Deletion mutants of human granulocyte-macrophage colony-stimulating factor

To study the structure-function relationship of the human granulocyte-macrophage colony-stimulating factor (GM-CSF), genes were constructed that encode its three deletion mutants: D1, a mutant with the deletion of six amino acid residues (37-42) some of which are a part of a beta-structural region; D2, a mutant with the deletion of the unstructured six-aa sequence of a loop (45-50); and D3, a mutant with the deletion of 14 aa residues (37-50) corresponding to the A-B loop and encoded by the second exon of the gmcsf gene. The expression products of these genes in E. coli were accumulated in a fraction of insoluble proteins. The secondary structures of the mutant proteins were similar to that of the full-size GM-CSF, but the biological activity of the deletion mutants was 130 times lower than that of the GM-CSF: they stimulated the proliferation of the TF-1 cell line at 3 ng/ml concentration. The resulting proteins displayed antagonistic properties toward the full-size GM-CSF, with the inhibition degree of its colony-stimulating activity being 27%. A decrease in the mutant activity in the row D2 > D1 > D3 implies the importance of the conserved hydrophobic residues involved in the formation of the beta-structure for the formation of the GM-CSF functional conformation.     

NMR assignment of human granulocyte-macrophage colony-stimulating factor

Human granulocyte-macrophage colony-stimulating factor (hGM-CSF), also known as sargramostim or molgramostin, is a cytokine that functions as a hematopoietic cell growth factor. Here we report a near complete assignment for the backbone and side chain resonances for the mature polypeptide.     

Characterization of the human granulocyte-macrophage colony-stimulating factor receptor

Human granulocyte-macrophage colony-stimulating factor (GM-CSF) is a cytokine derived from activated T cells, endothelial cells, fibroblasts, and macrophages. It stimulates myeloid and erythroid progenitors to form colonies in semisolid medium in vitro, as well as enhancing multiple differentiated functions of mature neutrophils, macrophages, and eosinophils. We have examined the binding of human GM-CSF to a variety of responsive human cells and cell lines. The most mature myelomonocytic cells, specifically human neutrophils, macrophages, and eosinophils, express the highest numbers of a single class of high affinity receptors (Kd approximately 37 pM, 293-1000 sites/cell). HL-60 and KG-1 cells exhibit an increase in specific binding at high concentrations of GM-CSF; computer analysis of the data is nonetheless consistent with a single class of high affinity binding sites with a Kd approximately 43 pM and 20-450 sites/cell. Dimethyl sulfoxide induces a 3-10-fold increase in high affinity receptors expressed in HL-60 cells, coincident with terminal neutrophilic differentiation. Finally, binding of 125I-GM-CSF to fresh peripheral blood cells from six patients with chronic myelogenous leukemia was analyzed. In three of six cases, binding was similar to the nonsaturable binding observed with HL-60 and KG-1 cells. GM-CSF binding was low, or in some cases, undetectable on myeloblasts obtained from eight patients with acute myelogenous leukemia. The observed affinities of the receptor for GM-CSF are consistent with all known biological activities. Affinity labeling of both normal neutrophils and dimethyl sulfoxide-induced HL-60 cells with unglycosylated 125I-GM-CSF yielded a band of 98 kDa, implying a molecular weight of approximately 84,000 for the human GM-CSF receptor.     

Clinical applications of human granulocyte-macrophage colony-stimulating factor.

Granulocyte-macrophage colony-stimulating factor (GM-CSF) is a hematopoietic growth factor that stimulates myeloid cell proliferation and maturation and enhances the function of terminally differentiated effector cells. Phase I and II clinical trials have demonstrated mild to moderate toxicities at doses of less than 30 micrograms/kg/day. These studies suggest a potential role for this growth factor to stimulate myelopoiesis in patients with aplastic anemia, myelodysplastic syndromes, AIDS, chemotherapy-induced myelosuppression, chronic neutropenia, and following bone marrow transplantation. The potential clinical uses of GM-CSF will depend on results of studies designed to optimize its therapeutic efficacy.     

