Measurement of human granulocyte-macrophage colony-stimulating factor (GM-CSF) by enzyme-linked immunosorbent assay

Summary An IgG monoclonal antibody against recombinant human granulocyte-macrophage colony-stimulating factor (GM-CSF), designated HGMI, was produced by fusion of immune mouse splenocytes with HAT-sensitive murine myeloma cells. A sandwich enzyme-linked immunosorbent assay (ELISA) for measurement of human GM-CSF was developed using this HGMI and a polyclonal antibody against GM-CSF raised in a rabbit. GM-CSF in culture supernatants of phytohemagglutinin (PHA)- or concanavalin A (Con A)-stimulated peripheral blood mononuclear cells (PBMC) were measured by this ELISA system and the conventional CFU-GM colony formation method. The data indicated that the ELISA was highly efficient and sensitive for the detection of as little as 50 pg/ml recombinant GM-CSF. The CFU-GM colony assay may be influenced by other cytokines which can enhance or suppress colony formation, and ELISA for GM-CSF is more useful for kinetic studies of precise levels of production from PBMC.     

Secretion and partial degradation of granulocyte-macrophage colony-stimulating factor (GM-CSF) of mouse L-P3 cells

Secretion of a granulocyte-macrophage colony-stimulating factor (GM-CSF) was accomplished by L-P3 cells in culture with a serum-free medium. Cell proliferation per se was not requisite for the production of GM-CSF; the cells continued secreting GM-CSF even after their growth had been suspended. The amount of GM-CSF accumulated in the conditioned medium was reasonably accounted by the daily rate of production, and the addition of a proteinase inhibitor such as leupeptin and pepstatin did not result in greater accumulation of GM-CSF in the culture. It is thus postulated that there is no significant proteolytic inactivation of the secreted GM-CSF in the culture. However, when partially purified GM-CSF preparation was chromatographed on a gel-filtration column in the presence of 0.1% Triton X-100, a derivative of the GM-CSF was yielded which had been diminished in the molecular weight and altered in the isoelectric point. On the other hand, when leupeptin was included in the solution during production and isolation of the factor, the yielded GM-CSF did not manifest such a detergent-induced transformation and maintained its isoelectric point at pH 3.5. It is thus assumed that, in the presence of the detergent, GM-CSF suffers deterioration by an endogenous proteinase and releases a sialoglycopeptide fragment without loosing its colony-stimulating activity.     

Improvement of pneumonia and arthritis in Felty's syndrome by treatment with granulocyte-macrophage colony-stimulating factor (GM-CSF)

A 63-year old man with Felty's syndrome and pneumonia of unknown origin was treated with GM-CSF. Granulocyte counts increased and arthritis-related symptoms improved under GM-CSF. Pneumonia was treated effectively with antibiotics only during or after GM-CSF application. This suggests, that antibiotic-resistant infections can be treated effectively in patients with Felty's syndrome when granulocyte counts are raised by GM-CSF.     

Up-regulation of granulocyte-macrophage colony-stimulating factor (GM-CSF) receptors in murine peritoneal exudate macrophages by both GM-CSF and IL-3.

Murine peritoneal exudate macrophages (PEM) display multiple CSF receptors. In this study, the expression of granulocyte-macrophage (GM)-CSF receptors in PEM was studied. PEM displayed over 5000 single type, high affinity GM-CSF receptors/cell with a Kd = 38 to 42 pM and an apparent molecular mass of 86,000 Da. Treatment of PEM with low, but not high, concentrations of recombinant murine (rMu) GM-CSF continuously for 24 h resulted in a marked up-regulation of GM-CSF receptors in PEM. A similar up-regulation of GM-CSF receptors also was detected in PEM cultures treated with rMuIL-3 (1-100 ng/ml) for 24 h or longer, regardless the doses of rMuIL-3 added in this case. Scatchard analysis of equilibrium binding showed that the enhanced binding activities in both cases were due to an increase in total number of GM-CSF receptors rather than changes in receptor affinity. Contrariwise, treatment with recombinant human macrophage-CSF (greater than 100-1000 ng/ml) partially inhibited the expression of GM-CSF receptors in PEM. Removal of rMuGM-CSF from culture medium 24 h after treatment led to a further up-regulation of GM-CSF receptors over a 4 to 24-h period, depending on the doses of initial treatment. On the other hand, removal of rMuIL-3 from culture medium after prolonged treatment did not result in further increase in GM-CSF receptors. The protein synthesis inhibitor cycloheximide abrogated GM-CSF receptor up-regulation induced by both rMuIL-3 and rMuGM-CSF, whereas actinomycin D inhibited only the second (8-24 h) phase of GM-CSF receptor up-regulation induced by exposure to high concentrations rMuGM-CSF (10 ng/ml). These findings suggest that rMuGM-CSF and rMuIL-3 up-regulate GM-CSF receptors in PEM in part through similar or identical metabolic pathways and provide further evidence of a close linkage between IL-3 and GM-CSF receptors.     

