Enhanced hematopoietic activity of a human granulocyte/macrophage colony-stimulating factor-interleukin 3 fusion protein.

Granulocyte/macrophage colony-stimulating factor-interleukin 3 (GM-CSF-IL-3) fusion proteins were generated by construction of a plasmid in which the coding regions of human GM-CSF and IL-3 cDNAs were connected by a synthetic linker sequence followed by subsequent expression in yeast. Both GM-CSF-IL-3 and IL-3-GM-CSF fusion proteins were purified to homogeneity and shown to bind to cell-surface receptors through either their GM-CSF or IL-3 domains. The fusion proteins exhibited enhanced receptor affinity, proliferative activity, and hematopoietic colony-stimulating activity compared with either IL-3 and/or GM-CSF alone. This suggests that GM-CSF-IL-3 fusion proteins may hold future promise as therapeutic agents.     

Prenatal identification of potential donors for umbilical cord blood transplantation for Fanconi anemia

Reported here are studies of Fanconi anemia fetal cells that led to the first use of umbilical cord blood for hematopoietic reconstitution in a clinical trial. Prenatal diagnosis and HLA typing were performed in fetuses at risk for Fanconi anemia (FA) to identify, prior to birth, those that were unaffected with the syndrome and were HLA-identical to affected siblings. Umbilical cord blood was harvested at the delivery of these infants; assays of progenitor cells indicated the presence of colony-forming units-granulocyte-macrophage (CFU-GM) in numbers similar to those of bone marrow CFU-GM that are associated with successful engraftment in HLA-matched allogeneic bone marrow transplantation. The possibility that umbilical cord blood from a single individual can be used as an alternative to bone marrow for hematopoietic reconstitution has now been demonstrated by the successful engraftment of two patients with FA. Progenitor cell assays of umbilical cord blood collected at the birth of a child affected with FA, who had been misdiagnosed on the basis of chorionic villus sampling (CVS) studies, indicated a profound deficiency in colony formation, consistent with previously reported abnormalities in the growth of FA cells in vitro. These results suggest that the hematopoietic disorder in FA is related to an underlying problem with cell proliferation.     

Human gamma interferon enhances release from phytohemagglutinin- stimulated T4+ lymphocytes of activities that stimulate colony formation by granulocyte-macrophage, erythroid, and multipotential progenitor cells

Human gamma interferon (HuIFN gamma) was evaluated for its effects on the release from human peripheral blood T lymphocytes (greater than 98% pure) stimulated by phytohemagglutinin (PHA) of activities that can stimulate granulocyte-macrophage (CFU-GM) colonies and clusters, erythroid (BFU-E) bursts, and mixed (CFU-GEMM) colonies. T lymphocytes did not release these activities in the absence of PHA with or without HuIFN gamma. In the presence of PHA, pure natural HuIFN gamma at concentrations of 0.1 to 100 U/mL significantly enhanced release of these colony-stimulating activities. Although enhanced release of granulocyte-macrophage colony-stimulating activities were noted when T lymphocytes were added to the conditioning medium in the presence of 0.1%, 0.5%, and 1% PHA, enhanced release of burst-promoting and mixed colony activities was seen only in the presence of 0.1% and 0.5% PHA. The enhanced release of colony-stimulating activities was not due to HuIFN gamma-suppression of the release from PHA-stimulated T lymphocytes of suppressor molecules. The enhancing effects of natural HuIFN gamma were neutralized with a monoclonal anti-natural HuIFN gamma, and recombinant HuIFN gamma mimicked the enhancing effects of the natural HuIFN gamma. This enhancing effect was noted only when HuIFN gamma was added with the T lymphocytes and PHA during the first 24 hours of incubation. T lymphocytes were separated into T4+, T8-, T8+, and T4- subsets (greater than 98% pure for the appropriate phenotypes) after incubation with OKT4- and OKT8- monoclonal antibodies and sorting on a fluorescence-activated cell sorter (FACS). All types of colony-stimulating activities were released from each population after stimulation with PHA, but enhanced release of these activities in the presence of HuIFN gamma was only detected with the T4+ or T8- subsets of lymphocytes. It cannot be concluded from these studies whether HuIFN gamma is enhancing the release of one or several types of colony-stimulating activities, but these studies suggest a role for HuIFN gamma and T4+ lymphocyte subsets in the regulation in vitro of the release of colony-stimulating activities.     

