Synergistic interactions between interleukin-11 and interleukin-4 in support of proliferation of primitive hematopoietic progenitors of mice.

Interleukin-11 (IL-11) is a newly identified lymphohematopoietic cytokine originally derived from the primate bone marrow stromal cell line, PU-34. Separately, we reported that IL-11 augments IL-3-dependent proliferation of primitive murine hematopoietic progenitors in culture. We have now examined the synergistic interactions between IL-11 and IL-4 in support of colony formation from marrow cells of mice treated 2 days before with 150 mg/kg 5-fluorouracil. Neither recombinant human IL-11 nor murine IL-4 alone was effective in the support of colony formation. When the two factors were combined, there was major enhancement of colony formation, including that of multilineage colony-forming cells. Serial observations (mapping studies) of development of multipotential blast cell colonies indicated that the synergy between IL-11 and IL-4 is due in part to shortening of the dormant period of the stem cells, an effect very similar to that of IL-6 and granulocyte colony-stimulating factor. The combination of IL-11 and IL-4 may be useful in the stimulation of dormant hematopoietic stem cells in vivo.

Multifactor stimulation of megakaryocytopoiesis: effects of interleukin 6.

We have previously demonstrated that interleukin 3 (IL-3), granulocyte-macrophage colony-stimulating factor (GM-CSF), and granulocyte colony-stimulating factor (G-CSF) stimulate various aspects of megakaryocytopoiesis. We have investigated the capacity of interleukin 6 (IL-6) to stimulate megakaryocyte colony formation from both normal Balb/C marrow and light-density marrow extensively depleted of adherent, pre-B, B and T cells. Human recombinant IL-6 (167 ng/ml) stimulated megakaryocyte colony formation from normal marrow (8.6 +/- 1 megakaryocyte colony-forming units [CFU-meg]/10(5) cells) as compared to control (1.5 +/- 4 CFU-meg/10(5) cells) in 16 determinations (p less than 0.01). IL-6 (167 ng/ml) also stimulated CFU-meg formation from depleted marrow (control, 10.8 +/- 4 CFU-meg/10(5) cells versus IL-6, 68 +/- 19 CFU-meg/10(5) cells in 12 determinations, p less than 0.01). IL-6 synergistically augmented IL-3-induced colony formation (139% IL-3 control, 120% calculated IL-3 plus IL-6 control, n = 11, p less than 0.01) in normal marrow and showed an additive effect in depleted marrow (133% IL-3 control, p less than 0.01, 114% of IL-3 plus IL-6, value not significant [NS] at 0.05 level). Studies with recombinant murine IL-6 gave similar results. There was an increasing level of megakaryocyte colony-stimulating activity from G-CSF (16,667 U/ml, 2.47 +/- 0.6 CFU-meg/10(5) cells, n = 17), to IL-6 (167 ng/ml, 8.47 +/- 0.96 CFU-meg/10(5) cells, n = 19), to GM-CSF (52 U/ml, 23 +/- 4 CFU-meg/10(5) cells, n = 14), to IL-3 (167 U/ml, 48 +/- 5 CFU-meg/10(5) cells, n = 20) as compared to media-stimulated marrow (range 1.29-1.86 CFU-meg/10(5) cells). A similar hierarchy was seen with depleted marrow. Combinations of factors (including IL-3, GM-CSF, G-CSF, and IL-6) tested against normal unseparated murine marrow did not further augment CFU-meg numbers over IL-3 plus IL-6 but did increase colony size. These data suggest that IL-6 is an important megakaryocyte regulator, that at least four growth factors interact synergistically or additively to regulate megakaryocytopoiesis, and that combinations of growth factors, possibly in physical association, might be critical in stimulating megakaryocyte stem cells.

IL-4 acts synergistically with IL-1 beta to promote lymphocyte adhesion to microvascular endothelium by induction of vascular cell adhesion molecule-1.