Attenuation of airway hyperresponsiveness in a murine asthma model by neutralization of granulocyte-macrophage colony-stimulating factor (GM-CSF)

Asthma is recognized as an inflammatory disease in which various cytokines are involved. Among these, granulocyte-macrophage colony-stimulating factor (GM-CSF) is known to play a critical role in the survival of eosinophils and in the activation of antigen-presenting cells (APC). We studied the effects of neutralization of GM-CSF in a murine model of asthma, to elucidate its role in enhanced airway responsiveness and in airway inflammation. A/J mice, which are genetically predisposed to acetylcholine hyperresponsiveness, were immunized with ovalbumin (OA) and alum. Thereafter, the mice were subjected to a two-week regimen of OA inhalation, during which either goat anti-mouse polyclonal GM-CSF antibody or isotype control goat IgG was administered intranasally. Pulmonary function was then analyzed using whole body plethysmography before and after acetylcholine (Ach) inhalation. Here we show that OA inhalation following OA immunization increased airway responsiveness to acetylcholine and induced GM-CSF as well as IL-4 and IL-5 mRNA expression in the lung. The administration of GM-CSF-neutralizing antibody during OA inhalation significantly reduced this increased airway hyperresponsiveness and also inhibited airway inflammation. Thus, endogenous GM-CSF plays an important role in the process of airway inflammation and airway hyperresponsiveness after antigen-specific immunity has been established.     

Stroma-mediated granulocyte-macrophage colony-stimulating factor (GM-CSF) control of myelopoiesis: spatial organisation of intercellular interactions

Granulocyte-macrophage colony-stimulating factor (GM-CSF) is one of the major cytokines involved in control of haemopoiesis both in bone marrow and in extramedullar sites. Its biological activity depends upon the composition and physicochemical properties of the microenvironment provided by the supporting stroma. GM-CSF activity is modulated and controlled by the stromal heparan-sulphate proteoglycans, but their optimal interaction occurs only at low pH. We questioned whether the microenvironment organisation of the interface between stroma and haemopoietic cells provides such conditions. We studied myeloid progenitor proliferation in contact with bone marrow-derived and extramedullar stromas using electron microscopy and selective labelling of pericellular components. We present evidence that, upon interaction, the two cell types reorganise their interface both in shape and molecular composition. Haemopoietic cells extend projections that considerably increase the area of intercellular contact, and stromal cells form lamellipodia and carry out a redistribution of membrane-associated sialylated glycoconjugates and proteoglycans. Such rearrangements lead to extensive capping of negatively charged molecules at the interface between the supporting stroma and the haemopoietic cells, leading potentially to a local decrease in pH. Our results indicate that the distribution of negative charges at the cellular interface may be responsible for the selectivity of cell response to GM-CSF.Publication of the Millennium Institute for Tissue Bioengineering. The study was supported by PRONEX, CNPq and FINEP grants from the Brazilian Ministry of Science and Technology and a FAPERJ grant from the Rio de Janeiro State Government.     

Effects of hematopoietic growth factor (GM-CSF: granulocyte-macrophage colony-stimulating factor) on thymocyte proliferation induced by interleukin-1 (IL-1)

A semi-purified fraction obtained from P388 D1 cell line conditioned medium (P388 D1 CM) which contains Interleukin-1 (IL-1) and Granulocyte-Macrophage Colony-Stimulating Factor (GM-CSF) stimulates murine thymocyte proliferation both in the absence and the presence of a suboptimal dose of phytohemagglutinin (PHA). Because this effect on thymocyte proliferation is always larger than that obtained with optimal concentrations of pure IL-1, we have investigated the possible involvement of GM-CSF in this semi-purified fraction mediated-thymocyte proliferation. We here show that the maximal level of thymocyte proliferation induced by the semi-purified fraction is comparable to that obtained by the co-addition of recombinant GM-CSF and IL-1. In addition, although GM-CSF alone induces no significant thymocyte proliferation, the presence of an anti-GM-CSF antiserum partially blocks the thymocyte proliferation induced by the semi-purified fraction. Thus, the capacity of the semi-purified fraction of P388 D1 to stimulate thymocyte proliferation appears to result from a synergistic action between GM-CSF and IL-1.     