Levels of human serum granulocyte colony-stimulating factor and granulocyte-macrophage colony-stimulating factor under pathological conditions

Levels of serum granulocyte colony-stimulating factor (G-CSF) and granulocyte-macrophage colony stimulating factor (GM-CSF) in patients with various leukocyte disorders were estimated by enzyme linked immunosorbent assay (ELISA). Some cases of acute myelogenous leukemia and aplastic anemia showed elevated serum levels of G-CSF and/or GM-CSF, whereas almost all of 23 healthy controls showed G-CSF and GM-CSF levels lower than 100 pg/ml. High levels of both types of CSF were noted in patients with granulocytosis due to infection. These levels became lower after resolution of the infection. Daily changes in serum CSF levels were also examined in a patient with autoimmune neutropenia, and it was found that the peripheral neutrophilic granulocyte count changed almost in parallel with the serum G-CSF level but not with GM-CSF, following the pattern with a delay of about 4–5 h, suggesting the possibility that G-CSF mainly regulates peripheral neutrophil circulation.     

Three-dimensional structure of recombinant human granulocyte-macrophage colony-stimulating factor.

The crystal structure of recombinant human granulocyte-macrophage colony-stimulating factor (rhGM-CSF) has been determined at 2.8 A resolution using multiple isomorphous replacement techniques. There are two molecules in the crystallographic asymmetric unit, which are related by an approximate non-crystallographic 2-fold axis. The overall structure is highly compact and globular with a predominantly hydrophobic core. The main structural feature of rhGM-CSF is a four alpha-helix bundle, which represents approximately 42% of the structure. The helices are arranged in a left-handed antiparallel bundle with two overhand connections. Within the connections is a two-stranded antiparallel beta-sheet. The tertiary structure of rhGM-CSF has a topology similar to that of porcine growth factor and interferon-beta. Most of the proposed critical regions for receptor binding are located on a continuous surface at one end of the molecule that includes the C terminus.     

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.     

Effect of granulocyte-macrophage colony-stimulating factor on chemiluminescence of human neutrophils

We investigated the capacity of recombinant human granulocyte-macrophage colony-stimulating factor (rhGM-CSF) to enhance the function of neutrophils. Neutrophil function was measured in terms of N-formyl-methionyl-leucyl-phenylalanine (fMLP)-induced luminol-dependent chemiluminescence (LDCL). LDCL of fMLP-stimulated neutrophils was enhanced up to 4.5 fold following preincubation with rhGM-CSF. This enhancement depended on the length of preincubation, reaching an optimal level at 120 min. The dose-response relationship for fMLP-induced LDCL of neutrophils preincubated with rhGM-CSF revealed that half-maximum enhancement was achieved at an approximately 20-fold higher concentration than that of colony-forming units in culture-derived colony formation. These results suggest that differences in dose dependency may be explained by differences in the distribution of receptor(s) for GM-CSF. This may also enable GM-CSF to affect the hematopoietic system, which contains cells at various levels of differentiation, thus mediating the host-defense mechanism.     

Immunoenzyme determination of human granulocyte-macrophage colony-stimulating factor using monoclonal antibodies

A set of seven hybridomas producing monoclonal antibodies (MAbs) to the human recombinant granulocyte-macrophage colony-stimulating factor (GM-CSF) was obtained. The properties of the monoclonal antibodies were characterized, and pairs of MAbs specific to different non-overlapping epitopes of GM-CSF were identified. A sensitive and simple method of two-site ELISA for GM-CSF was developed on the basis of two MAbs. According to this method, one MAb is absorbed onto a microtiter plate and another is labeled with biotin and used for the detection of GM-CSF bound to the first MAb. MAb labeled with biotin, in its turn, was visualized with the streptavidin-horseradish peroxidase conjugate. The sensitivity of this test was no less than 0.5 ng/ml, and a linear dose-response relationship was observed within a concentration interval from 0.5 to 32 ng/ml. No cross-reactivity was found with human tumor necrosis factor-alpha, granulocyte colony-stimulating factor, interleukin-2, or interleukin-3 in this test system.     