TGF-beta combined with M-CSF and IL-4 induces generation of immune inhibitory cord blood dendritic cells capable of enhancing cytokine-induced ex vivo expansion of myeloid progenitors

Tolerogenic dendritic cells (DCs) may be valuable in transplantation for silencing immune reaction. Macrophage colony-stimulating factor (M-CSF)/IL-4 induces differentiation of cord blood (CB) monocytes into DCs (M-DCs) with tolerogenic phenotype/function. We assessed whether factors produced by tolerogenic DCs could modulate hematopoiesis. TGF-beta1 added to CB M-DC cultures induced bona fide DC morphology (TGF-M-DCs), similar to that of DCs generated with TGF-beta and granulocyte-macrophage colony-stimulating factor (GM-CSF)/IL-4 (TGF-GM-DCs). Of conditioned media (CM) produced from TGF-M-DCs, TGF-GM-DCs, M-DCs, and GM-DCs, TGF-M-DC CM was the only one that enhanced SCF, Flt3 ligand, and TPO expansion of myeloid progenitor cells ex vivo. This effect was blocked by neutralizing anti-M-CSF Ab, but protein analysis of CM suggested that M-CSF alone was not manifesting enhanced expansion of myeloid progenitors. LPS-stimulated TGF-M-DCs induced T-cell tolerance/anergy as effectively as M-DCs. TGF-M-DCs secreted significantly lower concentrations of progenitor cell inhibitory cytokines and were less potent in activating T cells than TGF-GM-DCs. Functional differences between TGF-M-DCs and TGF-GM-DCs included enhanced responses to LPS-induced ERK, JNK, and P38 activation in TGF-M-DCs and their immune suppressive-skewed cytokine release profiles. TGF-M-DCs appear unique among culture-generated DCs in their capability for silencing immunity while promoting expansion of myeloid progenitors, events that may be of therapeutic value.     

Regulation of Hematopoiesis by Chemokine Family Members

Chemokines, originally designated as chemoattractant cytokines, comprise a large family of molecules that have been implicated in a number of different functions mediated through chemokine receptors. Among these functions are regulatory roles in hematopoiesis that encompass effects on the proliferation, survival, and homing/migration of myeloid progenitor cells. This article reviews the field of chemokine regulation of hematopoiesis at the level of myeloid progenitor cells.     

Activities derived from established human myeloid cell lines reverse the suppression of cell line colony formation by lactoferrin and transferrin

Myeloid cell lines were evaluated for the release of substances needed for colony formation by their own colony-forming cells (CFC) and by other myeloid cell lines. Dialyzed U937 conditioned medium (CM) had no effect on the cloning efficiency of U937 cells, whether or not U937 CFC had been induced for MHC class-II antigens by preincubation of these cells for 72 h with indomethacin and human gamma interferon (HuIFN gamma). Dialyzed U937 CM, however, restored colony formation of HuIFN gamma-induced U937 cells suppressed by lactoferrin (LF) or transferrin (TF). Dialyzed U937 CM did not restore colony formation of U937 cells suppressed by acidic isoferritins (AIF) or prostaglandin E2 (PGE2). Detection of the growth-restoring effects of U937 CM required that U937 CM be prepared in the presence of indomethacin or that the CM be dialyzed to remove inhibitors of U937 colony formation. Dialyzed U937 CM did not inactivate LF. Dialyzed U937 CM did not stimulate or enhance colony formation of normal human bone marrow granulocyte-macrophage (CFU-GM), erythroid (BFU-E), or multipotential (CFU-GEMM) progenitor cells, but did contain potent inhibitory activity against these progenitor cells. HL-60, EM2, EM3, and K562 cells were also evaluated. HL-60-, EM3-, and K562-CFC that were not preincubated with HuIFN gamma did not express MHC class-II antigens, and colony formation by these cells was not influenced by LF, TF, or AIF. Noninduced EM2-CFC constitutively expressed MHC class-II antigens, and colony formation by these cells was suppressed by LF, TF, and AIF. After induction of MHC class-II antigens on HL-60- and EM3-CFC by HuIFN gamma, colony formation by these cells was suppressed by LF, TF, and AIF. Colony formation by HuIFN gamma-induced EM2 cells was more responsive to inhibition by LF, TF, and AIF than was colony formation by noninduced EM2 cells. K562 cells were not induced into a responsive state to LF, TF, or AIF by HuIFN gamma. Dialyzed CM from HL-60, EM2, and EM3 cells contained activities that restored colony formation by their own LF-suppressed CFC. The activities present in dialyzed CM from U937, HL-60, EM2, and EM3 cells may be similar since they could each restore LF-suppressed colony formation of U937, HL-60, EM2, or EM3 cells.(ABSTRACT TRUNCATED AT 400 WORDS)     

SDF-1/CXCL12 enhances in vitro replating capacity of murine and human multipotential and macrophage progenitor cells

