Activity of receptors coupled to guanine nucleotide binding regulatory protein in doxorubicin induced cardiomyopathy.

STUDY OBJECTIVE--The aim was to study the activity of receptors coupled to guanine nucleotide binding regulatory proteins (G proteins) in doxorubicin induced cardiomyopathy, with special attention to G proteins, beta adrenoceptors, muscarinic receptors, and adenylyl cyclase. DESIGN--Messenger RNA of G proteins, densities and high affinity agonist binding of beta adrenoceptors and muscarinic receptors, activity of adenylyl cyclase, calcium influx, and in vivo lipid peroxidation were determined before, in the early stage, and in the later stage of doxorubicin cardiomyopathic heart failure. SUBJECTS--Sprague-Dawley rats between 150-200 g were used. Doxorubicin was given intravenously at two doses of 4 mg.kg-1 and 6 mg.kg-1 every third week (1st, 4th, 7th week) for nine weeks. Doxorubicin treated rats plus corresponding controls were killed at 3 weeks (n = 7), 6 weeks (n = 7), and 9 weeks (n = 6), respectively. MEASUREMENTS AND MAIN RESULTS--Northern blot and dot blot hybridisations of the total RNA revealed that messenger RNA of both stimulatory and inhibitory G proteins were identical between doxorubicin treated rats and controls. No alterations in the densities of beta adrenoceptors and muscarinic receptors were observed, neither did the high affinity agonist binding of beta adrenoceptors and muscarinic receptors change. Furthermore, modulation of adenylyl cyclase was unimpaired. In contrast, Ca(2+)-ATPase and serum water soluble fluorescent substance, a product of in vivo lipid peroxidation, were shown to increase dramatically in doxorubicin treated rats (4 mg.kg-1 for 6 and 9 weeks, 6 mg.kg-1 for 3, 6 and 9 weeks) as compared with corresponding controls. CONCLUSIONS--The findings suggest that, despite increased calcium influx and lipid peroxidation in doxorubicin induced cardiomyopathy, the activity of receptors coupled to G proteins remained normal.     

Reciprocal inhibition of binding between interleukin 3 and granulocyte-macrophage colony-stimulating factor to human eosinophils

125I-labeled recombinant human interleukin 3 (IL-3) bound, at 4 degrees C, to a single class of high-affinity receptors on human eosinophils with an apparent dissociation constant (Kd) of 470 pM, but it did not bind to human neutrophils. 125I-labeled recombinant human granulocyte-macrophage colony-stimulating factor (GM-CSF) also bound to a single class of high-affinity receptors on eosinophils with an apparent Kd of 44 pM and on neutrophils with an apparent Kd of 70 pM. These binding characteristics were consistent with the biological activities of IL-3 and GM-CSF on eosinophils and with the lack of stimulation of neutrophil function by IL-3. Specificity studies under conditions shown to prevent receptor internalization showed that the binding of 125I-labeled IL-3 to eosinophils was partially inhibited by GM-CSF but not by other cytokines. Reciprocal experiments with 125I-labeled GM-CSF showed that IL-3 but not other cytokines partially inhibited binding to eosinophils. In contrast, the binding of 125I-labeled GM-CSF to neutrophils was not inhibited by IL-3 or other cytokines tested. Quantitative inhibition binding experiments on eosinophils showed that the reciprocal inhibition between IL-3 and GM-CSF was not complete up to a concentration of heterologous ligand of 100 nM. These results show that (i) IL-3 binds to eosinophils but not neutrophils and (ii) IL-3 and GM-CSF specifically interact on the surface of eosinophils, providing a possible mechanism for the overlapping activities of IL-3 and GM-CSF on these cells.     

High-affinity binding of granulocyte-macrophage colony-stimulating factor to normal and leukemic human myeloid cells.

Purified natural and biosynthetic (recombinant) human granulocyte-macrophage colony-stimulating factor (GM-CSF) stimulate colony formation by myeloid progenitor cells and enhance the function of mature neutrophils. Both of these actions occur at concentrations between 1 and 100 pM, with half-maximal stimulation at 10-20 pM. We have examined specific binding of 125I-labeled GM-CSF to responsive target cells in this range of concentrations. The results show a low number (50-250) of high-affinity (15-30 pM) binding sites on GM-CSF-responsive leukemic cells (KG-1, HL-60), as well as on peripheral blood neutrophils from normal donors. This high-affinity binding component was absent from unresponsive cell lines (KG-1a, K562). These results suggest that this binding site mediates the biological activities of GM-CSF on both proliferation and function of myeloid cells.     

