Granulocyte-macrophage Colony-stimulating Factor Supplementation Research Articles

Granulocyte-Macrophage Colony-Stimulating Factor Cytokine Addition After the Freeze-Thawing Process Improves Human Sperm Motility and Vitality in Asthenoteratozoospermia Patients.

The cryopreservation-thawing process of spermatozoa cells has negative impacts on their structure, function, and fertility parameters, which are known as cryoinjury. Asthenozoospermia patients are more susceptible to cryoinjury. Granulocyte-macrophage colony-stimulating factor (GM-CSF) increases sperm glucose uptake via the induction of glucose transporters, resulting in increased sperm motility. This study aimed to investigate the efficiency of GM-CSF supplementation of the cryopreservation media for semen samples of asthenoteratozoospermia patients. The study was carried out on 20 semen samples from infertile men referred to diagnosing semen analysis. To avoid subjective bias, two main sperm motility parameters, including velocity along the curvilinear path and velocity along the straight-line path were considered by the computer-assisted sperm analysis system. Afterward, each semen sample was divided into three equal aliquots and randomly assigned to one of the following groups: group I (control, freezing media only), group II (+GM-CSF, freezing medium supplemented with 2 μL/mL GM-CSF), or group III (GM-CSF added after thawing and washing). Following semen thawing, standard parameters, mitochondrial membrane potential (MMP), and the DNA Fragmentation Index were analyzed. Total sperm motility (progressive and non-progressive) improved significantly in group III samples after a 30-minute incubation with GM-CSF compared with the control group (26.5% ± 3.1% vs. 17.51% ± 2.59%). However, no differences in progressive motility or sperm morphology were found among the three thawed samples. The percentage of vitality was significantly higher in group III compared with the other two groups (28.38% ± 3.4% vs. 22.4% ± 3.08% and 22.14% ± 2.77%, respectively) (p < 0.05). JC-1 levels (a marker of MMP) were not significantly different between the examined groups (44.95% ± 8.26% vs. 36.61% ± 6.95% vs. 46.67% ± 7.7%, for control, group II, and group III, respectively) (p > 0.05). GM-CSF may be advantageous as an additive after freezing, improving total motility and viability after 30 minutes of post-thaw incubation; however, when supplied to the freezing media before cryopreservation, it is unable to protect against cryoinjury.

Short communication: Effect of granulocyte-macrophage colony-stimulating factor on neonatal calf peripheral blood neutrophil function in vitro

Neutrophils are innate immunity cells that represent the first line of cellular defense against invading pathogens. Dairy calves, however, experience neutrophil dysfunction during the first weeks of age, contributing to increased disease susceptibility during this period. Granulocyte-macrophage colony-stimulating factor (GM-CSF) is a cytokine that improves neutrophil function in neonates of other species and mature cows. However, its capability to improve neonatal calf neutrophil function is unknown. Therefore, our objective was to evaluate the effect of GM-CSF on the functional capabilities of neutrophils of neonatal calves in vitro. We hypothesized that supplementation of neonatal neutrophils with GM-CSF would increase microbicidal functions to levels comparable with those of mature immunocompetent cattle. For this, we isolated blood neutrophils from 12 healthy 2- to 3-d-old Holstein calves, and neutrophils from 6 mid-lactation Holstein cows were used as a reference of robust neutrophil function. Subsequently, neutrophils from both calves and cattle were incubated for 9 h with 4 concentrations (0, 0.005, 0.05, or 0.5 µg/mL) of GM-CSF, and microbicidal function of neutrophils was assessed in terms of phagocytosis, respiratory burst, myeloperoxidase (MPO) activity, and extracellular trap formation. Mixed models with Tukey pairwise comparisons were used to identify differences among treatment and age groups. Supplementation of GM-CSF in vitro increased phagocytosis and MPO activity of calf and cow neutrophils, although not in a concentration-dependent manner. Respiratory burst and extracellular trap formation were not affected by GM-CSF supplementation. All the microbicidal capacity functions assessed were lower in neutrophils from calves, but supplementation with GM-CSF increased phagocytosis and MPO activity of calf neutrophils to levels comparable with unsupplemented cow neutrophils. Collectively, our results demonstrated that in vitro supplementation of calf neutrophils with GM-CSF enhanced some functional microbicidal capabilities to levels comparable with immunocompetent cattle. Hence, it may be possible to augment the functional capacity of calf neutrophils in vivo through the therapeutic application of GM-CSF and consequently enhance calves' resistance to infections. This should be tested in future in vivo studies.

