Granulocyte-macrophage colony stimulating factor (GM-CSF) and macrophage colony stimulating factor (CSF-1) synergize to stimulate progenitor cells with high proliferative potential

The ability to expand a population of bone marrow progenitors capable of forming macrophage colonies of high proliferative potential (HPP-CFC) was achieved using a culture system and signal requirements not previously shown to support the growth of HPP-CFC. Using bone marrow cells from untreated animals (not 5-FU-treated), culture conditions designed primarily for the detection of GM-CFC or M-CFC progenitors, and a more stringent criteria for HPP-CFC colony size (greater than or equal to 2 mm diameter), HPP-CFC progenitor expansion was demonstrated following simultaneous addition of rGM-CSF and CSF-1 to bone marrow cultures. Examination of the sequence and temporal requirements for rGM-CSF and CSF-1 addition necessary for the development of HPP-CFC-like colonies revealed that addition of the second factor could be delayed for up to 5 days and still result in the development of significant numbers of HPP-CFC colonies. Based on a comparison with human spleen cell conditioned medium (HSCM) and interleukin 1 (rIL 1), as sources of "synergistic activity" (SA) for the development of HPP-CFC-like colonies in combination with CSF-1, the combination of GM-CSF and CSF-1 appears to represent a novel pathway for stimulating the expansion of HPP-CFC progenitors with high proliferative potential.     

Ethnic differences in plasma levels of interleukin-8 (IL-8) and granulocyte colony stimulating factor (G-CSF)

Ethnic neutropenia is common in people of African descent. As interleukin-8 (IL-8) and granulocyte colony stimulating factor (G-CSF) bind to receptors on neutrophils, ethnic differences in neutrophil counts are hypothesized to result in different plasma levels of these cytokines. A prospective study was conducted in 72 healthy young volunteers. Neutrophil counts were 60% higher in Caucasians (P<0.00001). Average IL-8 and G-CSF levels were about 50% and 70% higher in African volunteers compared with Caucasian volunteers (P=0.0008 and P=0.00005, respectively). Additionally, oxidative burst capacity in stimulated neutrophils was significantly lower in volunteers of African descent (P=0.03 between both groups). In sum, lower neutrophil counts are associated with higher levels of IL-8 and G-CSF in Africans.     

Effects of granulocyte-macrophage colony stimulating factor (GM-CSF) in vivo on cytokine production and proliferation by spleen cells

GM-CSF is a potent stimulator of haematopoietic cells as well as some functions of granulocytes and macrophages. GM-CSF has many clinical uses; however, little is known about the effects of GM-CSF treatment in vivo on the responses of tissue lymphocytes in terms of secretion of Th-1 and Th-2 cytokines. We investigated this issue by measuring the responses of spleen cells from mice 24 h after treatment i.p. with saline or rmGM-CSF. GM-CSF at 16.7-50.0 microg/kg significantly increased (P < 0.01) spleen cellularity 2-2.5-fold and enhanced proliferative responses of non-stimulated (no mitogen) as well as concanavalin A (Con A)-stimulated spleen cells. Secretion of IFN-gamma by Con A (2.5 microg/ml)-stimulated spleen cells was significantly (P < 0.01) increased from 1.8 microg/ml by control spleen cells to 5.2 microg/ml by GM-CSF spleen cells. IL-10 production was greater (0.25 microg/ml, P < 0.05) by Con A-stimulated spleen cells from GM-CSF-treated mice compared to control spleen cells (0.06 microg/ml). By contrast, there were no significant differences in IL-4 production by Con A-stimulated spleen cells from the different groups. These results show that GM-CSF treatment increases spleen cellularity and primes lymphocytes for enhanced responses. The enhanced production of Th-1 cytokines by primed lymphocytes may partially explain the beneficial role of in vivo administration of GM-CSF in several clinical situations.     

Ultrastructural effects of granulocyte colony stimulating factor (G-CSF) and macrophage colony stimulating factor (M-CSF) on rat neutrophils and monocytes

Human granulocyte colony stimulating factor (G-CSF) and macrophage colony stimulating factor (M-CSF) were administered intravenously to rats, and their effects on neutrophils and monocytes were examined by electron microscopy. G-CSF increased the number of cytoplasmic granules in neutrophils. It also enhanced maturation of the nuclear shape in the neutrophils, while chromatin condensation and peroxidase distribution remained immature. M-CSF induced proliferation of monocytes in peripheral blood and bone marrow, but did not affect morphology or distribution of peroxidase reactivity. This study was presented at the 25th Annual Meeting of the Clinical Electron Microscopy Society of Japan, Matsumoto, September 28–30, 1993.     

