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.     

Pretreatment with granulocyte colony-stimulating factor reduces myelopoiesis in irradiated mice

The purpose of this study was to investigate effects of the treatment prior to irradiation with granulocyte colony-stimulating factor (G-CSF) on hematopoiesis in B10CBAF1 mice exposed to a sublethal dose of 6.5 Gy of 60Co gamma radiation. G-CSF was administered in a 4-day regimen (3 microg/day); irradiation followed 3 h after the last injection of G-CSF. Such a treatment was found to stimulate granulopoiesis, as shown by increased counts of granulocyte-macrophage progenitor cells (GM-CFC) and of granulocytic cells in the femoral marrow and spleen at the time of irradiation. However, postirradiation counts of GM-CFC and granulocytic cells in the marrow of mice pretreated with G-CSF were reduced up to day 18 after irradiation. Interestingly, the D0 values for marrow GM-CFC determined 1 h after in vivo irradiation were 1.98 Gy for controls and 2.47 Gy for mice pretreated with G-CSF, indicating a decreased radiosensitivity of these cells after drug treatment. The inhibitory effects of the pretreatment with G-CSF on the postirradiation granulopoiesis could be attributed to the phenomenon of "rebound quiescence" which can occur after cessation of the treatment with growth factors. Postirradiation recovery of erythropoiesis in the spleen of mice pretreated with G-CSF exhibited a dramatic increase and compensated for the decreased erythropoiesis in the marrow at the time of irradiation. This complexity of the hematopoietic response should be taken into account when administering G-CSF in preirradiation regimens.     

Circular permutation of granulocyte colony-stimulating factor

The sequence of granulocyte colony-stimulating factor (G-CSF) has been circularly permuted by introducing new chain termini into interhelical loops and by constraining the N- and C-terminal helices, either by direct linkage of the termini (L0) or by substitution of the amino-terminal 10-residue segment with a seven-residue linker composed of glycines and serines (L1). All the circularly permuted G-CSFs (cpG-CSFs) were able to fold into biologically active structures that could recognize the G-CSF receptor. CD and NMR spectroscopy demonstrated that all of the cpG-CSFs adopted a fold similar to that of the native molecule, except for one [cpG-CSF(L1)[142/141]] which has the new termini at the end of loop 34 with the shorter L1 linker. All of the cpG-CSFs underwent cooperative unfolding by urea, and a systematically lower free energy change (DeltaGurea) was observed for molecules with the shorter L1 linker than for those molecules in which the original termini were directly linked (the L0 linker). The thermodynamic stability of the cpG-CSFs toward urea was found to correlate with their relative ability to stimulate proliferation of G-CSF responsive cells. Taken together, these results indicate that the G-CSF sequence is robust in its ability to undergo linear rearrangement and adopt a biologically active conformation. The choice of linker, with its effect on stability, seems to be important for realizing the full biological activity of the three-dimensional structure. The breakpoint and linker together are the ultimate determinants of the structural and biological profiles of these circularly permuted cytokines. In the following paper [McWherter, C. A., et al. (1999) Biochemistry 38, 4564-4571], McWherter and co-workers have used circularly permuted G-CSF sequences to engineer chimeric dual IL-3 and G-CSF receptor agonists in which the relative spatial orientation of the receptor agonist domains is varied. Interpreting the differences in activity for the chimeric molecules in terms of the connectivity between domains depends critically on the results reported here for the isolated cpG-CSF domains.     

Characterization of peripheral blood stem cell grafts mobilized by granulocyte colony-stimulating factor and plerixafor compared with granulocyte colony-stimulating factor alone