Adhesion of lymphocytes to endothelial cells (EC) is the requisite first element in the multistep process of transmigration from blood across the postcapillary venules. Selective expression of cell adhesion molecules (CM) by microvascular EC in lymphoid organs (e.g., lymph nodes) and during tissue inflammation modulates this traffic in a site-directed manner. CAM synthesis by EC is regulated in turn by cytokines released in the local microenvironment. Studies done largely with human umbilical vein EC have implicated IL-1, IFN-gamma, and TNF-alpha as cytokines which promote leukocyte adhesion to EC. In the work reported here, the responses of cultured microvascular EC derived from macaque lymph nodes to IL-1beta, IL-2, IFN-gamma, and IL-4 were examined. Increases in lymphocyte adhesion after preculture of microvascular EC in IL-1beta or IFN-gamma were typically 2-to 4-fold above controls and comparable to those reported for human umbilical vein EC. IL-2 had no effect. In contrast, IL-4 markedly enhanced adhesion to microvascular EC. IL-4-induced adhesion was observed as early as 4 h after induction, plateaued by 24 h, was stable through 72 h of culture, but decayed to basal levels within 72 h after removal of IL-4 from the cultures. IL-1beta, but not IL-2 or IFN-gamma, synergistically enhanced the action of IL-4 on cultured microvascular EC to promote lymphocyte binding. Adhesion triggered in this manner required de novo protein synthesis. However, the avidity of IL-4-activated microvascular EC for lymphocytes, and analyses of kinetics, cation and temperature dependence, and/or lack of blockade with mAb to endothelial leukocyte adhesion molecule-1, intra-cellular adhesion molecule-1, and MECA-79 indicated that these CAM were not central to the phenomenon. To aid identification of the relevant CAM, mAb specific to IL-4-induced microvascular EC were produced. One of these, 6G10, blocked up to 90% of lymphocyte adhesion to IL-4-induced microvascular EC, immunoprecipitated an IL-4-induced cell-surface molecule of 110-kDa molecular mass, and reacted specifically with Chinese hamster ovary cells transfected with human vascular cell adhesion molecule-1. Our results suggest that IL-4 may have potent effects on lymphocyte recirculation in vivo.

Stimulation of canine hematopoiesis by recombinant human granulocyte-macrophage colony-stimulating factor.

The effect of recombinant human granulocyte-macrophage colony-stimulating factor (rhGM-CSF) on canine hematopoiesis was evaluated. rhGM-CSF stimulated granulocyte-macrophage colony formation of canine marrow depleted of accessory cells up to tenfold. Stimulation of colony formation was abrogated by anti-rhGM-CSF antiserum or heat inactivation. rhGM-CSF also stimulated in vivo canine hematopoiesis both when given as continuous i.v. infusion and as intermittent s.c. injections. Neutrophil, monocyte, and lymphocyte counts were increased three- to eightfold above controls, whereas values for eosinophils, reticulocytes, and hematocrits were not changed. Bone marrow histology after 2 weeks of treatment with rhGM-CSF showed hypercellularity with myeloid hyperplasia and left-shifted granulocytopoiesis. After discontinuation of rhGM-CSF, peripheral leukocyte counts returned to control level within 3-7 days. Platelet counts decreased rapidly after starting rhGM-CSF, to 5000-15,000 platelets/mm3, and increased within 24 h after stopping rhGM-CSF treatment, whereas marrow histology after 2 weeks of rhGM-CSF application showed the normal number and morphology of megakaryocytes.

Interleukin-3, GM-CSF, and G-CSF receptor expression on cell lines and primary leukemia cells: receptor heterogeneity and relationship to growth factor responsiveness.