Blockade of collagen-induced arthritis post-onset by antibody to granulocyte-macrophage colony-stimulating factor (GM-CSF): requirement for GM-CSF in the effector phase of disease

There is mounting evidence for a role of the growth factor granulocyte-macrophage colony-stimulating factor (GM-CSF) in inflammatory disease, including arthritis. In the present study, we examined the effectiveness of treatment of collagen-induced arthritis (CIA) with a neutralizing mAb to GM-CSF. DBA/1 mice were immunized for the development of CIA and treated at different times, and with different doses, with neutralizing mAb to GM-CSF or isotype control mAb. Anti-GM-CSF mAb treatment prior to the onset of arthritis, at the time of antigen challenge, was effective at ameliorating the ensuing disease. Modulation of arthritis was seen predominantly as a reduction in overall disease severity, both in terms of the number of limbs affected per mouse and the clinical score of affected limbs. Importantly, anti-GM-CSF mAb treatment ameliorated existing disease, seen both as a reduction in the number of initially affected limbs progressing and lower numbers of additional limbs becoming affected. By histology, both inflammation and cartilage destruction were reduced in anti-GM-CSF-treated mice, and the levels of tumor necrosis factor-a and IL-1? were also reduced in joint tissue washouts of these mice. Neither humoral nor cellular immunity to type II collagen, however, was affected by anti-GM-CSF mAb treatment. These results suggest that the major effect of GM-CSF in CIA is on mediating the effector phase of the inflammatory reaction to type II collagen. The results also highlight the essential role of GM-CSF in the ongoing development of inflammation and arthritis in CIA, with possible therapeutic implications for rheumatoid arthritis.     

A quantitative focus assay for titration of retroviruses that encode human granulocyte-macrophage colony-stimulating factor (GM-CSF).

Granulocyte-macrophage colony-stimulating factor (GM-CSF) is a haematopoietic growth factor that regulates proliferation, differentiation, and effector functions of monocyte-macrophages and granulocytic cells. Because of the ability of this cytokine to enhance immune functions of antigen-presenting cells, retroviruses encoding GM-CSF have been used to transduce GM-CSF into murine and human tumour cells as part of autologous tumour vaccine strategies. We have previously shown that NIH 3T3 cells engineered to express functional human GM-CSF receptors (hGMR-NIH 3T3), become fully transformed when these cells are incubated in the presence but not in the absence of human GM-CSF. In this study we have used these hGM-CSF conditional transformants to devise a sensitive focus assay to titrate retroviruses encoding hGM-CSF, using MFG-hGM-CSF/Psi-CRIP as our model virus. This helper-free amphotropic retrovirus, which has been frequently used to transduce hGM-CSF into tumour cells, was quite transforming in our indicator cell line, exhibiting virus titres well above 10(5)FFU/ml. The transformation-based assay described here allows rapid determination of the titre of hGM-CSF-viruses, and may serve as a model for development of quantitative assays for other cytokine-encoding viruses of clinical importance.     

Growth of an interleukin 2/interleukin 4-dependent T cell line induced by granulocyte-macrophage colony-stimulating factor (GM-CSF)

Lymphokine activities in conditioned medium from activated helper T cell lines are most commonly defined by the proliferation of "specific" lymphokine-dependent cell lines. Various sublines of IL 2-dependent (and ostensibly specific) HT-2 and CTLL cells have now been shown to proliferate in response to BSF-1/IL 4 as well. After activation with antigen or mitogen, D10.G4.1, an antigen-specific cloned T helper cell that has recently been shown to produce IL 4 but not IL 2, secretes two distinct cytokines that induce the growth of HT-2 cells. These "T cell growth factors" (TCGF) can be separated by reversed phase high-performance liquid chromatography (RP-HPLC). The TCGF activity of one of these factors can be blocked by 11B11, an antibody specific for IL 4. The second TCGF activity is not affected by 11B11 or by antibodies specific for IL 2. This TCGF activity can be neutralized by a goat polyclonal antibody to granulocyte-macrophage colony-stimulating factor (GM-CSF), and has a RP-HPLC elution profile identical to that of recombinant GM-CSF. Recombinant GM-CSF induces both proliferation and long-term growth of HT-2 but not CTLL cells, and this activity can be neutralized by the same antibody to GM-CSF. GM-CSF is best known as a factor that induces the maturation and growth of granulocytes and macrophages from bone marrow-derived hematopoietic precursor cells. The ability of GM-CSF to induce the growth of certain T cell lines indicates that this molecule may play a role in T cell-mediated immune responses, either as an autocrine growth factor or a paracrine stimulus from both lymphoid and nonlymphoid tissues that produce this cytokine.     