Relationship between granulocyte–macrophage colony-stimulating factor, embryo quality, and pregnancy outcomes in women of different ages in fresh transfer cycles: a retrospective study

This study aimed to explore the effects of low-concentration (0.6 ng/mL) granulocyte–macrophage colony-stimulating factor (GM-CSF) supplementation on human embryo quality and pregnancy outcomes in patients with fresh transfer cycles. The data were retrospectively collected from 719 hyperstimulation cycles of 631 patients divided into two groups: GM-CSF supplementation (0.6 ng/mL, n = 399) and control (n = 320). The embryo quality and pregnancy outcomes were compared. GM-CSF addition significantly increased the available embryo rate (52.0 vs. 48.1%, p < .05). In patients >38 years, it significantly enhanced cleavage (99.4 vs. 97.8%, p < .05) and blastocyst formation (45.7 vs. 34.9%, p < .05) rates but not pregnancy outcomes, including clinical pregnancy (power = 0.160) and implantation (power = 0.204) rates. The lack of a statistically significant difference could be related to low study power. These results suggest that low-concentration GM-CSF addition contributes to embryo quality improvement, especially in patients >38 years.IMPACT STATEMENTWhat is already known on this subject? Granulocyte–macrophage colony-stimulating factor (GM-CSF) has a beneficial effect on the development of human embryos in assisted reproductive technology.What do the results of this study add? Adding 0.6 ng/mL GM-CSF significantly increased the available embryo rate. In patients over 38 years of age, it statistically significantly enhanced the cleavage rate (99.4 vs. 97.8%, p < .05) and blastocyst formation rate (45.7 vs. 34.9%, p < .05).What are the implications of these findings for clinical practice and/or further research? GM-CSF benefits embryos with poorer development potential and a randomised clinical trial with a larger sample size should be performed.

GM-CSF Grown Bone Marrow Derived Cells Are Composed of Phenotypically Different Dendritic Cells and Macrophages.

Granulocyte-macrophage colony stimulating factor (GM-CSF) has a role in inducing emergency hematopoiesis upon exposure to inflammatory stimuli. Although GM-CSF generated murine bone marrow derived cells have been widely used as macrophages or dendritic cells in research, the exact characteristics of each cell population have not yet been defined. Here we discriminated GM-CSF grown bone marrow derived macrophages (GM-BMMs) from dendritic cells (GM-BMDCs) in several criteria. After C57BL/6J mice bone marrow cell culture for 7 days with GM-CSF supplementation, two main populations were observed in the attached cells based on MHCII and F4/80 marker expressions. GM-BMMs had MHCIIlowF4/80high as well as CD11c+CD11bhighCD80−CD64+MerTK+ phenotypes. In contrast, GM-BMDCs had MHCIIhighF4/80low and CD11chighCD8α− CD11b+CD80+CD64−MerTKlow phenotypes. Interestingly, the GM-BMM population increased but GM-BMDCs decreased in a GM-CSF dose-dependent manner. Functionally, GM-BMMs showed extremely high phagocytic abilities and produced higher IL-10 upon LPS stimulation. GM-BMDCs, however, could not phagocytose as well, but were efficient at producing TNFα, IL-1β, IL-12p70 and IL-6 as well as inducing T cell proliferation. Finally, whole transcriptome analysis revealed that GM-BMMs and GM-BMDCs are overlap with in vivo resident macrophages and dendritic cells, respectively. Taken together, our study shows the heterogeneicity of GM-CSF derived cell populations, and specifically characterizes GM-CSF derived macrophages compared to dendritic cells.

Intranodal Immunization With a Vaccinia Virus Encoding Multiple Antigenic Epitopes and Costimulatory Molecules in Metastatic Melanoma

Recombinant vaccinia virus (rVV) encoding tumor-associated antigens (TAAs) and adhesion or costimulatory molecules may represent important immunogenic reagents for cancer immunotherapy. Recently, intranodal (IN) antigen administration was suggested to be more immunogenic than intradermal (ID) vaccination. However, IN rVV administration has not been attempted so far. We used a rVV encoding gp100(280-288), Melan-A/MART-1(27-35) and tyrosinase(1-9) HLA-A0201 restricted epitopes and CD80 and CD86 costimulatory molecules in stage III and IV melanoma patients in a phase 1/2 trial. Of 15 patients initiating treatment, including two cycles of IN immunization, each comprising one rVV administration and three recall injections of the corresponding peptides, accompanied by subcutaneous granulocyte macrophage-colony stimulating factor supplementation, five withdrew due to progressing disease. Of 10 remaining patients seven showed evidence of induction of cytotoxic T lymphocytes (CTLs) directed against at least one epitope under investigation, as detectable by limiting dilution analysis (LDA) of specific precursors and multimer staining. Adverse reactions were mild (National Cancer Institute (NCI) grade 1-2) and mainly represented by fever, skin rashes, and pruritus. These data indicate that IN administration of rVV encoding melanoma-associated epitopes and costimulatory molecules is safe and immunogenic.

