an adherent 52-kD protein without other adherent bands. A K562 subclone with reduced adherence to BMFs showed reduced amounts of adherent 52-kD and 35- to 37-kD proteins. These proteins may be responsible for the adhesion of malignant and normal hematopoietic progenitor cells to the BM microenvironment.

Characterization of TGF‐β1‐binding proteins in human bone marrow stromal cells

The proliferation and differentiation of haemopoietic progenitor cells is dependent on their close relation with bone marrow stromal cells, which constitute a source of cytokines as well as expressing receptors for both the cytokines and progenitor cell adhesion molecules necessary for regulated haemopoiesis. We have generated human bone marrow stromal cell cultures and analysed the TGF-beta 1 receptor components expressed by these cells. [125I]TGF-beta 1-affinity labelling experiments showed the involvement of type I and II receptors in the binding of TGF-beta 1, as demonstrated by specific immunoprecipitation of [125I]TGF-beta 1-receptor complexes. In addition, large TGF-beta 1-labelled complexes displaying an electrophoretic mobility similar to betaglycan were also observed in these experiments. Endoglin, another component of the TGF-beta receptor system, was detected by flow cytometry on the surface of cultured marrow stromal cells, and in the human bone marrow stromal cell line Str-5, and was immunoprecipitated from surface-iodinated cells. Endoglin on the stromal cells was able to bind TGF-beta 1, as demonstrated by specific immunoprecipitation of [125I]TGF-beta 1-endoglin complexes using anti-endoglin antibodies. The results presented provide evidence that bone marrow stromal cells are fully capable of responding to TGF-beta 1. Given the important role of TGF-beta as a regulator of the synthesis of cytokines and cytokine receptors, as well as cell adhesion molecules, these data indicate that the binding of TGF-beta 1 by stromal cells might represent an important step in the regulation of the proliferation and differentiation of haemopoietic progenitor cells.

Adhesion of Human Hematopoietic Progenitor Cells to Bone-Marrow-Derived Stromal Cells Is Enhanced by Antibodies to CD44

It has been suggested that CD44 mediates adhesive interactions between hematopoietic progenitor cells and the stromal microenvironment. Ligands of CD44 include several extracellular matrix components, such as hyaluronic acid and fibronectin. Antibodies against CD44 have been shown to induce homotypic T cell aggregation, and to stimulate T and natural killer cell activity. We hypothesized that CD44 could similarly amplify interactions between blast-colony-forming cells and bone marrow stromal cells (BMSCs). Indeed, we have previously found that the anti-CD44 antibody NKI-P2 enhanced VLA-4-dependent interactions. Here, we studied an additional panel of nineteen anti-CD44 antibodies from the 5th Workshop on Leukocyte Differentiation antigens, to find out whether amplification was associated with a particular CD44 epitope. None of these antibodies showed inhibitory activity, whereas nine significantly increased the number of blast colonies more than 2-fold. Seven of these recognized epitope 1, and two epitope 2. More than 4-fold enhancement was only observed with epitope 1 antibodies: 4.C3 (4.4-fold), 212.3 (6.3-fold), L178 (9.1-fold), and NIH44-1 (9.2-fold). Our data suggest that primarily epitope 1 is associated with enhancement of colony formation. Furthermore, the findings support a role for CD44 as an amplifier in progenitor-BMSC interactions.

Influence of interferon-alpha on cytokine expression by the bone marrow microenvironment--impact on treatment of myeloproliferative disorders.

Myeloproliferative disorders (MPD) are characterized by several common clinical and biological features, although at the molecular level, each disease entity exhibits distinct abnormalities. IFN-alpha exerts beneficial therapeutic effects in chronic myelogenous leukemia, polycythemia vera and essential thrombocythemia, resulting in control of hematopoietic hyperplasia and, in a minority of patients, in induction of cytogenetic remission. The mechanism of action of IFN-alpha in MPD is poorly defined. Recently published in vitro findings suggest that IFN-alpha interacts with the regulation of hematopoiesis by multiple ways. Its antiproliferative activity is well known for more than a decade, however, substantial growth inhibition is achieved only at relatively high concentrations. Defective adhesion of hematopoietic progenitor cells in CML to bone marrow stromal cells is corrected by IFN-alpha, which might expose CML progenitors to inhibitory cytokines produced by the bone marrow microenvironment. Recent work from our group demonstrated, that IFN-alpha potently interacts with the production of hematopoietic cytokines in bone marrow stromal cells. Expression of stimulatory cytokines, such as GM-CSF, G-CSF, IL-1 and IL-11 is inhibited by IFN-ct, whereas the production of negative regulators, such as IL-1RA and MIP-1 alpha, is stimulated. The combined action of IFN-alpha on paracrine expression of cytokines suggests an indirect antihematopoietic effect, which might contribute to its clinical activity in MPD.

