Regulation of megakaryocytopoiesis in an in vitro stroma model: preferential adhesion of megakaryocytic progenitors and subsequent inhibition of maturation

OBJECTIVE: Studies of megakaryocytic progenitor cell interactions have focused on single receptor-ligand interactions using isolated components of the extracellular matrix. To approach a physiologic condition, we studied megakaryocytic development of human progenitor cells cultured on two stromal cell lines and on human bone marrow stroma. MATERIALS AND METHODS: Human CD34(+) cells were cocultured with stromal layers in the presence of thrombopoietin. Megakaryocytes were quantified by monoclonal antibodies against glycoprotein (GP) IIb/IIIa (CD41) and GPIX (CD42a). Megakaryocytic clonogenic capacity (burst-forming unit-megakaryocyte and colony-forming unit-megakaryocyte) was determined using fibrin clot assays. RESULTS: After 6 days, a higher percentage of megakaryocytes and more megakaryocytic colonies were recovered from the adherent cell fraction compared to the nonadherent cell fraction. In contrast, significantly more granulocytic and erythroid colonies were recovered from the nonadherent cell fraction. Repeated replating of nonadherent cells onto fresh stroma showed a decline in megakaryocytic recovery of the remaining adherent cells, pointing toward selective adhesion of megakaryocytic progenitors. This was supported further by the finding that burst-forming unit and colony-forming unit megakaryocytes were preferentially recovered from the adherent cell fraction at 24 hours. No effect of blocking the beta(1) integrins VLA-4 and VLA-5 on human progenitor cells was observed. A higher expression of CD42a antigen and a higher percentage of morphologically recognizable polyploid megakaryocytes were found when cells were grown in noncontact cultures compared to when grown adhered to stroma. CONCLUSION: In contrast to granulocytic and erythroid progenitors, both very early and more mature megakaryocytic progenitors are preferentially located in the adherent fraction in an in vitro stromal model, leading to inhibition of maturation of megakaryocytes. This suggests that the presence of stroma components in ex vivo expansion cultures, aimed at preservation and expansion of megakaryocytic progenitors, might be a prerequisite.     

Adhesive interactions of normal and leukemic human CD34+ myeloid progenitors: role of marrow stromal, fibroblast, and cytomatrix components.

Adhesive interactions between CD34+ myeloid progenitors, cytomatrix components, and marrow fibroblast and stromal monolayers are described and compared to the binding interactions of the CD34+ myeloid leukemic cell lines KG1a and KG1. Both normal precursors and their leukemic counterparts showed adhesion to marrow stroma and fibroblasts. CD34+ myeloid progenitors bound to the extracellular matrices of marrow stromal cell and fibroblast monolayers and to laminin and fibronectin to a lesser extent than to cellular stromal layers. These adhesive interactions were not inhibited by polyclonal antibodies to laminin or fibronectin, nor by 1 mM Arg-Gly-Asp-Ser (RGDS)-containing peptides. Also, although both normal and leukemic cells expressed the CD18 antigen, binding of these cells to stroma was not inhibited by blocking anti-CD18 monoclonal antibodies. Finally, KG1a adhesion was not blocked in the presence of anti-CD54 (ICAM) antibody, nor was it blocked when galactosyl or mannosyl pyranosides were added. KG1a binding was trypsin sensitive and enhanced in the presence of neuraminidase. These studies serve to characterize adhesive properties of normal and leukemic myeloid progenitors and begin to establish interactions important for the lodgement of early progenitor cells in human marrow.     

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.     

Ligation of CD44 with low-molecular-weight hyaluronan and a monoclonal antibody leads to inhibition of drug-induced apoptosis in a human myeloid cell line

Abstract

Adhesive interactions between hematopoietic progenitor cells and extracellular matrix can improve progenitor cell survival. These mechanisms involve a number of different molecules. CD44 is one such molecule, although its molecular basis has not been elucidated. In this study, we investigated the effect of CD44 monoclonal antibodies and hyaluronan, which is a ligand of CD44, on drug-induced apoptosis in human myeloid cell line KG1. Preincubation with anti-CD44 monoclonal antibody J173 or a lower-molecular-weight form of hyaluronan (LMW-HA) could reduce drug-induced apoptosis in a dose-dependent manner from 23·0 ± 1·4% to 5·9 ± 5·0% (p<0·01) or 9·7 ± 1·8% (p<0·01) respectively. On the other hand, another anti-CD44 monoclonal antibody L178 and the native high-molecular-weight polymer of hyaluronan had no effect on drug-induced apoptosis. Furthermore, J173 and LMW-HA induced a rapid increase in tyrosine phosphorylation of intracellular proteins. Genistein, a protein tyrosine kinase inhibitor, abrogated the inhibition of drug-induced apoptosis promoted by J173 and LMW-HA. These results suggest that the anti-apoptotic effect by ligation of CD44 was mediated by tyrosine phosphorylation of intracellular proteins. These data indicate that tyrosine phosphorylation via CD44 is involved in the survival of primitive myeloid cells.     