Granulocyte‐Macrophage Colony‐Stimulating‐Factor Increases Interferon‐τ Protein Secretion in Bovine Trophectoderm Cells

Uterine-derived factors are required for optimal conceptus development and secretion of the maternal recognition of pregnancy factor, interferon-tau (IFN-tau). Identifying these factors may lead to the development of schemes for increasing pregnancy success in cattle. The objectives were to examine the effects of granulocyte-macrophage colony-stimulating-factor (GM-CSF) on trophectoderm proliferation rates and IFN-tau production, and verify the appropriateness of using an in vitro model of bovine trophectoderm (CT-1 cell). Rate of [(3)H]-thymidine incorporation into DNA was increased by supplementation of CT-1 medium with 10 or 100 ng/mL porcine (po) GM-CSF. GM-CSF supplementation to CT-1 medium also increased IFN-tau secretion. When results were normalized to account for number of CT-1 cells, 10 and 100 ng/mL poGM-CSF increased antiviral activity and IFN-tau concentrations (using an IFN-tau-specific enzyme-linked immunosorbent assay) in CT-1 conditioned medium compared with controls. These findings indicate that GM-CSF increases proliferation and IFN-tau production in bovine trophectoderm.

Enhanced interleukin-8 production in THP-1 human monocytic cells by lipopolysaccharide from oral microorganisms and granulocyte-macrophage colony-stimulating factor.

Granulocyte-macrophage colony-stimulating factor (GM-CSF) has been used to assist in bone marrow recovery during cancer chemotherapy. Interleukin-8 (IL-8) plays an important role in macrophage mediated inflammatory processes including exacerbation of periodontal diseases, one of the most common complications in GM-CSF receiving cancer patients. The effect of GM-CSF supplementation on IL-8 production was investigated in a human monocyte cell line THP-1, stimulated with lipopolysaccharide extracted from two oral microorganisms, Porphyromonas gingivalis and Fusobacterium nucleatum. Resting THP-1 cells were treated with lipopolysaccharide (1 microgram/ml) of P. gingivalis or F. nucleatum and/or GM-CSF (50 IU/ml) for varying time periods. The production of IL-8 in THP-1 cells was measured by a solid-phase enzyme-linked immunosorbent assay (ELISA). A very low level of the cytokine IL-8 was produced constitutive in THP-1 cells. Starting from 8 h of treatment and afterwards GM-CSF alone significantly increased IL-8 production in THP-1 cells. Lipopolysaccharide (1 microgram/ml) extracts from either F. nucleatum or P. gingivalis amplified IL-8 production 500-800 times in comparison to resting THP-1 cells. When lipopolysaccharide of F. nucleatum or P. gingivalis was supplemented with 50 IU/ml of GM-CSF, there was a statistically significant enhanced production of IL-8 by THP-1 cells after 1 day to 7 days of treatment as compared with lipopolysaccharide treatment alone. GM-CSF (50 IU/ml) also significantly increased IL-8 production from 2-7 days of treatment of THP-1 cells when supplemented with a positive control, phorbol-12-myristate-13 acetate (PMA), as compared to PMA treatment alone. These investigations using the in vitro THP-1 human monocyte cell model indicate that there may be an increase in the response on a cellular level to oral endotoxin following GM-CSF therapy as evidenced by enhanced production of the tissue-reactive inflammatory cytokine, IL-8.

Interleukin-6 production by human monocytes treated with granulocyte-macrophage colony-stimulating factor in the presence of lipopolysaccharide of oral microorganisms.