Interleukin-10 contributes development of macrophage suppressor activities by macrophage colony-stimulating factor, but not by granulocyte-macrophage colony-stimulating factor

Macrophages are known to possess suppressor activities in immune responses. To determine the effects of GM-CSF and M-CSF on the expression of macrophage suppressor activities, monocyte-derived macrophages cultured with GM-CSF (GM-Mphis) were compared with those cultured with M-CSF (M-Mphis) for antigen-specific proliferation and interferon-gamma (IFN-gamma) production by lymphocytes. Both GM-Mphis and M-Mphis equally suppressed lymphocyte proliferation, but only M-Mphis suppressed IFN-gamma production in response to purified protein derivative (PPD). M-Mphis, but not GM-Mphis, released IL-10 not only in the course of macrophage differentiation but also in response to PPD after maturation to macrophages. From the results that (i) exogenous IL-10 suppressed IFN-gamma production, but not proliferation of lymphocytes, and that (ii) neutralizing antibody to IL-10 reversed suppressor activities of M-Mphis on IFN-gamma production, but not lymphocyte proliferation, it appeared that IL-10 was the major factor responsible for suppression of IFN-gamma production. Thus, these results suggest that only M-CSF augments IL-10-dependent suppressor activity of macrophages on IFN-gamma production and that both GM-CSF and M-CSF induce IL-10-independent macrophage suppressor activity on lymphocyte proliferation.     

Role of carbohydrate in the function of human granulocyte-macrophage colony-stimulating factor

cDNA clones for the human hematopoietic regulator granulocyte-macrophage colony-stimulating factor (hGM-CSF) were isolated from a lamba gt11 cDNA library prepared from RNA of COS cells transiently expressing the gene for hGM-CSF. As the RNA was a rich source of hGM-CSF mRNA, approximately 0.1% of the clones of this library contained hGM-CSF sequences. All of the clones analyzed were full length and were correctly processed. When subcloned into an expression vector and transfected into COS cells, the cDNA clones direct the synthesis of higher levels of the growth factor than the gene from which they were derived. The cDNA for native hGM-CSF was used to generate structural mutants which lack N-linked carbohydrate, O-linked carbohydrate, or both. Although the mutant proteins had differing specific activities, the nonglycosylated forms reproduce many, if not all, of the physiologic functions of authentic hGM-CSF. The role of carbohydrate in the secretion and function of hGM-CSF is discussed.     

Expression cloning of a receptor for human granulocyte-macrophage colony-stimulating factor.

Two cDNA clones encoding a receptor for human granulocyte-macrophage colony-stimulating factor (hGM-CSF-R) were isolated by expression screening of a library made from human placental mRNA. Pools of recombinant plasmid DNA were electroporated into COS cells which were then screened for their capacity to bind radioiodinated hGM-CSF using a sensitive microscopic autoradiographic approach. The cloned GM-CSF-R precursor is a 400 amino acid polypeptide (Mr 45,000) with a single transmembrane domain, a glycosylated extracellular domain and a short (54 amino acids) intracytoplasmic tail. It does not contain a tyrosine kinase domain nor show homology with members of the immunoglobulin super gene family, but does show some significant sequence homologies with receptors for several other haemopoietic growth factors, including those for interleukin-6, erythropoietin and interleukin-2 (beta-chain) and also to the prolactin receptor. When transfected into COS cells the cloned cDNA directed the expression of a GM-CSF-R showing a single class of affinity (KD = 2(-8) nM) and specificity for human GM-CSF but not interleukin-3. Messenger RNA coding for this receptor was detected in a variety of haemopoietic cells known to display hGM-CSF binding, and cross-linking experiments revealed a similar size for the glycosylated receptors in transfected COS and haemopoietic cells.     

Characterization of the soluble human granulocyte-macrophage colony-stimulating factor receptor complex.

The human granulocyte-macrophage colony-stimulating factor (GM-CSF) receptor (GM-R) is expressed on both hematopoietic and non-hematopoietic tissues. Although the receptor has been identified by cross-linking studies as an 84,000-dalton protein, very little is known about its biochemistry. In this report, we describe a soluble binding assay for the human GM-R which allowed us to characterize the receptor complex from various sources, including plasma membranes of placenta, neutrophils, and human myeloid leukemia cell lines. Preparation of membranes as well as solubilization by Triton X-100 and N-octylglucoside resulted in a 5-10-fold lower affinity of the receptor for GM-CSF. The Kd decreased from 20 to 80 pM in intact cells to 200-500 pM in both intact and solubilized membranes. Binding in solution was rapid, specific for GM-CSF, and best fit a "one-site" model with an approximate Kd of 500 pM. The dissociation rate constant for the soluble GM-R was very similar to that of intact cells (k2 = 0.013 min-1 versus 0.017 min-1, respectively). As expected, solubilized membranes obtained from those cells expressing the highest number of GM-R (neutrophils and dimethyl sulfoxide-induced HL-60 cells; approximately 500-800 sites/cell) possessed the highest concentration of soluble GM-R (approximately 2-3 x 10(8) GM-R/micrograms). Cross-linking of 125I-GM-CSF to soluble GM-R resulted in the appearance of two specifically labeled complexes. A major 110-kDa receptor-ligand complex is found when cross-linking is performed with intact cells; both 110- and 200-kDa species are seen when cross-linking is performed with either intact membranes or soluble GM-R. These studies define methods by which intact GM-R can be solubilized and measured in solution, permitting a more complete biochemical characterization of the intact GM-R complex.     