Effect of melatonin on lymphocyte proliferation and production of interleukin-2 (IL-2) and interleukin-1 beta (IL-1 beta) in mice splenocytes

The influence of Melatonin (MLT) on the modulation of the immune system has been described. In previous studies an increment of cell proliferation and an increase or a decrease of cytokines have been reported. Other workers have found inhibitory effects or no effect in the immune functions. Because of this controversy, and for the purpose of studying the mechanism by which MLT performs its functions, we evaluated its effect on murine splenocytes's proliferation after a mitogenic stimulation, and quantified the levels of IL-2 and IL-1 beta in the absence or presence of Phitohemaglutinin (PHA) in supernatants of mice splenocytes cell culture treated or not with MLT. The lymphoproliferative response was assessed using tritiated thimidine in the splenocytes of mice treated with 500 micrograms of MLT/Kg b.w. and in cell cultures containing 5, 50 and 100 micrograms MLT/mL. The production of IL-2 and IL-1 beta was detected by the ELISA test. An increase in the proliferation (p < 0.01) of spleen cells treated with 50 and 100 micrograms MLT/mL an optimal dose of PHA, was detected. The in vivo or in vitro treatment with MLT increased the levels of IL-2 and IL-1 beta in the absence or the presence of PHA, maintaining the increase in the concentration of IL-1 beta up to the to ninth day of treatment. These results suggest that MLT acts directly on cell proliferation probably by binding to high affinity receptors located on spleen cells, that stimulates the production of IL-2 and IL-1 beta giving rise to an increment of cell immunity.     

Haematological response of dogs to canine recombinant granulocyte colony stimulating factor (rcG-CSF)

Three beagle dogs were given 5 g canine recombinant granulocyte colony stimulating factor (rcG-CSF)/kg/day for 42 days. One day after the first dose the neutrophil count exceeded the pretreatment counts by 3- to 4-fold. A steady and rapid increase occurred during days 1 to 12. In two of the dogs the counts continued to increase, but at a slower rate, from day 14 to 28. The neutrophil count in the third dog decreased steadily from day 14 to 28, although the count remained above those of the presample period and exceeded the reference range for dogs established at the Veterinary Medical Teaching Hospital, University of California, Davis. After day 28 that dog had a rapid increase in neutrophil count that reached similar numbers to the other two dogs at 34 days. At no time was a significant left shift found, although an occasional band neutrophil was observed. In addition, a moderate increase in lymphocyte and monocyte counts were found. The degree of these increases was much less than that of the neutrophils. The leukocyte counts decreased rapidly after the last dose, and 10 days later the counts were similar to those of the pretrial period.Concomitant with the marked neutrophilia, bone marrows showed myeloid hyperplasia. The myeloid: erythroid ratios increased from 1.03–1.65 to 2.77–7.03, and the marrow cellularity increased from approximately 30%–50% to about 85%–100%. Clinical evaluation and serum chemistry panels revealed no adverse affects. We conclude that rcG-CSF effectively sustains an increased neutrophil count without producing significant adverse effects.     

Effect of granulocyte macrophage-colony stimulating factor (GM-CSF) on 5-FU-induced ulcerative mucositis in hamster buccal pouches.

The present study was performed to investigate the protective effects of granulocyte macrophage-colony stimulating factor (GM-CSF) against ulcerative mucositis in hamster buccal pouch. GM-CSF was topically administered to the buccal pouches of hamsters with two different doses of 5 and 20 microg/ml. The treatment of GM-CSF led to rapid healing effects in gross and histopathological findings. It decreased expression of pro-inflammatory cytokine mRNA levels in the mucosal tissue of buccal pouches. Also GM-CSF-treated animals showed high numbers of Ki-67 positive cells in basal cell layer. These results suggest that GM-CSF provided excellent healing effects to ulcerative mucositis in the buccal pouch of hamster.     