Roles for integrin very late activation antigen-4 in stroma-dependent erythropoiesis

Adhesion 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.     

Tissue- and epitope-specific mechanisms account for the diverse effects of anti-CD44 antibodies on the maintenance of primitive hematopoietic progenitors in vitro

The identification of rare stromal cells that support high levels of stem cells has opened avenues to identify molecules that contribute to the maintenance of these cells. We show that the maintenance of long-term culture initiating cells (LTC-IC) in stromal cell-supported cultures can be modulated via mAbs specific for CD44. mAb IM7.8.1 suppressed while mAb RAMBM44 enhanced LTC-IC levels in culture. Genetic polymorphisms in CD44 were used to show that the stromal cell compartment is targeted by mAb RAMBM44 and the hematopoietic compartment by mAb IM7.8. Neither of the CD44-specific mAbs inhibited adhesion of LTC-IC to the stroma, suggesting alternative mechanisms of action. In support of this interpretation, we show that mAb RAMBM44 directly induces signal transduction in the stromal cell line S17 but not in hematopoietic cells. Conversely, mAb IM7.8 elicited the appearance of phosphorylated bands in hematopoietic cells, but not in stromal cells. Collectively, the data indicate that the opposing effects of CD44-mediated regulation can be explained by different cellular programs that are elicited in distinct cell compartments. The binding of the enhancing mAb RAMBM44 to CD44 is specifically inhibited by collagen IV, while binding of the suppressive mAb IM7.8.1 is inhibited by a substance contained in the supernatant of the stromal cell line AC3.U. Thus, the CD44 epitopes defined by the mAbs bind distinct ligands and the ligands provide a potential physiological counterpart for the regulatory actions of the mAbs.     

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.     

Involvement of CD44 variant isoform v10 in progenitor cell adhesion and maturation

CD44 has been described repeatedly to be involved in hematopoiesis. Here, we addressed the question of functional activity of CD44 variant isoform v10 (CD44v10) in progenitor cell maturation by in vivo and in vitro blocking studies with a monoclonal antibody and a receptor globulin. We became interested in this question by the observation that CD44v10 is expressed, although at a low level, on a subpopulation of bone marrow cells. Flow cytometry revealed that 15%-20% of hematopoietic cells in the fetal liver and 25%-35% of bone marrow cells in adult mice were CD44v10 positive. The majority of CD44v10+ cells was HSA+/J11d+ and CD43+. CD44v10 was not detected on CD4+, CD8+, IgM+, or IgD+ cells. A CD44v10 receptor globulin did not bind to hematopoietic progenitor cells, but to stromal elements. The CD44v10-CD44v10 ligand interaction had a major impact on the adhesion of progenitor cells to stromal elements. When healthy animals received repeated injections of either anti CD44v10 or the CD44v10 receptor globulin, committed progenitors were mobilized and significantly augmented numbers were recovered in the spleen and the peripheral blood. Furthermore, the CD44v10-CD44v10 ligand interaction, which had no impact on progenitor expansion, influenced progenitor maturation, particularly of the B-cell lineage. Although the nature of the CD44v10 ligand remains to be explored, the supportive role of CD44v10 in progenitor maturation and, importantly, the efficient mobilization of progenitor cells by anti-CD44v10 and a CD44v10 receptor globulin could be of clinical benefit in peripheral blood stem cell transplantation.     

Expression of CD21 antigen on myeloma cells and its involvement in their adhesion to bone marrow stromal cells