Recombinant human granulocyte-macrophage (GM) colony-stimulating factor (GM-CSF), G-CSF, and interleukin-3 (IL-3) labeled with 125I were used to study the characteristics and distribution of receptors for these factors on in vitro cell lines and on cells from patients with acute nonlymphocytic leukemia (ANL) and acute lymphocytic leukemia (ALL). Receptors for GM-CSF and G-CSF were restricted to a subset of myelomonocytic cell lines whereas IL-3 receptors were also found on pre-B- or early B-cell lines. Receptors for all three CSFs were broadly distributed on ANL cells, with considerable variability in levels of expression. Measurement of the colony-forming ability of ANL cells in response to the CSFs showed that there was no direct correlation between the ability of the cells to respond to a growth factor and the absolute number of receptors expressed for that growth factor. Binding of radiolabeled IL-3 and GM-CSF to ANL cells produced complex biphasic curves. Further analysis showed that both IL-3 and GM-CSF were able to partially compete for specific binding of the heterologous radiolabeled ligand to cells from several ANL patients, suggesting that heterogeneity may exist in human CSF receptors. These results provide new insights into the complex role that CSFs may play in ANL.

Murine B-cell stimulatory factor-1 (BSF-1)/interleukin-4 (IL-4) is a multilineage colony-stimulating factor that acts directly on primitive hemopoietic progenitors.

Interleukin-4 (IL-4), which was originally identified as a B-cell growth factor, has been shown to produce diverse effects on hemopoietic progenitors. The present study investigated the effects of purified recombinant murine IL-4 on early hemopoetic progenitors in methylcellulose culture. IL-4 supported the formation of blast cell colonies and small granulocyte/macrophage (GM) colonies in cultures of marrow and spleen cells of normal mice as well as spleen cells of mice treated with 150 mg/kg 5-fluorouracil (5-FU) 4 days earlier. When the blast cell colonies were individually picked and replated in cultures containing WEHI-3 conditioned medium and erythropoietin (Ep), a variety of colonies were seen, including mixed erythroid colonies, indicating the multipotent nature of the blast cell colonies supported by IL-4. To test whether or not IL-4 affects multipotent progenitors directly, we replated pooled blast cells in cultures under varying conditions. In the presence of Ep, both IL-3 and IL-4 supported a similar number of granulocyte/erythrocyte/macrophage/megakaryocyte (GEMM) colonies. However, the number of GM colonies supported by IL-4 was significantly smaller than that supported by IL-3. When colony-supporting abilities of IL-4 and IL-3 were compared using day-4 post-5-FU spleen and day-2 post-5-FU marrow cells, IL-4 supported the formation of fewer blast cell colonies than did IL-3. IL-4 and IL-6 revealed synergy in support of colony formation from day 2 post-5-FU marrow cells. These results indicate that murine IL-4 is another direct-acting multilineage colony-stimulating factor (multi-CSF), similar to IL-3, that acts on primitive hemopoietic progenitors.

Heterogeneity in human interleukin-3 receptors. A subclass that binds human granulocyte/macrophage colony stimulating factor.

125I-Labeled recombinant human interleukin-3 (IL-3) was used to study the characteristics and distribution of receptors for IL-3 on human cells. Receptors were found on primary monocytes, on some strains of KG-1 cells, and on pre-B cell lines. Binding was rapid at 37 degrees C, while requiring several hours to reach equilibrium at 4 degrees C. Equilibrium binding studies indicated that IL-3 bound to a single class of high affinity receptor (less than 500 receptors/cell) with a Ka of approximately 1 x 10(10) M-1. Inhibition studies revealed that human granulocyte/macrophage colony stimulating factor partially inhibited the binding of 125I-IL-3 to human monocytes but not JM-1 cells. Additional analysis showed that on KG-1 cells, both IL-3 and GM-CSF partially competed specific binding of heterologous radiolabeled ligand, with approximately equivalent capacities. This competition occurred at both 37 and 4 degrees C. These results suggest heterogeneity in the binding sites for IL-3 and GM-CSF in which a subset of receptors binds only IL-3, a subset only GM-CSF, and another subset can bind both, all with high affinity. Additional heterogeneity was suggested by equilibrium binding of 125I-IL-3 to KG-1 cells which revealed a biphasic Scatchard plot containing a low affinity component not observed on monocytes and JM-1 cells.

In vivo biologic activities of recombinant human granulocyte-macrophage colony-stimulating factor.