Granulocyte-macrophage colony-stimulating factor and human neutrophils: role of guanine nucleotide regulatory proteins.

The addition of granulocyte-macrophage colony-stimulating factor (GM-CSF) to human neutrophils causes a rapid increase in the basal and fMet-Leu-Phe-stimulated Na+ influx and an increase in intracellular pH. The increase can be seen as early as 5 min after the addition of GM-CSF. Changes produced by GM-CSF are totally inhibited by amiloride and are significantly reduced in pertussis toxin-treated cells. The stimulation of the Na+/H+ exchange mechanism by GM-CSF inhibits further stimulation of this system with either fMet-Leu-Phe or phorbol 12-myristate 13-acetate. In addition, membrane preparations isolated from GM-CSF-treated neutrophils have higher basal and stimulated GTPase activities. The basal and the fMet-Leu-Phe- or platelet-activating factor-stimulated GTPase activities are reduced in pertussis toxin-treated cells. Cells pretreated with GM-CSF accumulate more radioactive phosphate than control cells, and this increase is diminished by pertussis toxin treatment. In addition, GM-CSF causes a rapid increase in the tyrosine phosphorylation levels of five proteins with molecular masses of 118 kDa, 92 kDa, 78 kDa, 54 kDa, and 40 kDa. These results clearly show that GM-CSF, on its own, can initiate several changes and that these changes are mediated in part by the pertussis toxin-sensitive guanine nucleotide regulatory protein.     

Differential and synergistic effects of human granulocyte-macrophage colony-stimulating factor and human granulocyte colony-stimulating factor on hematopoiesis in human long-term marrow cultures.

The ability of granulocyte-macrophage colony-stimulating factor (GM-CSF) and G-CSF to influence hematopoiesis in long-term cultures (LTC) of human marrow was studied by cocultivating light density normal human marrow cells with human marrow fibroblast feeders engineered by retroviral infection to constitutively produce one of these growth factors. Feeders producing stable levels of 4 ng/mL GM-CSF or 20 ng/mL G-CSF doubled the output of mature nonadherent cells. The numbers of both colony forming unit-GM (CFU-GM) and erythroid burst forming unit (BFU-E) in the G-CSF LTC were also increased (twofold and fourfold, respectively, after 5 weeks in culture), but this effect was not seen with the GM-CSF feeders. At the time of the weekly half medium change 3H-thymidine suicide assays showed primitive adherent layer progenitors in LTC to be quiescent in both the control and GM-CSF cultures. In contrast, in the G-CSF cultures, a high proportion of primitive progenitors were in S-phase. A single addition of either recombinant GM-CSF or G-CSF to LTC in doses as high as 80 ng/mL and 150 ng/mL, respectively, failed to induce primitive progenitor cycling. However, three sequential daily additions of 150 ng/mL G-CSF did stimulate primitive progenitors to enter S-phase and a single addition of 5 or 12.5 ng/mL of G-CSF together with 10 ng/mL GM-CSF was able to elicit the same effect. Thus, selective elevation of G-CSF in human LTC stimulates proliferation of primitive clonogenic progenitors, which may then proceed through to the terminal stages of granulopoiesis. In contrast, the effects of GM-CSF in this system appear limited to terminally differentiating granulopoietic cells. However, when both GM-CSF and G-CSF are provided together, otherwise biologically inactive doses show strong stimulatory activity. These findings suggest that the production of both of these growth factors by normal stromal cells may contribute to the support and proliferation of hematopoietic cells, not only in LTC, but also in the microenvironment of the marrow in vivo.     