Background aimsThis study aimed to characterize the immune effectors contained in apheresis samples obtained from patients with grafts mobilized with plerixafor and granulocyte colony-stimulating factor (G-CSF) (P+G) compared with grafts mobilized with G-CSF alone (G).MethodsAliquots of apheresis samples were obtained from 36 patients with malignant diseases after mobilization with G (n = 18) or P+G (n = 18). The phenotype and cytokine secretion profile of T cell and dendritic cell subsets were characterized by multicolor cytometry including intracellular cytokine staining.ResultsIn grafts collected after mobilization with P+G, there was a significantly higher percentage of CD3⁺ T cells compared with samples collected after mobilization with G alone. On a functional level, a significant increase of interferon-γ and tumor necrosis factor-α secreting CD8⁺ T cells was observed in the P+G group compared with the G group. CD4⁺Foxp3⁺ regulatory T cells were similar in both groups but exhibited a lower expression of inducible costimulatory molecule and a significantly higher expression of CD127 in the P+G group. Myeloid dendritic cells (MDCs) and BDCA3⁺ dendritic cells were similar in both groups. In contrast, plasmacytoid dendritic cells (PDCs) (CD123⁺BDCA2⁺HLA-DR⁺) were significantly increased in the P+G grafts, leading to a higher PDC-to-MDC ratio. PDCs mobilized by P+G displayed different functional markers—a higher percentage of ILT7⁺ PDCs and decreased expression of CD86—suggesting a potential regulatory capacity of PDCs mobilized by P+G.ConclusionsGrafts mobilized with P+G exhibited major different functional features compared with grafts mobilized with G alone, suggesting that such grafts may have an impact on patient outcome after autologous stem cell transplantation.     

Production of granulocyte colony-stimulating factor by head and neck carcinomas

Detectable levels of G-CSF by enzyme-linked immunosorbent assay (ELISA) were found in sera of 4 out of 15 patients with head and neck carcinomas. Also cells prepared from the tumors of these 4 patients secreted G-CSF. The supernatants of cells derived from all 15 patients did not contain granulocyte-monocyte CSF, monocyte CSF, tumor necrosis factor-, transforming growth factor- ₁, epidermal growth factor, interleukin (IL)-1 and IL-6. These findings suggest that leukocytosis in patients with carcinomas might be due to the production of G-CSF by tumor cells.Abbreviations CSF colony stimulating factor - EGF epidermal growth factor - ELISA Enzyme-linked immunosorbent assay - G granulocyte - GM granulocyte-monocyte - IL interluekin - M monocyte - TGF transforming growth factor - TNF tumor necrosis factor     

Production of granulocyte/macrophage colony-stimulating factor by cultured astrocytes

We investigated the production of interleukin-3 (IL-3)-like factor by murine astrocytes. Supernatants from lipopolysaccharide (LPS)-stimulated astrocytes induced proliferation of IC-2, an IL-3- and granulocyte/macrophage colony-stimulating factor (GM-CSF)-dependent cell line. This activity was completely neutralized by the antibody against GM-CSF but not by the anti-IL-3 monoclonal antibody. Northern blot analysis revealed the expression of GM-CSF mRNA, but not of IL-3 mRNA, in cultured astrocytes. These results indicate that with proper stimuli murine astrocytes produce GM-CSF.     

Reengineering granulocyte colony-stimulating factor for enhanced stability

Granulocyte colony-stimulating factor is a long-chain cytokine that has both biological and therapeutic applications. It is involved in the production and maturation of neutrophilic progenitor cells and neutrophils and is administered to stimulate the production of white blood cells to reduce the risk of serious infection in immunocompromised patients. We have reengineered granulocyte colony-stimulating factor to improve the thermodynamic stability of the protein, focusing on enhancing the alpha-helical propensity of residues in the antiparallel 4-helix bundle of the protein. These redesigns resulted in proteins with substantially enhanced stability while retaining wild-type levels of biological activity, measured as the ability of the reengineered proteins to stimulate the proliferation of murine myeloid cells transfected with the granulocyte colony-stimulating factor receptor.     

Discrimination of granulocyte colony-stimulating factor isoforms by high-performance capillary electrophoresis