Phagocytosis and killing of Staphylococcus aureus by granulocytes in mice after granulocyte-macrophage colony stimulating factor (GM-CSF) injection

GM-CSF is a hematopoietic growth factor. In vitro it stimulates the proliferation of myeloid progenitors and formation of granulocyte and macrophage colonies. It was found that GM-CSF in vitro is also stimulated the function of mature granulocytes, but we have no information about such influence in vivo. The purpose of this investigation was the evaluation in vivo of the GM-CSF effect on phagocytosis, bactericidal activity, and lysosome enzyme activities in granulocytes. GM-CSF was injected into mice subcutaneously during 5 consecutive days in the dose of 1 microgram/kg/d. The examination of the percent of cell phagocytizing bacteria (Staphylococcus aureus), NBT test, bactericidal activity and activation of acid phosphatase, alkaline phosphatase, peroxidase and esterase was performed every day and an evident increase of the tested parameters was found. These results prove in vivo activation of granulocytes by GM-CSF.     

Effects of recombinant murine granulocyte-macrophage colony-stimulating factor in cyclophosphamide-treated mice

To evaluate the efficacy of recombinant murine granulocyte-macrophage colony-stimulating factor (rGM-CSF) in attenuating the myelosuppression associated with chemotherapy, the effects of 100 and 300 ng rGM-CSF, administered twice daily by intraperitoneal injection for 6 consecutive days to mice 24 hours after a dose of 200 mg/kg cyclophosphamide, were measured. Six days after the initial injection of rGM-CSF, a significant increase occurred in the absolute myeloid count compared to that of vehicle-treated animals. The difference was most pronounced on day 7, attaining levels of 327% and 428% of the control; these increases slowly declined to that of the control level by day 19. No significant effect was produced by rGM-CSF on the packed red cell volume or on the platelet count. Furthermore, the administration of rGM-CSF did not alter bone marrow cellularity or increase the number of marrow-derived hematopoietic stem cells. In contrast, a significant splenomegaly occurred, starting on day 6 and continuing until day 17. This was characterized by a pronounced increase in splenic-derived granulocyte (CFU-G), granulocyte-macrophage (CFU-GM), macrophage (CFU-M), megakaryocyte (CFU-MK), and erythroid (BFU-E, CFU-E) stem cells. The increases occurred between days 6 and 9 following the initial administration of rGM-CSF. These findings indicated that the administration of rGM-CSF to cyclophosphamide-treated animals causes an absolute increase in circulating myeloid cells and that these increases are derived from the spleen. The use of recombinant hematopoietic growth factors may permit the administration of more intensive chemotherapy through amelioration of chemically induced leukopenia.     

The effects of recombinant human granulocyte-macrophage colony-stimulating factor (rhGM-CSF) on human osteoblast-like cells

The activity of human osteoblast-like cells cultured in vitro is regulated by a number of factors, which include systemic hormones as well as agents that can be produced locally within bone. Several cytokines and growth factors have been demonstrated to be produced by osteoblasts themselves, and this includes granulocyte-macrophage colony-stimulating factor (GM-CSF). In this report we show that recombinant human GM-CSF (rhGM-CSF) modulates the activities of osteoblast-like cells derived from human trabecular bone in vitro. rhGM-CSF stimulated the proliferation of the cultured human osteoblast-like cells, but antagonised the induction by 1,25(OH)2D3 of osteocalcin synthesis and alkaline phosphatase activity, two characteristic products of osteoblasts. rhGM-CSF however, had no appreciable effect on the production of prostaglandin E2, or on the plasminogen activator activity associated with human osteoblast-like cells. These results are the first report of which we are aware of an apparently direct action of GM-CSF on cells of the osteoblast phenotype. These studies indicate that GM-CSF represents another haematological factor that can potentially exert regulatory actions on human osteoblast-like cells. GM-CSF may therefore be a potential paracrine/autocrine regulator of osteoblast activity.     