The role of stromal cell heparan sulphate in regulating haemopoiesis.

Intimate contact between haemopoietic progenitor cells and elements of the bone marrow stroma is required for progenitor cell proliferation and differentiation. It is believed that the stroma provides particular niches for the development of haemopoietic cells of different lineages. Cytokines, stromal cell surface molecules and molecules of the stromal extracellular matrix all contribute to defining these microenvironmental niches. Data obtained using an in vitro model of haemopoiesis support the view that progenitor cell adhesion to stroma is mediated by multiple receptor-ligand interactions. The possibility of a tethering step, mediated by the engagement of stromal cell heparan sulphate with its ligands on the progenitor cells, preceding stable cell adhesion is discussed. The role of stromal cell heparan sulphate is likely to include cytokine presentation to progenitors as well as the tethering of progenitors to stroma. It is proposed that intracellular signals induced by progenitor cell adhesion to stroma act in association with cytokine induced signals to regulate progenitor cell proliferation and differentiation.

Modulation of the adhesion of hemopoietic progenitor cells to the rgd site of fibronectin by interleukin 3

The integrins are a class of adhesion molecules which have been implicated in the homing of hemopoietic stem cells and in their restriction within the bone marrow. Integrins function as mediators of cell-extracellular matrix (ECM) interactions amd also of cell-cell interactions. They are unique membrane receptors which are capable of activation, change in affinity, and change in expression. Because of their broad potential for modulation we examined the effect of a cytokine growth factor which is present constitutively in the marrow, interleukin 3 (IL3), on integrin-mediated adherence of hemopoietic progenitor cells to the matrix component fibronectin (FN). The multipotential murine cell line B6Sut and the committed granulocyte progenitor cell line FDCP-1 were used. Both of these cell lines have been shown to bind to FN-coated dishes and to dishes coated with the 120 kDa and 40 kDa chymotryptic fragments of FN. It was found that after a brief withdrawal of IL3 the cells lost 80% adherence to the 120 kDa FN fragment containing the RGD cell binding site. This loss of binding was not related to a loss of viability, appeared unrelated to the growth/survival activity of IL3, and was quickly reversible by readdition of the growth factor. Adhesion of these cells to the RGD site was likely mediated by alpha 5 beta 1 integrin which was identified in the cell membrane of both cell lines, but present in low copy number in B6Sut cells. Two antibodies against the external and internal domains of alpha 5 and one antibody against beta 1 were used to study expression of the integrin. By flow cytometry the expression of alpha 5 was found to decrease in both cell lines by 4 h in the absence of IL3. The relative mean fluorescence intensity for B6Sut cells decreased from 1.0 (control cells always in the presence of IL3) to 0.6 over 4 h, and for FDCP-1 cells the decrement was from 1.0 to 0.8. The loss of RGD-mediated adhesion in the absence of IL3 appeared to proceed through this decrement in expression of the integrin; a loss of affinity of the receptor for its substrate was not detected. The general modulation of integrin activity by growth factors is of great interest because of its potential negative impact on the endothelium in cytokine-treated patients, and also because of its potential positive impact on engraftment during clinical bone marrow transplantation.

Adhesion of committed human hematopoietic progenitors to synthetic peptides from the C-terminal heparin-binding domain of fibronectin: cooperation between the integrin alpha 4 beta 1 and the CD44 adhesion receptor

Close interaction of human hematopoietic progenitors with the bone marrow microenvironment is important for the ordered progression of human hematopoiesis. Progenitor cell adhesion to stroma has a complex molecular basis, involving various cell-extracellular matrix and cell- cell interactions. We have previously shown that adhesion of colony- forming cells (CFC) to fibronectin, present in stromal extracellular matrix, involves multiple sites, including two heparin-binding synthetic peptides (FN-C/H I and FN-C/H II) and the alpha 4 beta 1 integrin-binding peptide CS1. These synthetic peptides are located in close proximity in the type III repeat 14 and the immediately adjacent type IIIcs region of fibronectin. In the current study, we evaluate receptors expressed by CFC responsible for their adhesion to fibronectin. We show that the alpha 4 beta 1 integrin mediates adhesion to CFC to the peptides FN-C/H I and CS1. Adhesion of CFC to fibronectin is also mediated by proteoglycans, because removal of cell surface chondroitin-sulfate proteoglycans resulted in decreased adhesion of CFC to FN-C/ I and FN-C/H II. The core protein of this proteoglycan was identified by immunoprecipitation as a 90-kD member of the CD44 group of adhesion molecules. Interestingly, although the proteoglycan core protein failed to adhere to FN-C/H II affinity columns, anti-CD44 monoclonal antibodies blocked CFC adhesion to FN-C/H II, indicating that these monoclonal antibodies may interfere with core protein- mediated intracellular signalling. Finally, we show that CD44 and alpha 4 beta 1 may cooperate in establishing progenitor adhesion, because anti-CD44 antibodies potentiated the adhesion-inhibitory effects of suboptimal concentrations of anti-alpha 4 or anti-beta 1 monoclonal antibodies. These results provide a working model for progenitor cell recognition of fibronectin (and possibly the marrow micro-environment) in which the coordinated action of integrins and cell surface proteoglycans is necessary for cell adhesion. This model can now be used to study the complex relationship between progenitor cell adhesion and the regulation of their proliferation and differentiation.