The mature myeloma cells express very late antigen 5 (VLA-5) and MPC-1 antigens on their surface and adhere to bone marrow (BM) stromal cells more tightly than the VLA-5-MPC-1- immature myeloma cells in vitro. The VLA-5 and MPC-1 antigens possibly function as two of the molecules responsible for interaction of mature myeloma cells with BM stromal cells. However, the immature myeloma cells do interact with BM stromal cells, and it is unclear which adhesion molecules mediate their interaction. In this study, we found that both immature and mature myeloma cells expressed CD21, an adhesion molecule known to bind to CD23. CD21 was also detected on normal plasma cells. To evaluate the role of CD21 expression on myeloma cells, two myeloma cell lines, NOP-2 (VLA-5-MPC-1-) and KMS-5 (VLA-5+MPC-1+), were used as representatives of immature and mature myeloma cell types, respectively, and an adhesion assay was performed between the myeloma cell lines and BM stromal cells. Antibody-blocking results showed that adhesion of the mature type KMS-5 to KM102, a human BM-derived stromal cell line, or to short-term cultured BM primary stromal cells was inhibited by monoclonal antibodies (MoAbs) against CD21, VLA-5, and MPC-1, and inhibition of adhesion of the immature type NOP-2 to KM102 by the anti- CD21 MoAb was observed as well. Furthermore, CD23 was detected on KM102. Treatment of KM102 with an anti-CD23 MoAb also inhibited adhesion of either KMS-5 or NOP-2 to KM102. Therefore, we propose that CD21 expressed on myeloma cells likely functions as a molecule responsible for the interaction of immature myeloma cells as well as mature myeloma cells with BM stromal cells, and CD23 may be the ligand on the stromal cells for the CD21-mediated adhesion.     

Functional activity of CD44 isoforms in haemopoiesis of the rat

Summary. Expression of CD44 is involved in the maturation as well as the homing of haemopoietic progenitor cells. Whether these processes are mediated by CD44 standard (CD44s) or variant (CD44v) isoforms is unknown. To assign functional activities of CD44 in haemopoiesis of the rat to distinct isoforms, ligand binding of haemopoietic progenitor cells was inhibited by monoclonal antibodies recognizing an epitope on CD44s (0x50) or CD44 exon v6 (1.1 ASML). The vast majority of rat bone marrow cells (BMC) as well as stromal cells and non-adherent cells in long-term bone marrow culture (LTBMC) expressed CD44s. Bone marrow cells and non-adherent cells in LTBMC, but not the stromal cells, also contained a population of large and granulated cells, which stained with anti-CD44v6. In vivo and in vitro reconstitution experiments revealed that homing of BMC as well as settlement on stromal elements was influenced exclusively by anti-CD44s, which also inhibited proliferation of progenitor cells. Anti-CD44v6 had no influence on homing and seeding, but interfered with stroma formation and progenitor maturation. Finally. restoration of functional activity of T-lineage cells was impaired in the presence of anti-CD44v6. The data indicate that CD44s and CD44v6 fulfilled distinct functions in haemopoiesis of the rat. Although CD44s facilitated homing and expansion of stem cells/progenitor cells, CD44v6 was involved in differentiation processes, particularly of lymphoid progenitor cells.     

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.     

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.     

VLA-4 and VCAM-1 are the principal adhesion molecules involved in the interaction between blast colony-forming cells and bone marrow stromal cells

Summary. The molecular basis and functional significance of interactions between haemopoietic progenitor cells and the stromal microenvironment is still poorly understood. Here we investigated a broad panel of surface adhesion molecules for their involvement. For this purpose, the colony-forming capacity of stroma-adherent Bl-CFC, BFU-E and GM-CFC was studied. Both mononuclear bone marrow cells (BMC) and bone marrow-derived stromal cells (BMSC) express a wide variety of adhesion molecules. However, only antibodies against β1-, α4-integrin (both chains of the very late activation antigen-4 (VLA-4)) and vascular cell adhesion molecule (VCAM-1) inhibited colony formation from stroma-adherent Bl-CFC by 50% or more. Antibodies against a panel of other adhesion molecules, including the α5-integrin chain, were without effect. Subsequent pretreatment experiments revealed that VLA-4 on progenitors interacted with stromal VCAM-1. The inhibitory antibodies did not interfere with the clonogenic capacity of but with adhesion of BFU-E and GM-CFC. Whether the inhibitory antibodies act similarly on progenitors which depend on BMSC for growth and/or differentiation, such as Bl-CFC, remains to be determined.     