Granulocyte macrophage-colony stimulating factor (GM-CSF) recruits immune cells to the pancreas and delays STZ-induced diabetes.

Granulocyte macrophage-colony stimulating factor (GM-CSF) is one of the most widely used growth factors for enhancing immune responses and is known to recruit and activate antigen-presenting cells (APCs). This study hypothesized that overexpression of this cytokine within the pancreatic beta-cells would recruit, expand, and activate APCs. The question was whether this would lead to tolerance or autoimmunity to pancreatic antigens. This possibility was tested by preparing transgenic mice (ins-GM-CSF) whose islets expressed murine GM-CSF. By 6-8 weeks of age, these mice developed a profound mononuclear cell infiltration that often overwhelmed the exocrine pancreas, although no changes in enzyme or hormone function were apparent. The majority of the mononuclear infiltrate within the pancreas was identified as F4/80+ macrophages. Transgenic ins-GM-CSF mice had splenomegaly due to a massive increase in the macrophage population. Additionally, mononuclear cells were found within the livers of transgenic mice, with F4/80+ cells also identified within the infiltrate, indicating that GM-CSF-activated mononuclear cells circulated to organs other than the pancreas. To assess the disease potential, this study tested whether macrophage recruitment to the pancreas might accelerate or protect the islets from diabetes. It was found that the induction of diabetes by low-dose streptozotocin (STZ) was delayed and reduced within ins-GM-CSF transgenic mice, in comparison with negative littermates. Together, these data highlight the role of GM-CSF in recruiting APCs such as macrophages. Advanced cellular infiltration does not overtly harm, and may even protect, pancreatic function, as seen with the delay in chemically induced diabetes.     

Morphological alterations and growth inhibition of Leishmania (L.)amazonensis promastigotes exposed to zidovudine (AZT)

Leishmania parasites cause a worldwide public health disease and its treatment is still based on pentavalent antimonials which present financial and toxicologic limitations. Some nucleosidic derivatives have demonstrated anti-leishmanial properties and this study aims to evaluate the in vitro morphologic alterations and growth inhibition of Leishmania (L.) amazonensis promastigotes exposed to zidovudine at several concentrations. The citotoxicity of zidovudine (AZT) to macrophages was determined by an MTT assay. After which the promastigotes were exposed to concentrations of AZT, ranging from 1 to 50 μM. The evaluation of survival and morphometry alterations were performed in two distinct phases of in vitro growth, on the third and sixth days, representing the logarithmic and stationary phases, respectively. Slides with the promastigotes were photographed and analyzed using Image J. A significant reduction of parasite number in the logarithmic phase of in vitro growth was observed when the parasites were submitted to 20, 30, 40, and 50 μM of AZT. Morphometric alterations were observed such as an increase in width of the body, cytoplasmic granulations and vacuolizations. These data indicate the toxicity of AZT which prevents the parasite's multiplication, indicating a promising use of AZT as an anti-leishmania drug.     

Effect of synthetic thymic humoral factor (THF-gamma 2) on T cell activities in immunodeficient ageing mice.

Immunodeficient ageing (C57BL/10 x DBA/2)F1 mice were treated by a single injection of synthetic thymic hormones and 4 days later their thymus and spleen cells were assayed in vitro for T cell activities. A few nanograms of THF-gamma 2 were found to raise the frequency of mitogen-responsive T cells in thymus and spleen cell populations as well as the frequency of cytokine-producing splenic T cells, up to the levels observed in young mice. Moreover, injection of THF-gamma 2 was found to restore T cell growth factor (TCGF) production by mitogen-stimulated spleen cells. Also, the helper activity of spleen cells was enhanced by this treatment and increased with increasing the THF-gamma 2 dose over a wide range. Similarly, the effects of thymopentin and thymosin-alpha 1 on T helper cell activity increased with increasing the injected dose, but the efficiencies of THF-gamma 2 and thymopentin were, respectively, 400-fold and eight-fold greater than that of thymosin-alpha 1.     

The effects of recombinant granulocyte-macrophage colony-stimulating factor (GM-CSF) and interleukin-3 on the secretory capacity of human blood eosinophils.