Adhesion of Committed Human Hematopoietic Progenitors to Synthetic Peptides From the C-Terminal Heparin-Binding Domain of Fibronectin: Cooperation Between the Integrin α4β1 and the CD44 Adhesion Receptor

Close interaction of human hematopoietic progenitors with the bone marrow microenvironment is important for the ordered progression of human hematopoiesis. Progenitor cell adhesion to stroma has a complex molecular basis, involving various cell-extracellular matrix and cell-cell interactions. We have previously shown that adhesion of colony-forming cells (CFC) to fibronectin, present in stromal extracellular matrix, involves multiple sites, including two heparin-binding synthetic peptides (FN-C/H I and FN-C/H II) and the alpha 4 beta 1 integrin-binding peptide CS1. These synthetic peptides are located in close proximity in the type III repeat 14 and the immediately adjacent type IIIcs region of fibronectin. In the current study, we evaluate receptors expressed by CFC responsible for their adhesion to fibronectin. We show that the alpha 4 beta 1 integrin mediates adhesion to CFC to the peptides FN-C/H I and CS1. Adhesion of CFC to fibronectin is also mediated by proteoglycans, because removal of cell surface chondroitin-sulfate proteoglycans resulted in decreased adhesion of CFC to FN-C/ I and FN-C/H II. The core protein of this proteoglycan was identified by immunoprecipitation as a 90-kD member of the CD44 group of adhesion molecules. Interestingly, although the proteoglycan core protein failed to adhere to FN-C/H II affinity columns, anti-CD44 monoclonal antibodies blocked CFC adhesion to FN-C/H II, indicating that these monoclonal antibodies may interfere with core protein-mediated intracellular signalling. Finally, we show that CD44 and alpha 4 beta 1 may cooperate in establishing progenitor adhesion, because anti-CD44 antibodies potentiated the adhesion-inhibitory effects of suboptimal concentrations of anti-alpha 4 or anti-beta 1 monoclonal antibodies. These results provide a working model for progenitor cell recognition of fibronectin (and possibly the marrow micro-environment) in which the coordinated action of integrins and cell surface proteoglycans is necessary for cell adhesion. This model can now be used to study the complex relationship between progenitor cell adhesion and the regulation of their proliferation and differentiation.

Isolation and Characterization of Human Bone Marrow Microvascular Endothelial Cells: Hematopoietic Progenitor Cell Adhesion

To examine potential mechanisms by which hematopoiesis may be regulated by endothelial cells within the bone marrow (BM) microenvironment, we have devised a technique for the in vitro study of the interaction of human BM microvascular endothelial cells (BMEC) with hematopoietic cells. Microvessels isolated by collagenase digestion of spicules obtained from filtered BM aspirate were plated on gelatin-coated plastic dishes, and colonies of endothelial cells grown from microvessel explants were further purified by Ulex europaeus lectin affinity separation. BMEC monolayers isolated by this technique grew in typical cobblestone fashion, stained positively with antibody to factor VIII/von Willebrand factor, and incorporated acetylated LDL. Immunohistochemical studies showed that BM microvessels and BMEC monolayers express CD34, PECAM, and thrombospondin. Incubation of resting BMEC with BM mononuclear hematopoietic cells resulted in the selective adhesion of relatively large numbers of CD34+ progenitor cells and megakaryocytes. The binding of purified BM-derived CD34+ progenitor cells to BMEC was dependent on divalent cations and was partially blocked by antibodies to CD34. IL-1 beta treatment of BMEC monolayers resulted in an increase of CD34+ progenitor cell adhesion by mechanisms independent of CD34 or divalent cations. BMEC exhibit specific affinity for CD34+ progenitor cells and megakaryocytes, suggesting that the BM microvasculature may play a role in regulating the trafficking, proliferation, and differentiation of lineage specific hematopoietic elements, and possibly of pluripotent stem cells within the CD34+ population.