Contact with fibronectin enhances preservation of normal but not chronic myelogenous leukemia primitive hematopoietic progenitors

OBJECTIVE: Coculture with stromal cells enhances preservation and self-renewal of primitive progenitor potential in hematopoietic cells during ex vivo culture with growth factors (GF). However, the respective roles of growth factors, stromal contact, and extracellular matrix (ECM) ligands in this effect are not clear. Here we investigated the role of direct contact with stroma and the ECM protein fibronectin (FN) in these effects, and investigated whether abnormal integrin receptor function in chronic myelogenous leukemia (CML) progenitors was associated with perturbation in these responses. METHODS: Normal bone marrow CD34+ cells were cultured in GF-containing medium with or without contact with stromal layers, glutaraldehyde-fixed stromal layers (stroma-contact), or integrin-binding FN fragments for 7 days. Progeny cells were assayed for primitive progenitors in week-6 long-term culture-initiating cell (LTC-IC) and week-10 extended LTC-IC (ELTC-IC) assays. RESULTS: Increased LTC-IC and ELTC-IC preservation was seen following coculture with stroma, and was also observed after culture in contact with fixed stromal layers and FN. Both alpha4beta1 and alpha5beta1-integrin binding FN fragments enhanced LTC-IC preservation. Analysis of single CD34+CD38- cells showed that coculture with FN resulted in significantly reduced cell division, but enhanced retention of LTC-IC capacity in divided cells. FN also increased LTC-IC frequency in undivided cells. CML progenitors demonstrate deficient integrin-mediated adhesion, migration, and signaling. Coculture of CML CD34+ cells with stroma and FN failed to enhance LTC-IC preservation. CONCLUSION: We conclude that beta1 integrin-FN interactions enhance normal primitive progenitor preservation with or without cell division, and that these mechanisms are impaired in CML primitive progenitors.     

Hematopoiesis: The Role of Stromal Integrin Interactions in Pro-B Cell Proliferation

Recent studies using long term bone marrow cultures have concluded that adherence of lymphoid precursors to the underlying stromal cells is required for normal B cell development. However, the function of specific integrin interactions in B cell development remains unresolved. In our laboratory, we observed that maximal proliferation of pro-B cells required the presence of stromal cells and that stromal cytokines alone could not replace the requirement for stromal cell contact. For that reason, we questioned whether integrin interactions play a role in regulating pro-B cell proliferation in the bone marrow. Murine pro-B cell line Cl.92 expressed VLA-4, CD44, and fibronectin-receptor. Abrogation of binding of these molecules to stromal cell ligands using blocking antibodies resulted in failure of pro-B cell adherence and significant decreases in pro-B cell proliferation. Disruption of single integrin interactions did not compromise either adhesion of pro-B cells to stromal cells or IL-7 stimulated proliferation. Taken together, these data suggest that normal pro-B cells interact with stromal cells through multiple integrin interactions and that integrin mediated potentiation of pro-B cell proliferation is functionally redundant and not affected by failure of single ligand interactions.     

Ligation of CD44 leads to killing activity in human peripheral mononuclear cells via MAP kinase and tyrosine kinases

CD44 is a widely distributed transmembrane glycoprotein associated with various lymphocyte functions, including lympho-hemopoiesis, adhesion to the extracellular matrix, and T cell activation. In this study, we examined the mechanisms of CD44 involvement in regulating the killing activity of human peripheral mononuclear cells (PMC). An anti-CD44 monoclonal antibody (mAb) J173 enhanced the killing activity of PMC against Daudi and K562 cells in a dose-dependent manner. The increased cytotoxicity peaked at mAb concentration of 1.25 microg/ml. Under this condition, triggering of CD44 enhanced the killing activity by 1.5- and 2.2-fold at an effector-to-target (E/T) ratio of 20 for Daudi and K562 cells, respectively. Cytotoxic activity was remarkably diminished by treatment of PMC with concanamycin A, suggesting that this PMC-mediated cytotoxicity is mainly exerted via the perforin pathway. Moreover, we found that ligation of CD44 transduced signals to PMC that led to the tyrosine phosphorylation of several intracellular proteins and activation of mitogen-activated protein (MAP) kinase. Genistein, an inhibitor of tyrosine phosphorylation, and PD98059, an inhibitor of MAP kinase, suppressed CD44-induced enhancement of cytotoxicity. These results suggest that the CD44 molecule, which is a main receptor for hyaluronan known to be expressed on the surface of tumor cells, plays an important role in PMC-mediated cytotoxicity, and that tyrosine kinases and MAP kinase are essential for CD44-mediated signaling in cytotoxicity.     