We evaluated the biologic effects of recombinant human granulocyte-macrophage colony-stimulating factor (GM-CSF) in 25 patients with malignancy and/or bone marrow failure of diverse etiologies. The continuous infusion of GM-CSF (15 to 500 micrograms/m2/day) elicited marked leukocytosis (2- to 70-fold increase), consisting primarily of neutrophils, eosinophils, and monocytes. Six patients with cytopenias experienced a multilineage response characterized by significant increases in platelet counts and improvement in erythropoiesis. Response in blood counts was accompanied by significant increases in bone marrow cellularity, myeloid:erythroid cell ratios, and frequency of cycling progenitors, indicating an effect at the stem cell level. By premature chromosome condensation analysis, neutrophils from patients with myeloid diseases were found to be derived from normal as well as abnormal clones. Side effects were generally mild and commonly included constitutional symptoms and bone pain. These results indicate that GM-CSF is a significant stimulus for hematopoiesis in vivo and might play an important role in several clinical arenas.

Production of recombinant human colony stimulating factors in yeast.

Efficient yeast expression and purification systems for production of recombinant human GM-CSF, IL-3 and G-CSF have been established. Though yeast-derived production of recombinant CSFs (through the use of secretion based system) allows for generation of native molecules which can then be readily separated from fermentation broth, in many instances, natural cDNAs have had to be altered to allow for efficient expression, as well as production of a less heterogeneous product. In the case of CSFs described herein, beneficial mutations (made through site-directed mutagenesis) have included elimination of potential N-linked glycosylation sites, removal of KexII protease recognition sites (notably alterations in dibasic sequences) and elimination of extraneous cysteine residues which might complicate isolation of a homogeneous product due to intermolecular disulfide bonding.

Human macrophage colony stimulating factor (M-CSF): alternate RNA splicing generates three different proteins that are expressed on the cell surface and secreted.

Human macrophage colony stimulating factor (M-CSF) cDNA clones were isolated from a pancreatic carcinoma cell line. Three different classes of M-CSF precursor protein (256, 554 and 438 amino acids in length) were predicted to be encoded by these cDNAs. Two of these, that we designate M-CSF alpha and M-CSF beta have already been described. The third, M-CSF gamma represents a novel class of M-CSF cDNA. All three precursors share a 32 amino acid signal sequence and the first 149 amino acids of the mature protein. At this position, M-CSF beta and gamma have insertions of 298 and 182 amino acids relative to M-CSF alpha. The first 182 amino acids of these insertions are shared between M-CSF beta and gamma. All three precursors share the C-terminal 75 amino acids that encode the transmembrane and cytoplasmic domains. Expression of all three cDNAs in COS-7 monkey kidney cells gave rise to soluble M-CSF activity, associated with proteins of subunit molecular weight 44 Kda (beta and gamma) or 28 Kda (alpha). In addition, M-CSF proteins could be detected on the surface of the transfected cells by indirect immunofluorescence. When the transmembrane and cytoplasmic domains of M-CSF alpha were removed by introducing a stop codon after amino acid 190, no membrane-bound M-CSF could be detected, but the truncated protein was secreted efficiently and was biologically active. This suggests that all three forms of M-CSF can exist as cell surface proteins, anchored by their hydrophobic transmembrane domains, and can be processed to soluble forms by proteolytic digestion. Although all soluble forms of M-CSF were biologically active in murine bone marrow colony and proliferation assays, they showed greatly reduced or no activity in similar assays using human bone marrow.

Recombinant granulocyte/macrophage-colony stimulating factor in human and murine bone marrow transplantation.

Recombinant granulocyte/macrophage-colony stimulating factor (rGM-CSF) is a protein that mediates a broad range of biological activities including inducing proliferation of bone marrow progenitor cells and up-regulating the functional capabilities of neutrophils, monocytes, and macrophages. We have administered rGM-CSF in vivo in the context of allogeneic (murine) and autologous (human) bone marrow transplantation (BMT). In a murine allogeneic model of T-cell depletion constructed to favor graft failure/rejection of donor marrow, ex vivo treatment of marrow with rGM-CSF significantly promoted engraftment. However, when rGM-CSF was infused in vivo, engraftment was significantly impaired. We have initiated a phase I clinical trial of rGM-CSF in human autologous BMT for patients with acute lymphoblastic leukemia (ALL). rGM-CSF has been administered for 14-21 days in a dose escalation study (16-128 micrograms/M2/day). At high doses of rGM-CSF, neutrophil recovery has returned earlier and to higher levels than previously observed in historical controls. rGM-CSF related toxicity in this study has been mild.