Isolation and characterization of human bone marrow microvascular endothelial cells: hematopoietic progenitor cell adhesion

To examine potential mechanisms by which hematopoiesis may be regulated by endothelial cells within the bone marrow (BM) microenvironment, we have devised a technique for the in vitro study of the interaction of human BM microvascular endothelial cells (BMEC) with hematopoietic cells. Microvessels isolated by collagenase digestion of spicules obtained from filtered BM aspirate were plated on gelatin-coated plastic dishes, and colonies of endothelial cells grown from microvessel explants were further purified by Ulex europaeus lectin affinity separation. BMEC monolayers isolated by this technique grew in typical cobblestone fashion, stained positively with antibody to factor VIII/von Willebrand factor, and incorporated acetylated LDL. Immunohistochemical studies showed that BM microvessels and BMEC monolayers express CD34, PECAM, and thrombospondin. Incubation of resting BMEC with BM mononuclear hematopoietic cells resulted in the selective adhesion of relatively large numbers of CD34+ progenitor cells and megakaryocytes. The binding of purified BM-derived CD34+ progenitor cells to BMEC was dependent on divalent cations and was partially blocked by antibodies to CD34. IL-1 beta treatment of BMEC monolayers resulted in an increase of CD34+ progenitor cell adhesion by mechanisms independent of CD34 or divalent cations. BMEC exhibit specific affinity for CD34+ progenitor cells and megakaryocytes, suggesting that the BM microvasculature may play a role in regulating the trafficking, proliferation, and differentiation of lineage specific hematopoietic elements, and possibly of pluripotent stem cells within the CD34+ population.

Roles for Integrin Very Late Activation Antigen-4 in Stroma-Dependent Erythropoiesis

AbstractAdhesion molecules are required for development of hematopoietic stem and progenitor cells in the respective hematopoietic microenvironments. We previously showed that development of the erythroid progenitor cells is dependent on their direct adhesion to the stroma cells established from the erythropoietic organs. In this stroma-dependent erythropoiesis, we examined the role of adhesion molecules in erythropoiesis by blocking antibodies. The development of the erythroid cells on stroma cells was inhibited by anti-very late activation antigen-4 (VLA-4 integrin) antibody, but not by anti-VLA-5 antibody, although the erythroid cells express both VLA-4 and VLA-5. Whereas high levels of expression of vascular cell adhesion molecule-1 (VCAM-1) and fibronectin, ligands for VLA-4, were detected in the stroma cells, the adhesion and development of the erythroid progenitor cells were partly inhibited by the blocking antibody against VCAM-1. VLA-5 and fibronectin could mediate adhesion of the erythroid progenitor cells to the stromal cells, but the adhesion itself may not be sufficient for the stroma-supported erythropoiesis. The stromal cells may support erythroid development by the adhesion through a new ligand molecule(s) for VLA-4 in addition to VCAM-1, and such collaborative interaction may provide adequate signaling for the erythroid progenitor cells in the erythropoietic microenvironment.

Cellular adhesion molecules.

NORMAL hematopoiesis in adult humans occurs within the bone marrow (BM) in the milleu of the marrow microenvironment. l Cellular,2 cytokine,3,4 and extracellular matrix (ECM)5 interactions facilitate normal proliferation and differentiation of hematopoietic progenitor cells. Long-term BM cultures provide an in vitro model for study of this in vivo microenvironment and show that an adherent stromal layer consisting of fibroblasts, macrophages, endothelial cells, and ECM is required to sustain hematopoiesis.6 In particular, early progenitor cells preferentially associate with the stromal layer: adhesion of early progenitor cells to marrow-derived adherent cells has been documented for erythroid, myeloid, and lymphoid precursors. 9 Moreover, the interactions of normal and malignant hematopoietic cells with one another, with the vascular endothelium, and with the subendothelial matrix are essential for a number of other physiological and pathologieal processes including lymphocyte migration, wound healing, inflammation, hemostasis, thrombosis, and tumor cell metastasis. These attachments require the concerted actions of a number of cell adhesion molecules. Cell adhesion molecules (CAMs) that mediate cell-cell and cell-matrix interactions belong to at least five families: cadherins, the immunoglobulin gene superfamily, integrins, selectins, and cell surface proteoglycans (Table l). A number of reviews of these families of CAMs delineate their role in normal hematopoietic and immunologie ontogeny, and in the growth and spread of tumor cells. 10-14 In this review, we briefly characterize the role of these five CAM families in normal cell adhesion and then focus on their role in the pathogenesis of hematologic malignancies.