The effects of recombinant human GM-CSF and interleukin-3 (IL-3) on human blood eosinophil survival, activation, and secretion were studied. Purified normal density eosinophils from patients with the idiopathic hypereosinophilic syndrome (HES) survived in culture for 7 days (50% viable) in the presence of 50 nM GM-CSF or 50 nM IL-3. Neutrophils did not survive after 4 days. No eosinophils survived in the absence of GM-CSF or IL-3. In two out of five patients studied, the cultured eosinophils became elongated with numerous processes. In all five patients the cells became adherent, but there were no morphological signs of degranulation. Both GM-CSF and IL-3 activated eosinophils, transforming the storage form of eosinophil cationic protein (ECP) into the secreted form. The proportion of activated cells increased from less than 20% to over 50% after 4 days in culture. However, GM-CSF and IL-3 did not induce secretion on their own. On the other hand, when GM-CSF/IL-3-activated eosinophils were exposed to known secretory stimuli, there was a six-fold increase in the amount of ECP released when the cells were stimulated with sepharose coated with C3b, and a two-fold increase when they were stimulated with sepharose-activated whole autologous serum. Eosinophils from patients taking steroids were unable to secrete their granule contents, even though they became activated by GM-CSF and IL-3. A novel finding was that sepharose-activated whole serum was an extremely potent secretory signal for ECP, releasing up to 50% of the total ECP content. These studies showed that GM-CSF and IL-3 prime eosinophil effector function by initiating granule solubilization which is the first step in the secretory event, without affecting the subsequent extracellular release of granule proteins.     

Effects of IL-5, granulocyte/macrophage colony-stimulating factor (GM-CSF) and IL-3 on the survival of human blood eosinophils in vitro.

The mechanisms that could affect the lifespan of eosinophils after they have left the bone marrow, and their capacity to respond to activation factors were studied by examining the effects of IL-5, GM-CSF and IL-3 on purified human blood eosinophils in culture. All three cytokines prolonged the lifespan of the majority of blood eosinophils. This effect was dose-dependent: IL-5 greater than GM-CSF greater than IL-3. Light density eosinophils from most patients had a longer lifespan in culture than did normal density eosinophils, with or without the three cytokines. Eosinophil death in the absence of these cytokines occurred by apoptosis. Eosinophils from two patients did not survive when cultured with IL-5, although they survived in the presence of IL-3 or GM-CSF. IL-5, GM-CSF and IL-3 induced the expression of the activation epitope on the eosinophil ribonucleases recognized by monoclonal antibody EG2. We conclude that small amounts of IL-5, GM-CSF and IL-3 prevented programmed cell death in human blood eosinophils and induced the expression of the activation forms of eosinophil ribonucleases. We suggest that differences in the capacity of normal and light density eosinophils to survive in culture, and in the ability of eosinophils from some patients to respond to IL-5 could account for variations in the severity of disease seen in patients with persistent eosinophilia.     

Effect of exogenous interleukin-18 (IL-18) and IL-12 in the course of Brucella abortus 2308 infection in mice

In this study we demonstrated that combined inoculation of interleukin-12 (IL-12) and IL-18 reduced the number of bacteria in the spleens of mice infected with Brucella abortus 2308 and that the effect of the treatment was mediated by an increased capability of spleen cells to produce gamma interferon at the early phase of infection.     

人粒细胞集落刺激因子在家蚕细胞及幼虫中的高效表达

含信号肽序列的ｈＧ－ＣＳＦｃＤＮＡ基因克隆于Ｍ１３ｍｐ１８中，测序表明其基因序列与高活性ｈＧ－ＣＳＦｃＤＮＡ一致。将ｈＧ－ＣＳＦ基因插入家蚕核型多角体病毒（ＢｍＮＰＶ）转移载体ｐＢＥ２８４，经与野生型病毒ＤＮＡ共转染家蚕细胞后，通过体内同源重组的方式构建了重组病毒ＢｍＮＰＶ－Ｇ－ＣＳＦ，Ｓｏｕｔｈｅｒｎ印迹杂交表明重组病毒ＤＮＡ中含Ｇ－ＣＳＦ基因片段。重组病毒感染单层ＢｍＮ细胞后第四天表达量达１． ２×１０６ＣＦＵ／ｍｌ培养液，Ｗｅｓｔｅｒｎ－ｂｌｏｔ分析可见分子量为１９×１０３左右一条带。家蚕幼虫感染重组病毒后第四天表达量达１．４×１０７ＣＦＵ／ｍｌ血淋巴（ｈｅｍｏｌｙｍｐｈ）。     