Granulocyte colony-stimulating factor (G-CSF) is a glycoprotein which acts primarily to stimulate the proliferation, differentiation and activation of committed progenitor cells of the neutrophil–granulocyte lineage into functionally mature neutrophils. The traditional biological assays employed to detect G-CSF are a myeloid bone marrow colony assay, a factor-dependent cell line specific for G-CSF and commercially available immunoassays. However, these methods will not distinguish between glycosylated and non-glycosylated forms of the molecule. In this study high-performance capillary electrophoresis (HPCE) was used to analyse glycosylated and non-glycosylated recombinant human granulocyte colony-stimulating factor (r-met-hG-CSF). Glycosylated G-CSF preparations contained human serum albumin (HSA), added as a protein carrier. Glycosylated and non-glycosylated G-CSFs were prepared in 40 mM Na₂HPO₄ buffer, pH 2.5, containing hydroxypropylmethylcellulose (HPMC) or 50 mM Na₂HPO₄ buffer, pH 9.0. Glycosylated G-CSF could be separated into two distinct glycoform populations at the lower pH studied. Differences in migration time and peak shape between glycosylated and non-glycosylated G-CSF were demonstrated. HPCE analysis of G-CSF produced using a baculovirus expression vector system revealed a further distinct G-CSF glycoform and demonstrated the resolving power of the technique.     

Protein tailoring of human granulocyte colony-stimulating factor

Summary Recombinant human granulocyte-colony stimulating factor (rhG-CSF) was modified by site-directed mutagenesis and chemical modification in order to improve its pharmacological activity and its thermostability. The mutant rhG CSF which 17th cysteine was substituted with alanine was chemically modified by activated polyethylene glycol. The chemically modified mutant rhG-CSF greatly increased both its biological activityin vivo and its thermostability. This is a successful example of protein tailoring in which site-directed mutagenesis and chemical modification were used at the same time.     

Involvement of protein kinase Cepsilon in the negative regulation of Akt activation stimulated by granulocyte colony-stimulating factor

Stimulation of cells with G-CSF activates multiple signaling cascades, including the serine/threonine kinase Akt pathway. We show in this study that G-CSF-induced activation of Akt in myeloid 32D was specifically inhibited by treatment with PMA, a protein kinase C (PKC) activator. PMA treatment also rapidly attenuated sustained Akt activation mediated by a carboxy truncated G-CSF receptor, expressed in patients with acute myeloid leukemia evolving from severe congenital neutropenia. The inhibitory effect of PMA was abolished by pretreatment of cells with specific PKC inhibitor GF109203X, suggesting that the PKC pathway negatively regulates Akt activation. Ro31-8820, a PKCepsilon inhibitor, also abrogated PMA-mediated inhibition of Akt activation, whereas rottlerin and Go6976, inhibitors of PKCdelta and PKCalphabetaI, respectively, exhibited no significant effects. Furthermore, overexpression of the wild-type and a constitutively active, but not a kinase-dead, forms of PKCepsilon markedly attenuated Akt activation, and inhibited the proliferation and survival of cells in response to G-CSF. The expression of PKCepsilon was down-regulated with G-CSF-induced terminal granulocytic differentiation. Together, these results implicate PKCepsilon as a negative regulator of Akt activation stimulated by G-CSF and indicate that PKCepsilon plays a negative role in cell proliferation and survival in response to G-CSF.     

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.     

Inhibition of polymorphonuclear leukocyte capping by a chemotactic factor.

Incubation of human polymorphonuclear leukocytes with colchicine and fluorescein-concanavalin A leads to the formation of a polarized cap of fluorescence not seen if cells are incubated with fluorescein-Con A along. When cells are preincubated with a chemotactic factor before colchicine treatment, the capping is inhibited in a dose-related manner. Studies with alpha-methylmannoside indicate that the caps represent extracellular fluorescein-Con A and are not areas of Con A internalization. Experiments utilizing an irreversible inhibitor of serine esterases suggest that a chemotactic factor-activated enzyme is involved in the inhibition of cap formation in the human neutrophil.     

Gene structure and function of granulocyte colony-stimulating factor

In the last few years, the molecular and genetic nature of the granulocyte colony-stimulating factor, which controls proliferation and differentiation of neutrophils, has been characterized. Recent clinical application of G-CSF proves that this hormone is effective in the treatment of patients suffering from neutropenia.     