Studies on hematopoietic growth factor receptors using human recombinant IL-3, GM-CSF, G-CSF, M-CSF, IL-1 and IL-4.

An increasing number of cytokines including GM-CSF, G-SCF, M-CSF, IL-3, IL-1 and IL-4, have been implicated in the control of the growth and differentiation of hematopoietic cells. The characterization of the cell surface receptors for these proteins is described. The binding of rh IL-3 to human monocytes is described in some detail, and the binding parameters measured for this cytokine are compared with those determined for the other cytokines. The range of cells to which rh IL-3, rh GM-CSF and rh G-CSF bind is more restricted than that observed for the cytokines IL-1 and IL-4, which bind to a diverse spectrum of cell types. Practical applications of receptor studies include the determination of receptor affinity as a measure of recombinant cytokine potency and the use of the receptor ligand interaction to establish sensitive radioreceptor assays for the detection of cytokines in solution. Finally, methods used for the molecular characterization of cytokine receptors are described and summarized for the CSF receptors, IL-4 and IL-1.

Human macrophage-colony stimulating factor: alternative RNA and protein processing from a single gene.

Macrophage-colony stimulating factor (M-CSF, CSF-1) has been reported to be required for the proliferation and differentiation of macrophages from hematopoietic progenitor cells. Recently, two human M-CSF cDNA clones were isolated encoding proteins of 256 and 554 amino acids. We report here the isolation of a third M-CSF cDNA that encodes a protein of 438 amino acids. The coding regions for the three cDNA clones share a common amino-terminus of 149 amino acids and a common carboxyl-terminus of 75 amino acids including a membrane spanning region. In addition, we isolated a genomic clone of human M-CSF. When each of the cDNA clones or the genomic clone were transfected into COS-7 monkey kidney cells, biologically active M-CSF was expressed as judged by the ability of transfected cell supernatants to stimulate proliferation and colony formation of murine bone marrow cells, as well as formation of monocytic colonies from human bone marrow cells. Surprisingly, proliferation of human bone marrow cells was not induced by recombinant human M-CSF. Analysis of the M-CSF proteins released by COS-7 cells revealed that monomer subunit proteins of 44 or 28 kDa were produced. In addition, we found that the membrane spanning region, present in all three forms of M-CSF cDNA, was not required for the synthesis of a biologically active protein. However, when the membrane spanning region was present in the three M-CSF cDNAs, cell surface associated forms of M-CSF could be readily detected.

In vitro and in vivo cytokine-induced facilitation of immunohematopoietic reconstitution in mice undergoing bone marrow transplantation.