Activation of lymphocytes induced by recombinant human granulocyte-macrophage colony-stimulating factor in patients with malignant lymphoma

To investigate effects of recombinant human granulocyte-macrophage colony-stimulating factor (rhGM-CSF) on lymphoid cells in vivo, we monitored changes in absolute lymphocyte counts, plasma concentrations of soluble interleukin-2 receptor (sIL-2R) and soluble cytotoxic/suppressor (sCD8) antigens, and phenotypic changes of surface membrane antigens of peripheral mononuclear cells from 14 patients with malignant lymphoma treated with rhGM-CSF. Eight of the 14 patients had relapsed or had refractory non-Hodgkin's lymphoma (NHL) and received rhGM-CSF after intensive chemotherapy with novantrone (NO) and high-dose Ara-C (AC) (NOAC) as salvage regimen. Six other patients with NHL or Hodgkin's disease (HD) were in complete remission and treated with rhGM-CSF to enhance peripheral hematopoietic progenitor cell harvest for autografting. An increase in absolute lymphocyte count at the zenith of leukocyte elevation and a drastic increase in concentration of sIL-2R from a median of 565 U/mL to 6,700 U/mL on rhGM-CSF infusion were found in all patients. There was also a moderate increase in sCD8 levels from a median of 277 U/mL to 470 U/mL. Ten patients were available for serial studies of phenotypic changes in surface membrane antigens. A significant increase in CD25+ (IL-2R+) (P = .0020) and CD4+ (P = .0137) lymphocytes was observed in all patients, but no significant change in CD3+, CD8+, TCR delta 1+, or CD19+ cells. Elevations in absolute lymphocyte counts or in concentrations of sIL-2R or sCD8 were not observed in four other patients during recovery from intensive chemotherapy without rhGM-CSF support. Our results provide evidence that administration of rhGM-CSF might activate lymphocytes in vivo. The impact of this activation on the remission rate and duration, as well as survival in patients with NHL, warrants further investigation.     

Synergistic effects of nerve growth factor and granulocyte-macrophage colony-stimulating factor on human basophilic cell differentiation

We have recently shown that nerve growth factor (NGF) promotes human granulopoiesis, specifically augmenting basophilic cell differentiation observed in methylcellulose hematopoietic colony assays of human peripheral blood. Because the NGF effect was seen in the presence of conditioned medium derived from a human T-cell line (Mo-CM) containing granulocyte-macrophage colony-stimulating factor (GM-CSF), we examined interactions of purified NGF and recombinant human GM-CSF (rhGM-CSF) on granulocyte growth and differentiation. rhGM-CSF stimulated a dose- dependent increase in methylcellulose colony growth at concentrations between 0.1 U/mL and 10 U/mL, and in the presence of NGF at 500 ng/mL this effect was enhanced. The number of basophilic cell colony-forming units (CFU-Baso) and histamine-positive colonies increased synergistically when NGF was added to rhGM-CSF. Furthermore, because Mo- CM acts with sodium butyrate to promote basophilic differentiation of alkaline-passaged myeloid leukemia cells, HL-60, we also examined the interaction of NGF and Mo-CM or rhGM-CSF using this assay. In the presence of NGF, Mo-CM at concentrations of 0.5% to 20% vol/vol, and rhGM-CSF at concentrations of 0.1 U/mL to 100 U/mL synergistically increased histamine production by butyrate-induced, alkaline-passaged HL-60 cells; this was associated with the appearance of metachromatic, tryptase-negative, IgE receptor-positive cells. The effects of rhGM-CSF or Mo-CM were completely abrogated by a specific anti-rhGM-CSF neutralizing antibody in methylcellulose, with or without NGF; the NGF synergy with rhGM-CSF in the HL-60 assay was also inhibited by either anti-rhGM-CSF or anti-NGF antibody. These studies support the notion that differentiation in the basophilic lineage may be enhanced by NGF acting to increase the number of GM-CSF-responsive basophilic cell progenitors.     

Immunization with recombinant human granulocyte-macrophage colony-stimulating factor as a vaccine adjuvant elicits both a cellular and humoral response to recombinant human granulocyte-macrophage colony-stimulating factor

Granulocyte-macrophage colony-stimulating factor (GM-CSF) is an important cytokine for the generation and propagation of antigen-presenting cells and for priming a cellular immune response. We report here that use of recombinant human GM-CSF (rhGM-CSF), administered as an adjuvant in a peptide-based vaccine trial given monthly by intradermal injection, led to the development of a T-cell and antibody response to rhGM-CSF. An antibody response occurred in the majority of patients (72%). This antibody response was not found to be neutralizing. In addition, by 48-hour delayed type hypersensitivity (DTH) skin testing, 17% of patients were shown to have a cellular immune response to the adjuvant rhGM-CSF alone. Thymidine incorporation assays also showed a peripheral blood T-cell response to rhGM-CSF in at least 17% of the patients. The generation of rhGM-CSF-specific T-cell immune responses, elicited in this fashion, is an important observation because rhGM-CSF is being used as a vaccine adjuvant in various vaccine strategies. rhGM-CSF-specific immune responses may be incorrectly interpreted as antigen-specific immunity, particularly when local DTH responses to vaccination are the primary means of immunologic evaluation. We found no evidence of hematologic or infectious complications as a result of the development of rhGM-CSF-specific immune responses.     