Interleukin 1 stimulates human endothelial cells to produce granulocyte-macrophage colony-stimulating factor and granulocyte colony-stimulating factor

Endothelial cells are a potent source of hematopoietic growth factors when stimulated by soluble products of monocytes. Interleukin 1 (IL 1) is released by activated monocytes and is a mediator of the inflammatory response. We determined whether purified recombinant human IL 1 could stimulate cultured human umbilical vein endothelial cells to release hematopoietic growth factors. As little as 1 U/ml of IL 1 stimulated growth factor production by the endothelial cells, and increasing amounts of IL 1 enhanced growth factor production in a dose-dependent manner. Growth factor production increased within 2 to 4 hr and remained elevated for more than 48 hr. To investigate the molecular basis for these findings, oligonucleotide probes for granulocyte-macrophage colony-stimulating factor (GM-CSF), granulocyte colony-stimulating factor (G-CSF), macrophage colony-stimulating factor (M-CSF), and multi-CSF were hybridized to poly(A)-containing RNA prepared from unstimulated and IL 1-stimulated endothelial cells. Significant levels of GM-CSF and G-CSF, but not M-CSF or multi-CSF, mRNA were detected in the IL 1-stimulated endothelial cells. Biological assays performed on the IL 1-stimulated endothelial cell-conditioned medium confirmed the presence of both GM- and G-CSF. These results demonstrate that human recombinant IL 1 can stimulate endothelial cells to release GM-CSF and G-CSF, and provide a mechanism by which IL 1 could modulate both granulocyte production and function during the course of an inflammatory response.     

Specific binding of radioiodinated granulocyte-macrophage colony-stimulating factor to hemopoietic cells

The hemopoietic growth factor granulocyte-macrophage colony-stimulating factor, GM-CSF, specifically controls the production of granulocytes and macrophages. This report describes the binding of biologically-active 125I-labeled murine GM-CSF to a range of hemopoietic cells. Specific binding was restricted to murine cells and neither rat nor human bone marrow cells appeared to have surface receptors for 125I-labeled GM-CSF. 125I-Labeled GM-CSF only appeared to bind specifically to cells in the myelomonocytic lineage. The binding of 125I-labeled GM-CSF to both bone marrow cells and WEHI-3B(D+) was rapid (50% maximum binding was attained within 5 min at both 20 degrees C and 37 degrees C). Unlabeled GM-CSF was the only polypeptide hormone which completely inhibited the binding of 125I-labeled GM-CSF to bone marrow cells, however, multi-CSF (also called IL-3) and G-CSF partially reduced the binding of 125I-labeled GM-CSF to bone marrow cells. Interestingly, the binding of 125I-labeled GM-CSF to a myelomonocytic cell line, WEHI-3B(D+), was inhibited by unlabeled GM-CSF but not by multi-CSF or G-CSF. Scatchard analysis of the binding of 125I-labeled GM-CSF to WEHI-3B(D+) cells, bone marrow cells and peritoneal neutrophils indicated that there were two classes of binding sites: one of high affinity (Kd1 = 20 pM) and one of low affinity (Kd2 = 0.8-1.2 nM). Multi-CSF only inhibited the binding of 125I-labeled GM-CSF to the high affinity receptor on bone marrow cells: this inhibition appeared to be a result of down regulation or modification of the GM-CSF receptor.(ABSTRACT TRUNCATED AT 250 WORDS)     

A novel human macrophage-activating factor: distinction from interferon-gamma (IFN-gamma) and granulocyte-macrophage colony-stimulating factor (GMCSF)

Culture supernatant from a human T-cell leukemia virus type I (HTLV-1)-infected cell line, DGA-1, contained a novel macrophage-activating factor (MAF). This MAF was antigenically and functionally distinct from interferon-gamma (IFN-gamma) and from granulocyte-monocyte colony-stimulating factor (GMCSF). Potential contaminants such as bacterial lipopolysaccharide (LPS), Mycoplasma spp, and HTLV-1 were not responsible for this MAF activity. The DGA-1 MAF was secreted constitutively and the cell line grew well in the absence of growth factors such as interleukin-2, mitogen, or antigen. This cell line should provide a good source of this MAF for further purification and characterization.     

Capillary leak syndrome during low dose granulocyte-macrophage colony-stimulating factor (rh GM-CSF) treatment of a patient in a continuous febrile state

A 20 years old man with peripheral primitive neuroectodermal tumor involving the bone marrow received 12 Gy fractionated total body irradiation, 140 mg/m2 melphalan, 1800 mg/m2 etoposide, and 1500 mg/m2 carboplatin for consolidation of first remission. Thereafter, 250 micrograms/m2/day recombinant human granulocyte-macrophage colony-stimulating factor (rh GM-CSF) (Behring Werke) were administered as continuous infusion 4 days after infusion of autologous bone marrow and peripheral stem cells to accelerate granulocyte reconstitution for control of a continued febrile state. The clinical picture of capillary leak syndrome developed with weight gain, pleural effusions and peripheral edema. The patient's condition stabilized after discontinuation of rh GM-CSF. Eight days later he died of invasive aspergillosis. The clinical course of our patient suggests a potentially fatal toxic effect of rh GM-CSF, even in low dose, in the setting of septicemia or fungemia.