Hematopoietic progenitor cells (HPCs) manifest a limited self-renewal capacity, as determined by a surrogate assay involving replating capacity of single colonies in vitro with generation of secondary colonies. Stromal cell-derived factor-1 (SDF-1/CXCL12), has been implicated in regulation of hematopoiesis through its modulation of hematopoietic stem cell (HSC) and HPC migration, homing, mobilization, and survival. We used bone marrow cells from SDF-1/CXCL12 transgenic and littermate control mice, and culture of normal mouse bone marrow and human cord blood cells plated in the presence or absence of recombinant SDF-1/CXCL12 to evaluate a role for SDF-1/CXCL12 in the replating capability in vitro of multipotential [colony-forming units (CFU)-GEMM] and macrophage (CFU-M) progenitor cells. Competitive repopulating capacity of mouse HSCs was assessed in lethally irradiated mice. Transgenic or exogenous SDF-1/CXCL12 significantly enhanced numbers of secondary colonies formed from primary CFU-GEMM or CFU-M colonies. In the limited setting of our in vivo studies, the SDF-1/CXCL12 transgene did not influence HSC competitive repopulation. However, the results suggest that SDF-1/CXCL12 enhances in vitro replating/self-renewal of HPCs, which may contribute to myelopoiesis in vivo. This information may be of value to ex vivo expansion of HPCs/HSCs.     

Identification of lactoferrin as the granulocyte-derived inhibitor of colony-stimulating activity production.

Lactoferrin (LF), the iron-binding protein present in the specific granules of mature granulocytes has been identified as colony inhibitory factor (CIF) which suppresses granulocyte--macrophage colony stimulating activity (CSA) production by monocytes and macrophages in vitro and rebound granulopoiesis in vivo. Separation of LF and CIF by isoelectric focusing confirmed that the regions of inhibitory activity corresponded in both to a pH of congruent to 6.5. In addition, the purified immunoglobulin fraction of rabbit anti-human LF antiserum, but not rabbit anti-transferrin (TF), inactivated the capacity of LF and CIF to inhibit CSA production, an effect blocked by prior incubation of anti-LF with neutralizing concentrations of LF. Suppression of CSA production correlated with the iron-saturation of LF; APO-LF (depleted of iron) was only active concentrations greater than 10(-7) M, native LF (8% iron saturated) was active at 10(-15) M, and fully iron-saturated LF inhibited at 10(-17) M. Suppression of CSA production occurred within a 1/2-h preincubation period with human blood monocytes but was reversed by bacterial lipopolysaccharide (LPS). This reversal was dependent on the relative concentrations of LF to LPS. Serum TF, a biochemically similar iron-binding protein which is antigenically distinct from LF, was only minimally active at concentrations greater than 10(-6) M. LF did not inhibit exogenously stimulated human granylocyte and macrophage colony-forming cells or erythropoietin-dependent human or murine erythroid colony- or erythroid burst-forming cells. Microgram quantities of LF acted in vivo to inhibit rebound granulopoiesis and CSA production in CD1 and C57Bl/6 mice pretreated with cyclophosphamide. These results strongly implicate LF as a physiological regulator of granulopoiesis.     

Membrane-bound Steel factor induces more persistent tyrosine kinase activation and longer life span of c-kit gene-encoded protein than its soluble form

Alternative splicing of exon 6 results in the production of two isoforms of Steel factor (SLF): the membrane-bound and soluble forms. To investigate differences in the kinetics of c-kit tyrosine kinase activated by these two isoforms, we used a stromal cell line (SI/SI4) established from SI/SI homozygous murine embryo fetal liver and its stable transfectants containing either hSCF248 cDNA (including exon 6; secreted form) or hSCF220 cDNA (lacking exon 6; membrane-bound form) as the source of each isoform. Interaction of factor dependent myeloid cell line MO7e with stromal cells producing either isoform resulted in activated c-kit tyrosine kinase and induction of the same series of tyrosine phosphorylated cellular proteins in MO7e cells. However, SI4- h220 (membrane-bound form) induced more persistent activation of c-kit kinase than SI4-h248 (soluble form) did. Flow cytometric analysis and pulse-chase studies using [35S]methionine showed that SI4-h248 induced rapid downmodulation of cell-surface c-kit expression and its protein degradation in MO7e cells, whereas SI4-h220 induced more prolonged life span of c-kit protein. Addition of soluble recombinant human SLF to SI4- h220 cultures enhanced reduction of cell-surface c-kit expression and its protein degradation. Because the kinetics of c-kit inactivation strikingly fits with the protein degradation rates of c-kit under the conditions described above, rapid proteolysis of c-kit protein induced by soluble SLF stimulation may function as a "turn-off switch" for activated c-kit kinase.