Modulation of integrin function in hematopoietic progenitor cells by CD43 engagement: possible involvement of protein tyrosine kinase and phospholipase C-gamma

Attachment of cells to extracellular matrix components is critical for the regulation of hematopoiesis. CD43 is a mucin-like transmembrane sialoglycoprotein expressed on the surface of almost all hematopoietic cells. A highly extended structure of extracellular mucin with negative charge may function as a repulsive barrier to hematopoietic cells. However, some investigators have shown that CD43 has proadhesive properties, and engagement of CD43 has been reported to upregulate integrin-mediated cell adhesion in T cells. We found that cross-linking of CD43 with monoclonal antibodies (MoAbs) enhanced integrin alpha4beta1 (very late antigen [VLA]-4) and alpha5 beta1 (VLA-5)-dependent adhesion of human cord blood CD34(+) cells to fibronectin. CD34(+) CD38(hi), but not CD34(+)CD38(-/low) cells responded significantly to the stimulus, suggesting that committed, but not stem and more immature progenitors are sensitive to CD43-mediated activation of integrin. To elucidate the molecular mechanism leading to integrin activation, we used the growth factor-dependent cell line MO7e. Cross-linking of CD43 induced tyrosine phosphorylation of several intracellular molecules including the protein tyrosine kinase Syk, the proto-oncogene product Cbl, and phospholipase C (PLC)-gamma2 in MO7e cells. Moreover, protein tyrosine kinase inhibitor herbimycin A and PLC inhibitor U73122 both blocked CD43-induced enhancement of adhesion to fibronectin. These results indicate that signals mediated through CD43 may increase integrin affinity to fibronectin via a pathway dependent on protein tyrosine kinase and PLC-gamma activation in hematopoietic progenitors.     

Ligation of CD44 leads to killing activity in human peripheral mononuclear cells via MAP kinase and tyrosine kinases

Abstract

CD44 is a widely distributed transmembrane glycoprotein associated with various lymphocyte functions, including lympho-hemopoiesis, adhesion to the extracellular matrix, and T cell activation. In this study, we examined the mechanisms of CD44 involvement in regulating the killing activity of human peripheral mononuclear cells (PMC). An anti-CD44 monoclonal antibody (mAb) J173 enhanced the killing activity of PMC against Daudi and K562 cells in a dose-dependent manner. The increased cytotoxicity peaked at mAb concentration of 1·25 μg/ml. Under this condition, triggering of CD44 enhanced the killing activity by 1·5- and 2·2-fold at an effector-to-target (E/T) ratio of 20 for Daudi and K562 cells, respectively. Cytotoxic activity was remarkably diminished by treatment of PMC with concanamycin A, suggesting that this PMC-mediated cytotoxicity is mainly exerted via the perforin pathway. Moreover, we found that ligation of CD44 transduced signals to PMC that led to the tyrosine phosphorylation of several intracellular proteins and activation of mitogen-activated protein (MAP) kinase. Genistein, an inhibitor of tyrosine phosphorylation, and PD98059, an inhibitor of MAP kinase, suppressed CD44-induced enhancement of cytotoxicity. These results suggest that the CD44 molecule, which is a main receptor for hyaluronan known to be expressed on the surface of tumor cells, plays an important role in PMC-mediated cytotoxicity, and that tyrosine kinases and MAP kinase are essential for CD44-mediated signaling in cytotoxicity.     

Functional receptor for C3a anaphylatoxin is expressed by normal hematopoietic stem/progenitor cells,and C3a enhances their homing-related responses to SDF-1

Complement has recently been implicated in developmental pathways and noninflammatory processes. The expression of various complement components and receptors has been shown in a wide range of circulating myeloid and lymphoid cells, but their role in normal hematopoiesis and stem cell homing has not yet been investigated. We report that normal human CD34(+) cells and lineage-differentiated hematopoietic progenitors express the complement anaphylatoxin C3a receptor (C3aR) and respond to C3a. Moreover, C3a, but not the biologically inactive desArg-C3a, induces calcium flux in these cells. Furthermore, we found that C3 is secreted by bone marrow stroma and that, although C3a does not influence directly the proliferation/survival of hematopoietic progenitors, it (1) potentiates the stromal cell-derived factor 1 (SDF-1)-dependent chemotaxis of human CD34(+) cells and lineage-committed myeloid, erythroid, and megakaryocytic progenitors; (2) primes SDF-1-dependent trans-Matrigel migration; and (3) stimulates matrix metalloproteinase-9 secretion and very late antigen 4 (VLA-4)-mediated adhesion to vascular cell adhesion molecule 1 (VCAM-1). Furthermore, we found that murine Sca-1(+) cells primed by C3a engrafted faster in lethally irradiated animals. These results indicate that normal human hematopoietic stem and progenitor cells express functional C3aR and that the C3aR-C3a axis sensitizes the responses of these cells to SDF-1 and thus may be involved in promoting their homing into the bone marrow via cross talk with the SDF-CXC chemokine receptor-4 (CXCR4) signaling axis. C3a is the first positive regulator of this axis to be identified.