This study focused on the effect of granulocyte-macrophage colony-stimulating factor (GM-CSF) and lipopolysaccharide of the putative periodontal pathogens Porphyromonas gingivalis or Fusobacterium nucleatum on IL-6 production by THP-1 cells (a human monocytic cell line). Resting THP-1 cells were alternatively treated with GM-CSF (50 IU/ml) and lipopolysaccharide of P. gingivalis or F. nucleatum, in varying concentrations for varying time periods. IL-6 production in supernatant fluids of treated cells was evaluated by an enzyme-linked immunosorbent assay (ELISA) and a reverse transcription polymerase chain reaction (RT-PCR) was used to evaluate gene expression. Untreated THP-1 cells did not produce IL-6 as determined by ELISA. RT-PCR also failed to detect IL-6 mRNA in untreated THP-1 cells, indicating that IL-6 was not constitutively produced. After stimulation of THP-1 cells with lipopolysaccharide of F. nucleatum or P. gingivalis, IL-6 was produced, peaking at 4 h (200-300 pg/ml) and thereafter sharply declining by 8 h. When GM-CSF was added together with lipopolysaccharide of P. gingivalis or F. nucleatum, there was a synergistic quantitative increase in production of IL-6 as measured by ELISA as compared with lipopolysaccharide alone. IL-6 mRNA was detected by RT-PCR, 15 min after stimulation with lipopolysaccharide of either P. gingivalis or F. nucleatum. GM-CSF supplementation with lipopolysaccharide of P. gingivalis shortened the transcription of IL-6 mRNA to 5 min, a shift which was not observed with lipopolysaccharide of F. nucleatum, possibly indicating a different mechanism of initiation of transcription. Production of IL-6 by GM-CSF-treated THP-1 cells in the presence of lipopolysaccharide of oral microorganisms may provide a model for studying the role of macrophages in acute and chronic periodontal diseases, including the clinical periodontal exacerbation as observed in chemotherapy patients receiving GM-CSF for bone marrow recovery.

Granulocyte-macrophage colony-stimulating factor amplification of interleukin-1beta and tumor necrosis factor alpha production in THP-1 human monocytic cells stimulated with lipopolysaccharide of oral microorganisms.

Cytokines, including granulocyte-macrophage colony-stimulating factor (GM-CSF), are used to assist in bone marrow recovery during cancer chemotherapy. Interleukin-1beta (IL-1beta) and tumor necrosis factor alpha (TNF-alpha) play important roles in inflammatory processes, including exacerbation of periodontal diseases, one of the most common complications in patients who undergo this therapy. A human monocyte cell line (THP-1) was utilized to investigate IL-1beta and TNF-alpha production following GM-CSF supplementation with lipopolysaccharide (LPS) from two oral microorganisms, Porphyromonas gingivalis and Fusobacterium nucleatum. LPS of P. gingivalis or F. nucleatum was prepared by a phenol-water extraction method and characterized by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and determination of total protein and endotoxin contents. Resting THP-1 cells were treated with LPS of P. gingivalis or F. nucleatum and/or GM-CSF (50 IU/ml) by using different concentrations for various time periods. Production of IL-1beta and TNF-alpha in THP-1 cells was measured by solid-phase enzyme-linked immunosorbent assay. Reverse transcription (RT)-PCR was used to evaluate the gene expression of resting and treated THP-1 cells. IL-1beta was not detected in untreated THP-1 cells. IL-1beta production was, however, stimulated sharply at 4 h. GM-CSF amplified IL-1beta production in THP-1 cells treated with LPS from both oral anaerobes. No IL-1beta-specific mRNA transcript was detected in untreated THP-1 cells. However, IL-1beta mRNA was detected by RT-PCR 2 h after stimulation of THP-1 cells with LPS from both organisms. GM-CSF did not shorten the IL-1beta transcriptional activation time. GM-CSF plus F. nucleatum or P. gingivalis LPS activated THP-1 cells to produce a 1.6-fold increase in TNF-alpha production at 4 h over LPS stimulation alone. These investigations with the in vitro THP-1 model indicate that there may be an increase in the cellular immune response to oral endotoxin following GM-CSF therapy, as evidenced by production of the tissue-reactive cytokines IL-1beta and TNF-alpha.

Granulocyte-macrophage colony-stimulating factor enhances macrophage accessory function in Con A-stimulated T-cell proliferation

Granulocyte-macrophage colony-stimulating factor (GM-CSF) has been reported to augment various macrophage (Mφ) functions, including antigen presentation in the antibody-producing response. We investigated the augmentative effect of GM-CSF on Mφ A-cell activity in concanavalin A-stimulated T-cell proliferation. Pretreatment with GM-CSF of peritoneal Mφ enhanced the T-cell proliferative response. This effect of GM-CSF was dose dependent and GM-CSF supplementation was needed at the beginning of Mφ culture. We observed that GM-CSF induced Mφ spreading and firm attachment accompanied with enlargement of the cytoplasm, but could not induce de novo expression of Ia antigen. GM-CSF treatment enabled Mφ to produce more interleukin (IL)-1 and IL-6 upon stimulation with lipopolysaccharides or polyinosinic-polycytidylic acid, but was unable to stimulate Mφ directly. This was confirmed by Northern blot analysis. These results indicate that GM-CSF augments Mφ A-cell activity through the enhancement of the capacity of Mφ to produce IL-1 and IL-6.