Thrombopoiesis-stimulating factor: its effects on megakaryocyte colony formation in vitro and its relation to human granulocyte-macrophage colony-stimulating factor

The effects of thrombopoiesis-stimulating factor (TSF) on human marrow megakaryocyte colony formation in vitro were studied by the plasma clot method. TSF was found to stimulate megakaryocyte as well as granulocyte-macrophage colony formation in vitro at optimal concentrations of 200-300 pg/ml of medium containing 2.5% horse serum. This colony-stimulating effect of TSF was not affected by polyclonal antibodies to human (h) interleukin 3 (IL-3) or to granulocyte colony-stimulating factor (G-CSF) but was neutralized by monoclonal or polyclonal antibodies to human granulocyte-macrophage colony-stimulating factor (hGM-CSF). In order to differentiate among cross-reactivity between TSF and hGM-CSF, induction of colony growth via release of GM-CSF, and presence of hGM-CSF in TSF preparations, TSF was tested on murine marrow cells, which are not responsive to hGM-CSF. TSF induced growth of murine megakaryocyte colony-forming units (CFU-MK) and granulocyte-macrophage colony-forming units (CFU-GM) in vitro with a dose response similar to that observed on human marrow cells; however, this effect could not be neutralized by antibodies to either human or murine GM-CSF. Using a double-antibody enzyme-linked immunosorbent assay, TSF preparations were found to contain 36 +/- 4 U of hGM-CSF per picogram of TSF protein. These findings indicate that hGM-CSF is responsible for the megakaryocyte colony-promoting effects of TSF on human marrow cells in vitro.     

Pharmacokinetics of human granulocyte-macrophage colony-stimulating factor using a sensitive immunoassay

A sensitive and reliable sandwich enzyme-linked immunosorbent assay (ELISA) has been developed for recombinant human granulocyte-macrophage colony-stimulating factor (hGM-CSF). The assay is quantitative between 100 pg/mL and 2.5 ng/mL for bacterially synthesized hGM-CSF in human serum and is more sensitive and specific than the semisolid agar bioassay. As part of a phase I study, the pharmacokinetics of intravenous (IV) bolus injection and subcutaneous (SC) administration of hGM-CSF were studied. Following a single IV dose, an initial high blood level of hGM-CSF occurred, followed by a rapid decrease occurring in two apparent phases with a half-life (t1/2)alpha of less than five minutes and a t1/2 beta of 150 minutes. After an SC injection, detectable serum levels occurred within 15 to 30 minutes, and serum levels were sustained for a variable time depending on the dose. At the highest SC dose (10 micrograms/kg), a serum level of greater than 1 ng/mL (65 pmol/L) was maintained for greater than 12 hours after a single injection. This corresponds to the concentration of hGM-CSF supporting near-maximum proliferation in vitro.     

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.     

Purification and characterization of a human T-lymphocyte-derived granulocyte-macrophage colony-stimulating factor

We have examined the biologic and physical properties of a human T- lymphocyte granulocyte-macrophage colony-stimulating factor (CSF). The source of the factor is a T-lymphoblast cell line (Mo) that was derived from a patient with a T-cell variant of hairy-cell leukemia. The Mo line constitutively produces a number of lymphokines that are normally produced by mitogen-stimulated T lymphocytes. Medium conditioned by Mo cells grown in the absence of serum is especially rich in CSF activity, and using this source we have purified the CSF to a specific activity of about 3.5 x 10(6) colonies per 10(5) Ficoll-Hypaque-separated human bone marrow cells plated per mg protein. The Mo CSF stimulates the formation of both granulocyte and macrophage colonies in vitro (in about equal numbers) and it has a relatively steep dose-response curve. Both the crude and purified preparations stimulated the formation of eosinophil as well as neutrophil colonies; it is unclear whether this is due to the presence of multiple factors with similar physical properties or a single factor with multiple activities. The CSF has little stimulating activity for mouse bone marrow progenitors. Physically, the Mo CSF is an acidic glycoprotein of molecular weight about 34,000. It binds to concanavalin A-Sepharose, is unusually resistant to denaturing agents and heat treatment, and is not inactivated in the presence of sulfhydryl reagents. The Mo CSF is distinct from factors stimulating erythroid colony formation and inhibiting neutrophil migration that are also produced by Mo cells. It differs in several physical and biologic properties from other human CSFs that have been characterized.     