人粒细胞巨噬细胞集落刺激因子在重组痘苗病毒中的 …

目的 观察人ＧＭ－ＣＳＦ（粒细胞－巨噬细胞集落刺激因子）对重组痘苗病毒免疫效果的影响。方法 大鼠体内观察。结果 从核酸和蛋白水平均证实重组痘苗病毒ＲＶＪＳＢ１１７５Ｄ／ＧＭ－ＣＳＦ可同时表达ＧＭ－ＣＳＦ及ＨＳＶ－１ｇＤ（单纯疱疹病毒ｇＤ糖蛋白），但ＲＶＪＳＢ１１７５Ｄ／ＧＭ－ＣＳＦ免疫大鼠产生的抗－ＨＳＶ１ｇＤ特异性抗体水平与ＲＶＪＳＢ１１７５Ｄ组相比无显著性差异。结论 人ＧＭ－ＣＳＦ在大鼠体内对     

Effects of tumour necrosis factor-alpha, interleukin-1 alpha, macrophage colony stimulating factor and transforming growth factor beta on trophoblastic matrix metalloproteinases.

The aim of this study was to determine the effects of tumour necrosis factor alpha (TNF), interleukin-1 alpha (IL-1alpha), macrophage colony-stimulating factor (MCSF) and transforming growth factor beta (TGFbeta) on the secretion of matrix metalloproteinases (MMP), human chorionic gonadotrophin (HCG) and fetal fibronectin (fFN) by purified first trimester cytotrophoblastic cells (CTB) in vitro. CTB were obtained from legal abortions and cultured in vitro in the presence or absence of the different cytokines. Secreted gelatinases were analysed in the culture supernatants by zymography, by measurements of the total gelatinolytic activity and by enzyme immunoassays. HCG and fFN were measured by commercially available immunoassays. TNF increased the total gelatinolytic activity by increasing MMP-9 activity (P = 0.025-0.0177) but decreased MMP-2 activity (P < 0.03) and immunoreactivity (P < 0.05), fFN (P < 0.02) and HCG (P < 0.01). IL-1alpha significantly increased the secretion of fFN (P < 0.02), the activity (P < 0.02) and immunoreactivity (P < 0.05) of MMP-9 but had no effect on the other parameters. MCSF increased MMP-9 immunoreactivity (P < 0.05) and moderately decreased HCG. TGFbeta inhibited total gelatinolytic activity, MMP-9 activity and immunoreactivity, but was without effect on MMP-2 concentrations and activity. TGFbeta decreased HCG (P < 0.041) and increased fFN (P < 0.042). Our results indicate that TGFbeta, TNF and IL-1alpha are important regulators of trophoblastic MMP secretion.     

Natural killer (NK) cell activity in human long-term bone marrow cultures (LTBMC): effects of interleukin-2 (IL-2) and granulocyte-macrophage colony-stimulating factor (GM-CSF) on the progenitor cells.

Human bone marrow-derived progenitor cells were studied in a long-term bone marrow culture system (LTBMC) dependent on an autologous stroma cell layer. The establishment of the stromal cell layer was facilitated by using marrow obtained from small pieces of sternum, which was cultured for 4 weeks without addition of exogenous growth factors. After this period, the response of LTBMC to two different cytokines [recombinant human interleukin-2 (rhIL-2) and recombinant human granulocyte-macrophage colony-stimulating factor (rhGM-CSF)] was investigated. Our results show proliferation in response to both cytokines and induction of differentiation of cells able to bind IL-2 and/or GM-CSF again. The two cytokines also generate cells responding to rhGM-CSF by colony formation. However, a difference with respect to morphology, phenotype and cytotoxic function of cells in the LTBMC, was noted between the two cytokines. Cells with large granular lymphocyte (LGL) morphology and cytotoxic activity against K562 and Daudi were generated only in the rhIL-2-supplemented LTBMC. This was compatible with a higher frequency of cells expressing the CD56+ phenotype in the IL-2-stimulated LTBMC as compared to the GM-CSF supplemented LTBMC. Our results also demonstrate the existence of a population of myeloid progenitor cells (CD33+) with ability to bind IL-2 in fresh bone marrow (BM).