The aim of this study was to test whether colony stimulating factors (CSF) and other cytokines facilitate the recovery of a variety of immunohematopoietic functions in lethally irradiated mice undergoing bone marrow transplantation (BMT). Two experimental systems were employed: (a) lethally irradiated mice transplanted with syngeneic or T cell-depleted semi-allogeneic bone marrow (BM) cells (0.1-10 x 10(6)), subsequently treated by multiple doses of cytokines; and (b) lethally irradiated mice transplanted with BM cells that had previously been cultivated with cytokines. The cytokines used were: pure natural mouse interleukin-3 (IL-3); recombinant mouse granulocyte-macrophage CSF (rGM-CSF); recombinant human interleukin-2 (rIL-2); and crude cytokine preparations obtained from the culture supernatants of murine leukemia WEHI-3b cells (containing mainly IL-3), and of phorbol myristate acetate (PMA)-stimulated EL4 leukemia cells and concanavalin A-stimulated rat splenocytes (each containing a multitude of cytokines). For BM cultures (1-9 days), the cytokines were used at a dosage of 1-100 U/ml; for in vivo treatment, 2 x 10(2)-5 x 10(4) units were administered intraperitoneally and subcutaneously at different schedules for varying periods (1-3 weeks). The following parameters were tested 1-10 weeks post-BMT: white blood cell count, colony formation in agar and in the spleen of lethally irradiated mice, proliferative responses to mitogens and alloantigens, allocytotoxicity and antibody production (serum agglutinins and plaque-forming cells) against sheep red blood cells. Under appropriate conditions, cytokine treatment either in vitro or in vivo significantly enhanced (2- to 50-fold compared with controls) most functions tested at 2-8 weeks post-BMT, and shortened the time interval required for full immunohematopoietic recovery by 2-5 weeks. In recipients of semi-allogeneic, T lymphocyte-depleted BM no evidence of graft-versus-host disease was found. It is suggested that judicious application in vitro and/or in vivo of certain pure cytokines (e.g. GM-CSF, IL-3) or cytokine 'cocktails' might be beneficial in enhancing hematopoiesis and in the treatment of immunodeficiency associated with BMT.

Stimulation of B-cell progenitors by cloned murine interleukin-7.

The events involved in the commitment and development of lymphoid lineage cells are poorly understood. We have used a recently described long-term culture system to establish a bioassay that can detect a novel growth factor capable of stimulating the proliferation of lymphoid progenitors. Using direct expression in mammalian cells we have isolated a complementary DNA clone encoding this novel haematopoietic growth factor, designated interleukin-7.

B cell precursor growth-promoting activity. Purification and characterization of a growth factor active on lymphocyte precursors.

We have used a biological assay system we developed to biochemically purify a previously uncharacterized murine lymphopoietic growth factor designated lymphopoietin 1 (LP-1). This factor is capable of stimulating the proliferation and extended maintenance of precursor cells of the B lineage. A stromal cell line producing LP-1 was established after transfection of primary stromal cultures with a plasmid encoding the transforming genes of SV40. This factor was purified to a single 25-kD species from the culture supernatant of an adherent stromal cell line. This material acts on immature lymphocytes, it binds to specific receptors on cells, and is distinct from previously described hematopoietic factors. LP-1 has been purified some 10(7)-fold with an overall recovery of 35%. The purified protein exhibits a specific activity of approximately 4 X 10(6) U/micrograms of protein and is active at a half-maximal concentration of 10(-13) M.

Augmentation of donor bone marrow engraftment in histoincompatible murine recipients by granulocyte/macrophage colony-stimulating factor.

T cell depletion of donor bone marrow can prevent graft v host disease (GVHD) in human and murine marrow graft recipients. However, engraftment in the recipient may be compromised as a consequence of donor marrow T cell depletion. The effect of recombinant murine granulocyte/macrophage colony-stimulating factor (rmu GM-CSF) on engraftment and hematologic reconstitution was evaluated in a murine allogeneic bone marrow transplantation (BMT) model involving T cell depletion of marrow. Before transplantation into irradiated mice differing at major and minor histocompatibility loci, rmu GM-CSF was preincubated with T cell-depleted donor marrow. When low degrees of engraftment were noted in control recipients, treatment of donor marrow with high concentrations of rmu GM-CSF led to enhanced engraftment. Ex vivo donor graft incubation with rmu GM-CSF or a single in vivo injection of rmu GM-CSF were both effective means of promoting engraftment. When the engraftment rate in control recipients was high, rmu GM-CSF did not have an identifiable effect. Only slight increases in hematologic recovery were detected regardless of the rate of engraftment. Neither post-BMT survival nor marrow stem cell capacity was affected by rmu GM-CSF incubation. Furthermore, growth factor administration did not have a significant effect on the incidence of GVHD in recipients of non-T cell-depleted bone marrow splenocyte preparations. In vitro natural killer-mediated target cell lysis was not altered by incubation of effector cells with rmu GM-CSF. These results demonstrate the potential of ex vivo rmu GM-CSF treatment of donor marrow to facilitate engraftment across extensive histo-compatibility barriers.