Structural studies on the murine granulocyte colony-stimulating factor

Granulocyte-colony stimulating factor (G-CSF) is a glycoprotein hemopoietic growth factor which regulates the production of granulocytes and macrophages. Reversed-phase microbore high-performance liquid chromatography was employed to purify a number of tryptic and Staphylococcus aureus V8 proteinase peptides generated from approximately 400 pmol G-CSF purified from medium conditioned by lungs from mice previously injected with endotoxin. N-Terminal amino-acid sequence analyses were performed on the parent polypeptide and on four tryptic peptides and one Staphylococcus aureus V8 protease peptide, yielding 68 unique amino-acid assignments; this corresponds to approximately 38% of the molecule.     

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.     

A single injection of polyethylene-glycol granulocyte colony-stimulating factor strongly prolongs survival of mice with systemic candidiasis

Systemic candidiasis is a life-threatening disease occurring in immunocompromized patients. Granulocyte colony-stimulating factor (G-CSF) reduces mortality in experimental invasive candidiasis. Covalent conjugation of polyethylene-glycol (peg) to proteins increases their stability and in vivo bioactivity. In this study, the effect of a single subcutaneous injection of peg-G-CSF on lethal candidiasis was assessed. This was performed in acute and chronic candidiasis models in non-neutropenic FVB/N mice. Peg-G-CSF rapidly increased circulating polymorphonuclear leukocyte (PMNL) numbers in mice, sustaining high for >4 days. Candida albicans outgrowth from kidneys of infected mice was strongly reduced after peg-G-CSF treatment (5.76 log cfu/g kidney vs 7.66 control), with absence of hyphal outgrowth and enhanced PMNL influx. Moreover, peg-G-CSF increased survival of C. albicans -infected mice, whereas efficacy of uncoupled G-CSF was obtained only after repeated treatment. These data document a potent in vivo biological effect of peg-G-CSF, resulting in strongly enhanced resistance against systemic candidiasis.     

Keratinocyte-derived granulocyte/macrophage colony-stimulating factor induces DNA synthesis by peritoneal macrophages

Keratinocytes have been demonstrated to produce a number of cytokines, including growth factors such as the CSF IL-3. Circulating blood monocytes and some elicited macrophages retain a significant proliferative potential in response to colony-stimulating activity. Because a macrophage response is prominent in a variety of cutaneous immune reactions, we have studied the ability of conditioned media (CM) from a transformed murine keratinocyte cell line (PAM 212) and from normal murine keratinocytes to induce growth of peritoneal macrophages. CM from both normal and transformed keratinocyte cultures induces [3H]thymidine incorporation by thioglycollate-elicited, but not resident, peritoneal macrophages. IEF of PAM 212 CM reveals peaks of activity at pI 4.8 and less than or equal to 4.2. Analysis of CM by reversed-phase HPLC demonstrates active fractions that elute at 46 to 48% and 53 to 55% acetonitrile. The Mr of the 46 to 48% acetonitrile factor is 25 to 30 kDa by gel filtration HPLC. Polyclonal anti-granulocyte/macrophage (GM) CSF antibody blocks the induction of macrophage [3H]thymidine incorporation by factors with pI 4.8 and eluting at 46 to 48% acetonitrile but does not reduce the activity of crude CM or the factor eluting at 53 to 55% acetonitrile. Based on both physiochemical criteria and antibody neutralization, keratinocytes produce GM-CSF. Keratinocyte-derived factors, including GM-CSF, may play an important role in regulating cutaneous macrophage responses.     

Histone acetylation induced by granulocyte colony-stimulating factor in a map kinase-dependent manner

Histone acetylation has been shown to affect chromatin structure and gene expression. The mitogen-activated protein (MAP) kinase pathway is activated by a number of cytokines and plays critical roles in hematopoietic cell survival, proliferation, and differentiation. We focused on the part of the MAP kinase cascade and granulocyte colony-stimulating factor (G-CSF)in histone acetylation at one of the critical myeloid differentiation-associated genes, myeloperoxidase (MPO). G-CSF caused rapid acetylation of histone H3 and H4 at the promoter of MPO as revealed by chromatin immunoprecipitation. In addition, CBP and p300 were recruited to the promoter in response to G-CSF. Furthermore, we showed that rapid histone acetylation induced by G-CSF is MAP kinase-dependent. These results illustrate how myeloid-differentiating signals via G-CSF may be coupled with histone acetylation during the process of gene expression.