Differential effects of granulocyte-macrophage colony-stimulating factor and granulocyte colony-stimulating factor in children with severe congenital neutropenia.

Severe congenital neutropenia (SCN) is a disorder of myelopoiesis characterized by severe neutropenia secondary to a maturational arrest at the level of promyelocytes. We treated five patients with SCN with recombinant human granulocyte-macrophage colony-stimulating factor (rhGM-CSF) for 42 days and subsequently, between 1 and 3 months later, with rhG-CSF for 142 days. The objective was to evaluate the safety and ability of these factors to elicit a neutrophil response. rhGM-CSF was administered at a dose of 3 to 30 micrograms/kg/d (30 to 60 minutes, intravenously). In all patients, a specific, dose-dependent increase in the absolute granulocyte counts was observed. However, in four patients this increase was due to an increase in eosinophils, and in only one patient it was due to an increase in the absolute neutrophil counts (ANC). Subsequently, all patients received rhG-CSF at a dose of 3 to 15 micrograms/kg/d subcutaneously. In contrast to rhGM-CSF treatment, all five patients responded to rhG-CSF during the first 6 weeks of treatment with an increase in the ANC to above 1,000/microL. The level of ANC could be maintained during maintenance treatment. In one patient, the increase in ANC was associated with an improvement of a severe pneumonitis caused by Peptostreptococcus and resistant to antibiotic treatment. No severe bacterial infections occurred in any of the patients during CSF treatment. All patients tolerated rhGM-CSF and rhG-CSF treatment without severe side effects. These results demonstrate the beneficial effect of rhG-CSF in SCN patients.     

Regulation of human monocyte adherence by granulocyte-macrophage colony-stimulating factor.

Recombinant human granulocyte-macrophage colony-stimulating factor (GM-CSF) was found to increase the adherence of purified peripheral blood monocytes to plastic surfaces and to monolayers of human umbilical vein endothelial cells. With plastic surfaces as a model 9-hr culture with GM-CSF was necessary for enhancement, and maximum levels were obtained after 24-hr stimulation. GM-CSF-stimulated adherence must require new RNA and protein synthesis because actinomycin D and cycloheximide abolished existing adherence and prevented further monocyte attachment. Interestingly, shorter incubations (1-2 hr) with cycloheximide increased adherence, suggesting a labile inhibitor. Formaldehyde fixation of monocytes but not of human vein endothelial cells abolished adherence, indicating the need for actively metabolizing monocytes. Thus, a hemopoietic growth factor, responsible for the proliferation and differentiation of monocytes, can also alter their adhesive characteristics. These observations may have important implications in pathological situations and in the in vivo use of GM-CSF.     

Kinetic resolution of two mechanisms for high-affinity granulocyte-macrophage colony-stimulating factor binding to its receptor

Granulocyte-macrophage colony-stimulating factor (GM-CSF) is an important hematopoietic cytokine that exerts its effects by interaction with the GM-CSF receptor (GMR) on the surface of responsive cells. The GM-CSF receptor consists of two subunits: GMRalpha, which binds GM-CSF with low affinity, and GMRbeta, which lacks intrinsic ligand-binding capability but complexes with GMRalpha to form a high-affinity receptor (GMRalpha/beta). We conducted dynamic kinetic analyses of GM-CSF receptors to define the role of GMRbeta in the interaction of ligand and receptor. Our data show that GMRalpha/beta exhibits a higher k(on) than GMRalpha, indicating that GMRbeta facilitates ligand acquisition to the binding pocket. Heterogeneity with regard to GM-CSF dissociation from GMRalpha/beta points to the presence of loose and tight ligand-receptor complexes in high-affinity binding. Although the loose complex has a k(off) similar to GMRalpha, the lower k(off) indicates that GMRbeta inhibits GM-CSF release from the tight receptor complex. The two rates of ligand dissociation may provide for discrete mechanisms of interaction between GM-CSF and its high-affinity receptor. These results show that the beta subunit functions to stabilize ligand binding as well as to facilitate ligand acquisition.     