Regulation of IFN-gamma-receptor expression in human monocytes by granulocyte-macrophage colony-stimulating factor

The regulation of human IFN-gamma receptor (IFN-gamma-R) expression by granulocyte-macrophage CSF (GM-CSF) was investigated. On monocytic cell lines (U937, HL60) and peripheral blood monocytes, IFN-gamma-binding capacity was down-regulated upon incubation with GM-CSF. Scatchard plot analyses revealed that down-regulation was caused by a decrease in IFN-gamma-R number rather than by a change in affinity. GM-CSF treatment did not reduce IFN-gamma-R-specific mRNA levels, but reduced the half-life of membrane-expressed IFN-gamma-R, indicating a post-translational control of IFN-gamma-R by GM-CSF. Because both IFN-gamma and GM-CSF are crucially involved in activation of monocytic function, the data presented suggest that down-regulation of IFN-gamma-R by GM-CSF may represent a potential negative feedback control of monocyte activation. Further studies of IFN-gamma binding characteristics and isolation of IFN-gamma-R by immunoprecipitation revealed that IFN-gamma binding to human peripheral blood monocytes is mediated by a receptor protein structurally and functionally identical to that previously characterized in several established cell lines of other tissue origin.     

Effect of recombinant human interleukin 3, granulocyte-macrophage colony-stimulating factor and granulocyte colony-stimulating factor on human BFU-e in serum-free cultures

The effects of recombinant human hemopoietic growth factors on early and late human erythroid progenitors (BFU-e and CFU-e) were investigated in serum-free cultures. Recombinant human erythropoietin (rhEpo) induced the formation of not only human CFU-e-derived colonies but also human BFU-e-derived bursts. Recombinant human interleukin 3 (rhIL-3) alone did not induce the formation of human BFU-e-derived bursts and human CFU-e-derived colonies. In the presence of rhEpo, rhIL-3 dose dependently increased the number of bursts stimulated by rhEpo, although rhIL-3 did not have the augmentative effect on human CFU-e growth. On the other hand, rhIL-3 did not stimulate the formation of murine BFU-e-derived bursts, and murine IL-3 did not stimulate the formation of human BFU-e-derived bursts. The results indicated that the burst-promoting activity of IL-3 was species-specific between human and murine cells. Recombinant human GM-CSF (rhGM-CSF) or recombinant human G-CSF (rhG-CSF) failed to induce human burst formation and did not augment the effect of rhEpo on human burst formation. The results of the present study suggest that in vitro, IL-3 can stimulate BFU-e in collaboration with Epo, but GM-CSF and G-CSF do not stimulate BFU-e growth in the presence or absence of Epo.     

Cross-linking of human FcgammaRIIIb induces the production of granulocyte colony-stimulating factor and granulocyte-macrophage colony-stimulating factor by polymorphonuclear neutrophils