A novel 37-Kd adhesive membrane protein from cloned murine bone marrow stromal cells and cloned murine hematopoietic progenitor cells

The adhesion of hematopoietic progenitor cells to bone marrow stromal cells is critical to hematopoiesis and involves multiple effector molecules. Stromal cell molecules that participate in this interaction were sought by analyzing the detergent-soluble membrane proteins of GBI/6 stromal cells that could be adsorbed by intact FDCP-1 progenitor cells. A single-chain protein from GBI/6 cells having an apparent molecular weight of 37 Kd was selectively adsorbed by FDCP-1 cells. This protein, designated p37, could be surface-radiolabeled and thus appeared to be exposed on the cell membrane. An apparently identical 37-Kd protein was expressed by three stromal cell lines, by Swiss 3T3 fibroblastic cells, and by FDCP-1 and FDCP-2 progenitor cells. p37 was selectively adsorbed from membrane lysates by a variety of murine hematopoietic cells, including erythrocytes, but not by human erythrocytes. Binding of p37 to cells was calcium-dependent, and was not affected by inhibitors of the hematopoietic homing receptor or the cell-binding or heparin-binding functions of fibronectin. It is proposed that p37 may be a novel adhesive molecule expressed on the surface of a variety of hematopoietic cells that could participate in both homotypic and heterotypic interactions of stromal and progenitor cells.

A Novel 37-Kd Adhesive Membrane Protein From Cloned Murine Bone Marrow Stromal Cells and Cloned Murine Hematopoietic Progenitor Cells

AbstractThe adhesion of hematopoietic progenitor cells to bone marrow stromal cells is critical to hematopoiesis and involves multiple effector molecules. Stromal cell molecules that participate in this interaction were sought by analyzing the detergent-soluble membrane proteins of GBI/6 stromal cells that could be adsorbed by intact FDCP-1 progenitor cells. A single-chain protein from GBI/6 cells having an apparent molecular weight of 37 Kd was selectively adsorbed by FDCP-1 cells. This protein, designated p37, could be surface-radiolabeled and thus appeared to be exposed on the cell membrane. An apparently identical 37-Kd protein was expressed by three stromal cell lines, by Swiss 3T3 fibroblastic cells, and by FDCP-1 and FDCP-2 progenitor cells. p37 was selectively adsorbed from membrane lysates by a variety of murine hematopoietic cells, including erythrocytes, but not by human erythrocytes. Binding of p37 to cells was calcium-dependent, and was not affected by inhibitors of the hematopoietic homing receptor or the cell-binding or heparin-binding functions of fibronectin. It is proposed that p37 may be a novel adhesive molecule expressed on the surface of a variety of hematopoietic cells that could participate in both homotypic and heterotypic interactions of stromal and progenitor cells.

Expression of integrins and examination of their adhesive function in normal and leukemic hematopoietic cells

Adhesion of hematopoietic progenitor cells to marrow-derived adherent cells has been noted for erythroid, myeloid, and lymphoid precursors. In this report, we have characterized very late antigen (VLA) integrin expression on normal CD34+ marrow progenitors, on leukemic cell lines, and on blasts from patients with acute myelogenous or monocytic leukemias. CD34+ progenitor cells expressed the integrin beta 1 chain (CD29), VLA-4 alpha (CD49d), and VLA-5 alpha (CD49e). The myeloid lines KG1 and KG1a also expressed CD49d and CD49e as did the Mo7e megakaryoblastic line. CD29, CD18, and CD11a were also present on each of these cell lines. Only the Mo7e line expressed the cytoadhesins GPIIbIIIa or GPIb. Binding of KG1a to marrow stroma was partially inhibited by antibodies to CD49d and its ligand, vascular cell adhesion molecule (VCAM-1). The majority of leukemic blasts studied expressed CD49d and CD49e as well. Blasts from patients with acute myelomonocytic leukemia consistently bound to stroma at levels greater than 20%, and adhesion to stroma could in some cases be partly inhibited by anti- CD49d. No role for glycosylphosphatidyl-inositol (GPI)-linked structures was demonstrated in these binding assays because the adhesion of leukemic blasts to stroma was not diminished after treatment with phosphatidylinositol-specific phospholipase C (PI-PLC). These studies indicate that CD34+ myeloid progenitors, myeloid leukemic cell lines, and leukemic blasts possess a similar array of VLA integrins. Their functional importance individually or in combination with other mediators of attachment in adhesion, transendothelial migration, and differentiation has yet to be fully elucidated.