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.     

Phosphorylase kinase from human polymorphonuclear leukocytes.

Phosphorylase kinase from human polymorphonuclear leukocytes was investigated in a gel filtered crude preparation (17,000 x g supernatant). It was found to exist in two forms, one (the phosphorylated form) more active than the other (the dephosphorylated form). Interconversion between the two forms was carried out by a cyclic AMP dependent protein kinase and phosphoprotein phosphatase, respectively. The ratio of activity measured at pH 8.0 and 6.0 was 0.36 for the non-activated and 0.83 for the activated form, which is in contrast to the behaviour of phosphorylase kinase from muscle. Km app for the substrate phosphorylase b was 650 U/ml and 85 U/ml for the non-activated and activated form, respectively, whereas Km app for ATP was 0.03 mM and identical for the two forms. The non-activated form of phosphorylase kinase was activated by Ca2+ in the range 10(-7)--5 . 10(-6) M, which may have physiological importance, whereas the activated form was insensitive to variations in Ca2+ concentration between 10(-9) and 10(-3) M.     

Regulation by the protein kinase C activator phorbol ester of calcium channels in polymorphonuclear leukocytes

The Quin fluorescence in gamma-hexachlorocyclohexane-stimulated polymorphonuclear leukocytes is rapidly increased, which points to the increase in Ca2+in concentration during leukotriene B4 synthesis in leukocytes. An addition of EGTA and calcium antagonists (nifedipine, verapamil, diltiazem) to cell suspensions does not affect the basal level of internal Ca2+ but results in the inhibition of the gamma-hexachlorocyclohexane-induced Ca2+ increase. Two mechanisms of calcium homeostasis regulation in neutrophils are proposed. One of them, cAMP regulation, is coupled with a potent inhibiting effect of prostacyclin, an adenylate cyclase activator, on Ca2+in increase in stimulated neutrophils. The other one is the activation of protein kinase C catalyzed by 4 beta-phorbol-12 beta-myristate-13 alpha-acetate. The experimental results suggest that such an activation blocks Ca2+ influx into the cells via the closure of Ca2+ channels. The synergism of action of the above mechanisms in the regulation of calcium homeostasis in neutrophils is demonstrated.     

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.     

Evidence that bacteria induce translocation of protein kinase C activity in human polymorphonuclear leukocytes

Particulate fraction associated protein kinase activity was studied in human polymorphonuclear leukocytes stimulated by bacteria. Staphylococcus aureus was found to increase particulate fraction associated protein kinase C activity in a time and concentration dependent manner. The increase comprised both the phospholipid dependent and independent kinase activity and was augmented by addition of serum. Similar observations were done using Staphylococcus epidermidis and Klebsiellae pneumoniae. However, Escherichia coli only increased the phospholipid independent kinase activity in the particulate fraction, which suggests the presence of protease activity.     

Regulation of expression of granulocyte-macrophage colony-stimulating factor in human bronchial epithelial cells: roles of protein kinase C and mitogen-activated protein kinases

GM-CSF has a major role in the immune and inflammatory milieu of the airway. Airway epithelial cells (AEC) are among the first targets of environmental stimuli and local cytokines, in response to which they can produce GM-CSF. The regulation of GM-CSF is only minimally understood in AEC. We hypothesized that GM-CSF expression in AEC would result from activation of protein kinase C (PKC) and subsequent activation of the extracellular signal-regulated kinase (MAPKerk1/2) pathway, so we investigated signal transduction pathways in human primary culture bronchial epithelial cells (HBECs). TNF-alpha, IL-1beta, and PMA induced the release of GM-CSF in HBECs. The robust response to PMA was not detected in SV40 adenovirus-transformed normal human bronchial epithelial cells (BEAS-2B). PMA and TNF-alpha stimulation of GM-CSF required activation of PKC (inhibition by staurosporine and bisindolylmaleimide I). GM-CSF expression was up-regulated by a nonphorbol PKC activator, but not by an inactive PMA analogue. PMA-induced GM-CSF production in HBECs did not require a Ca2+ ionophore and was not inhibited by cyclosporin A. Activation of MAPKerk1/2 via PKC was associated with and was required for GM-CSF production induced by PMA and TNF-alpha. The data demonstrate regulation of GM-CSF in HBECs by PKC pathways converging on the MAPKerk1/2 pathway and further define cell-specific regulation critical for local airway responses.     

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.     