We have reported that human autoantibodies reacting with the polymorphonuclear neutrophil (PMN)-anchored FcgammaRIIIb (CD16) protect these cells from spontaneous apoptosis. In this study, we used anti-CD16 F(ab')(2) to delineate the mechanism(s) whereby the PMN life span is extended. As documented using four methods, CD16 cross-linking impeded spontaneous apoptosis, whereas anti-CD18 F(ab')(2) exerted no effect. Incubation of PMNs with anti-CD16 prevented the up-regulation of beta(2) integrins, particularly CD11b, which is the alpha-chain of complement receptor type 3, but also CD18, which is its beta-chain, as well as CD11a and CD11c. Anti-CD16-conditioned supernatant of PMNs diminished the percentage of annexin V-binding fresh PMNs after another 18 h in culture, whereas the negative control anti-CD18 had no effect. The expression of mRNA for G-CSF and GM-CSF was induced by anti-CD16, followed by the release of G-CSF and GM-CSF in a dose-dependent manner. Anti-G-CSF and anti-GM-CSF mAbs abrogated the antiapoptotic effect of the related growth factors. The delay in apoptosis was accompanied by a down-regulated expression of Bax, and a partial reduction of caspase-3 activity. These data suggest an autocrine involvement of anti-CD16-induced survival factors in the rescue of PMNs from spontaneous apoptosis. Thus, apoptosis of aged PMNs can be modulated by signaling through FcgammaRIIIb, which may occur in patients with PMN-binding anti-FcgammaRIIIb autoantibodies.     

Plasmacytoma growth factor activity of murine granulocyte-macrophage colony-stimulating factor

Mouse plasmacytoma FLOPC21 was adapted to culture in the presence of a mouse Th cell supernatant. A stable factor-dependent cell line was derived from this culture and the factor responsible for its growth was identified as granulocyte-macrophage colony-stimulating factor.     

Site-specific PEGylation of engineered cysteine analogues of recombinant human granulocyte-macrophage colony-stimulating factor

Granulocyte macrophage colony-stimulating factor (GM-CSF) stimulates proliferation of hematopoietic cells of the macrophage and granulocyte lineages and is used clinically to treat neutropenia and other myeloid disorders. Because of its short circulating half-life, GM-CSF is administered to patients by daily injection. We describe here the engineering of highly potent, long-acting human GM-CSF proteins through site-specific modification of GM-CSF cysteine analogues with a cysteine-reactive poly(ethylene glycol) (PEG) reagent. Thirteen cysteine analogues of GM-CSF were constructed, primarily in nonhelical regions of the protein believed to lie away from the major receptor binding sites. The GM-CSF cysteine analogues were properly processed but insoluble following secretion into the Escherichia coli periplasm. The proteins were refolded and purified by column chromatography. Ten of the cysteine analogues could be modified with a 5-kDa maleimide PEG, and seven of the mono-PEGylated proteins were purified by ion-exchange column chromatography. Biological activities of the 13 cysteine analogues and 7 PEGylated cysteine analogues were comparable to that of wild-type GM-CSF in an in vitro cell proliferation assay using human TF-1 cells. One cysteine analogue was modified with larger 10-, 20-, and 40-kDa PEGs, with only minimal loss of in vitro bioactivity. Pharmacokinetic experiments in rats demonstrated that the PEGylated proteins had up to 47-fold longer circulating half-lives than wild-type GM-CSF. These data demonstrate the utility of site-specific PEGylation for creating highly potent, long-acting GM-CSF analogues and provide further evidence that the nonhelical regions of human GM-CSF examined are largely nonessential for biological activity of the protein.     

Evaluation of human N-linked glycosylation sites in murine granulocyte-macrophage colony-stimulating factor.

Nonglycosylated murine and human granulocyte-macrophage colony-stimulating factor have a molecular mass of approximately 14.5 kDa predicted from the primary amino acid sequence. The expression of both proteins in COS cells leads to a heterogeneous population of molecules that differ in the degree of glycosylation. Both human and murine molecules contain two N-linked glycosylation sites that are situated in nonhomologous locations along the linear sequence. Despite this difference both proteins show a similar size distribution among the glycosylation variants. These studies analyze the effects of introducing in the murine protein novel N-linked glycosylation sites corresponding to those sites found in the human molecule. A panel of molecules composed of various combinations of human N-linked glycosylation sites in either the presence or the absence of murine N-linked glycosylation was compared. Substitution of a proper human N-linked glycosylation consensus sequence at Asn 24 did not result in N-linked glycosylation, nor was there any considerable effect on bioactivity. Replacement of the N-linked glycosylation consensus sequence at Asn 34 results in glycosylation similar to that found in the human molecule and causes a significant decrease in bioactivity. These data suggest that the position of N-linked glycosylation is critical for maximal bioactivity in a particular species and that the changes in position of these sites in different species probably occurred during evolution in response to changes in their receptors.