T lymphocytes downregulate granulocyte-macrophage colony-stimulating factor secretion from stimulated monocytes by increasing the secretion of monocyte-derived interleukin-10

In previous studies we characterized the cytokine regulation of granulocyte-macrophage colony-stimulating factor (GM-CSF) and granulocyte colony-stimulating factor (G-CSF) secretion by endothelial cells and monocytes and found differences in secretion pattern within and between these cell systems. In this study, the regulatory effect of T lymphocytes on CSF secretion was examined. T lymphocytes had no effect on CSF secretion by endothelial cells. In contrast, the addition of T lymphocytes significantly and dose dependently downregulated GM-CSF, but not G-CSF, secretion by monocytes. In one of our previous studies it was shown that interleukin-4 (IL-4) and interleukin-10 (IL-10) were the most potent inhibitory cytokines of CSF secretion by monocytes. Both these cytokines are produced by T lymphocytes. However, the downregulating effect on monocyte GM-CSF secretion was not due to increased secretion of T-lymphocyte-derived IL-4 or IL-10. Instead, the presence of T lymphocytes increased the secretion of monocyte-derived IL-10. It was shown earlier than IL-10 regulates CSF secretion by monocytes in an autocrine manner. Our data indicate that T lymphocytes might interfere with this autocrine regulation and thereby influence monocyte function in immune response and cell proliferation.     

Three conserved motifs in the extracellular domain of the human granulocyte-macrophage colony-stimulating factor receptor subunit are essential for ligand binding and surface expression

The receptor for the human granulocyte-macrophage colony-stimulating factor (GM-CSF) (GM-R) is a heterodimeric complex consisting of two subunits, GM-R alpha and GM-R beta. Structural analyses have shown a number of highly conserved amino acid motifs present in both GM-R alpha and GM-R beta. These motifs include QYFLY, CXW, XW, and WSXWS motifs in the extracellular domain; a conserved cysteine in the transmembrane domain; and the entire cytoplasmic domain, including the LXVLX box in the carboxy terminal region of the cytoplasmic domain. We have investigated the role of these motifs in GM-R alpha by examining the effects of specific motif mutations on ligand binding and surface expression. Transient expression of these mutant GM-R alpha subunits in COS cells shows that these extracellular motis are essential for ligand binding. Alterations of the cytoplasmic region of GM-R alpha do not alter GM-CSF binding or the reconstitution of high-affinity receptors when coexpressed with GM-R beta. Permeabilization and immunostaining of cells transfected with mutant GM-R alpha subunits yields data suggesting that each of the mutant subunits is present in the cytoplasm. Immunostaining of both intact and permeabilized COS cells transiently transfected with wild-type or mutant GM-R alpha s showed that extracellular domain mutants accumulated in the cytoplasm and were not efficiently transported to the cell surface.     

Recombinant human granulocyte-macrophage colony-stimulating factor promotes megakaryocyte maturation in nonhuman primates

Megakaryocytes are responsive to several nonlineage-specific cytokines in vitro. In this study, we examined the in vivo effects of recombinant human granulocyte-macrophage colony-stimulating factor (rh GM-CSF) on late stages of megakaryocytopoiesis in the rhesus monkey. Four rhesus monkeys were given 10 micrograms/kg body weight/day of rh GM-CSF s.c. in two divided doses daily for 8 days. Megakaryocyte maturation was evaluated serially by measuring nuclear ploidy and cytoplasmic size. GM-CSF-treated monkeys developed significant shifts in ploidy distribution from days 3 through 15 (p less than or equal to 0.001), with increased frequencies of 64N and 128N megakaryocytes. Mean megakaryocyte size increased 92.5% on day 9, paralleling the increase in DNA content, although megakaryocyte size within ploidy groups did not increase. Megakaryocyte number remained unchanged following rh GM-CSF treatment. The platelet count responses were variable, and mean platelet volume did not change. The present study demonstrates that therapeutic doses of rh GM-CSF stimulate megakaryocyte endomitosis and increase mean size. The data indicate that GM-CSF is effective in promoting the maturation stage of megakaryocyte development but does not result in a consistent thrombopoietic response.