Priming of calcium mobilization in human neutrophils by granulocyte-macrophage colony-stimulating factor: Evidence for an involvement of phospholipase D-derived phosphatidic acid

Human neutrophils pre-incubated with granulocyte-macrophage-colony-stimulating factor (GM-CSF) exhibit an enhanced mobilization of calcium in response to secondary stimuli such as chemotactic factors. The mechanisms underlying this priming effect of GM-CSF were examined. It was first demonstrated that the additional calcium mobilized by chemotactic factors in GM-CSF-treated cells was derived from intracellular stores and was associated neither with an increased permeability to calcium nor with production of inositol 1,4,5-trisphosphate. These results indicated that GM-CSF called upon a novel mechanism in order to enhance the mobilization of calcium in human neutrophils. The growth factor has recently been shown to prime phospholipase D leading to an enhanced activation by chemotactic factors and an augmented production of phosphatidic acid. Furthermore the ability of exogenous phosphatidic acid to mobilize calcium in cell types other than neutrophils has been previously demonstrated. Therefore, we examined the potential involvement of phospholipase D in the priming of the calcium response by GM-CSF in human neutrophils. Inhibition of the production of the fMet-Leu-Phe-stimulated production of phosphatidic acid by ethanol or wortmannin had only marginal effects on the concurrent mobilization of calcium. However, the priming of the mobilization of calcium by GM-CSF was greatly decreased in cells treated with either ethanol or wortmannin. These results provide strong support for the hypothesis that the production of phosphatidic acid, which is enhanced in GM-CSF-treated cells, is linked to an increased mobilization of intracellular calcium. These results may have relevance to the mechanism of action of GM-CSF in mature haematopoeitic cells as well to the mitogenic activity of other growth factors.     

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.     

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.     

Biological activities of human granulocyte-macrophage colony-stimulating factor

Granulocyte-macrophage colony-stimulating factor (GM-CSF) has emerged as an important regulation for hematopoietic cell development and function. Within the myeloid lineages, GM-CSF serves as a growth and developmental factor for intermediate-stage progenitors between early, interleukin 3-responsive and late granulocyte colony-stimulating factor-responsive precursors. GM-CSF also serves as an activator of circulating effector cells. The ability of GM-CSF to induce monocyte expression of tumor necrosis factor, interleukin 1 and other factors, further ties this hormone into a network of cytokines that interact to regulate many hematologic and immunologic responses. The availability of large quantities of recombinant GM-CSF now provides the opportunity and challenge not only for unraveling the mechanisms regulating hematopoiesis, but also for developing new therapies for enhancement of host defense against infection that were not previously possible.     

Intracellular calcium changes induced by the endozepine triakontatetraneuropeptide in human polymorphonuclear leukocytes: role of protein kinase C and effect of calcium channel blockers

Background  

The endozepine triakontatetraneuropeptide (TTN) induces intracellular calcium ([Ca⁺⁺]_i) changes followed by activation in human polymorphonuclear leukocytes (PMNs). The present study was undertaken to investigate the role of protein kinase (PK) C in the modulation of the response to TTN by human PMNs, and to examine the pharmacology of TTN-induced Ca⁺⁺ entry through the plasma membrane of these cells.     

Stimulation of hexose transport by human polymorphonuclear leukocytes: a possible role for protein kinase C

The protein C kinase activators 1-O-oleoyl, 2-O-acetylglycerol, 12-O-tetradecanoyl phorbol-13-acetate, and mezerein, stimulated deoxyglucose uptake in human neutrophils. The responses were stimulus specific since no effect was noted with the diether analogues 1-O-hexadecyl-2-O-ethylglycerol, 1-O-palmitoyl-2-O-acetyl or 1-O-palmitoyl-3-O-acetyl diesters of propanediol, or with 1,2-diolein. Stimulation of deoxyglucose uptake had the characteristics of carrier facilitated hexose transport. Stimulated uptake of deoxy-glucose was inhibited by trifluoperazine (10-30 microM). Activation of protein kinase C therefore appears to trigger events involved in hexose transport.     

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.     

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.     

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.     

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.     

Ethanol increases superoxide anion production stimulated with 4beta-phorbol 12-myristate 13-acetate in human polymorphonuclear leukocytes. Involvement of protein kinase C.

Stimulation of human polymorphonuclear leukocytes (PMNs) with PMA initiates a cascade of events leading to the production and release of superoxide anion (O-2), a major component in anti-bacterial defense. Generation of O-2 by PMA-stimulated PMNs occurs through the translocation and activation of protein kinase C (PKC). In this study, using freshly isolated PMNs, we examined the effect of ethanol on this response to PMA. Our results show that the basal production of O-2 was not affected by ethanol. In contrast, the response induced by PMA was potentiated by ethanol. This potentiation was observed even at high doses of PMA (200 nM) which alone had stimulated the O-2 response maximally. This enhanced response was not due to an increase of PMA uptake by PMNs. The maximal effect was obtained when the cells were preincubated with 80 mM of ethanol before PMA stimulation. Measurement of PKC activity in the cytosolic and membrane fractions showed that pretreatment of PMNs with ethanol increased twofold the PMA-stimulated PKC activity in the membrane fraction. Furthermore, Western blot analysis verified that this increase in PKC activity in the membrane fraction was linked to an increase in the translocation of PKC-alpha and -beta isoforms to the membrane. These results suggest that ethanol potentiates PMA-induced O-2 production through increasing PKC translocation and activity in PMNs.     

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.