Bovine Granulocyte-macrophage Colony-stimulating Factor Research Articles

Protective effects of engineered Lactobacillus johnsonii expressing bovine granulocyte-macrophage colony-stimulating factor on bovine postpartum endometritis.

Postpartum endometritis is a prevalent reproductive disorder in bovines, leading to a prolonged open period, infertility, and other complications. While Lactobacillus strains can mitigate these conditions by reducing uterine inflammation, their effectiveness is limited due to a lack of direct anti microbial action and extended treatment duration. This study aimed to construct a recombinant Lactobacillus johnsonii strain expressing bovine Granulocyte-macrophage colony-stimulating factor (GM-CSF) to evaluate its potential in reducing postpartum uterine inflammation. The recombinant Lactobacillus johnsonii strain was engineered to express bovine GM-CSF and administered to pregnant mice via vaginal perfusion. Postpartum endometritis was induced using E. coli infection, and the protective effects of the engineered strain were assessed. Inflammatory markers (IL-6, IL-1β, TNF-α), myeloperoxidase (MPO) activity, and nitric oxide (NO) concentration were measured. Histological examination was performed to evaluate uterine morphology and pathological damage. The recombinant L. johnsonii strain expressing GM-CSF significantly reduced inflammation levels induced by E. coli infection in the uterus. This reduction was evidenced by decreased expression of IL-6, IL-1β, TNF-α, as well as reduced MPO activity and NO concentration. Histological examination revealed improved uterine morphology and reduced pathological damage in mice treated with the recombinant GM-CSF strain. Crucially, the recombinant strain also exerts beneficial effects on bovine endometritis by reducing levels of inflammatory cytokines, suggesting a beneficial effect on clinical bovine endometritis. The recombinant Lactobacillus johnsonii expressing GM-CSF demonstrated protective effects against postpartum endometritis in bovines by reducing inflammatory cytokines. The findings indicate the potential clinical application of this engineered strain in preventing postpartum uterine inflammation, offering a novel and effective protective option for related disorders and improving bovine reproductive efficiency.

A Novel Quadruple Gene-Deleted BoHV-1-Vectored RVFV Subunit Vaccine Induces Humoral and Cell-Mediated Immune Response against Rift Valley Fever in Calves.

Rift Valley fever virus (RVFV) is considered to be a high biodefense priority based on its threat to livestock and its ability to cause human hemorrhagic fever. RVFV-infected livestock are also a significant risk factor for human infection by direct contact with contaminated blood, tissues, and aborted fetal materials. Therefore, livestock vaccination in the affected regions has the direct dual benefit and one-health approach of protecting the lives of millions of animals and eliminating the risk of severe and sometimes lethal human Rift Valley fever (RVF) disease. Recently, we have developed a bovine herpesvirus type 1 (BoHV-1) quadruple gene mutant virus (BoHV-1qmv) vector that lacks virulence and immunosuppressive properties due to the deletion of envelope proteins UL49.5, glycoprotein G (gG), gE cytoplasmic tail, and US9 coding sequences. In the current study, we engineered the BoHV-1qmv further by incorporating a chimeric gene sequence to express a proteolytically cleavable polyprotein: RVFV envelope proteins Gn ectodomain sequence fused with bovine granulocyte-macrophage colony-stimulating factor (GMCSF) and Gc, resulting in a live BoHV-1qmv-vectored subunit vaccine against RVFV for livestock. In vitro, the resulting recombinant virus, BoHV-1qmv Sub-RVFV, was replicated in cell culture with high titers. The chimeric Gn-GMCSF and Gc proteins expressed by the vaccine virus formed the Gn-Gc complex. In calves, the BoHV-1qmv Sub-RVFV vaccination was safe and induced moderate levels of the RVFV vaccine strain, MP12-specific neutralizing antibody titers. Additionally, the peripheral blood mononuclear cells from the vaccinated calves had six-fold increased levels of interferon-gamma transcription compared with that of the BoHV-1qmv (vector)-vaccinated calves when stimulated with heat-inactivated MP12 antigen in vitro. Based on these findings, we believe that a single dose of BoHV-1qmv Sub-RVFV vaccine generated a protective RVFV-MP12-specific humoral and cellular immune response. Therefore, the BoHV-1qmv sub-RVFV can potentially be a protective subunit vaccine for cattle against RVFV.

Effect of intramammary infusion of recombinant bovine GM-CSF and IL-8 on CMT score, somatic cell count, and milk mononuclear cell populations in Holstein cows with Staphylococcus aureus subclinical mastitis.

The effect of intramammary infusion of recombinant bovine granulocyte-macrophage colony-stimulating factor (rbGM-CSF) and interleukin-8 (rbIL-8) on mononuclear cell populations in quarters, somatic cell count (SCC) and the California Mastitis Test (CMT) score were investigated. From the selected cows with naturally occurring Staphylococcus aureus subclinical mastitis, one quarter of each cow were selected for the infusions of rbGM-CSF (400μg/5mL/quarter, n=9), rbIL-8 (1mg/5mL/quarter, n=9), and phosphate-buffered saline (5mL/quarter, n=7). The CMT score of both cytokines post infusion temporarily increased between days 0 and 1 and significantly decreased between days 7 and 14 compared to the preinfusion level. The SCC on day 14 after infusions of rbGM-CSF tended to be lower than that of the control group. The percentage of CD14+ cells increased on days 1 and 2 post infusion of rbGM-CSF. The percentage of CD4+ and CD8+ cells also increased on days 2 and 3, suggesting that the infusion of rbGM-CSF enhanced cellular immunity in the mammary gland. In contrast, the percentage of CD14+ cells decreased on days 0.25 and 1 post infusion of rbIL-8. No significant changes in the percentages of CD4+ and CD8+ cells in milk after infusion of rbIL-8 were evident during the experimental period, which suggested that rbIL-8 had little effect on the function of T cells in the mammary gland. These results indicated that rbGM-CSF and rbIL-8 decreased the CMT score by a different mechanism and may have a potential as therapeutic agents for subclinical mastitis.

Dairy cows produce cytokine and cytotoxic T cell responses following vaccination with an antigenic fraction from Streptococcus uberis

Streptococcus uberis is a major cause of mastitis in dairy cows worldwide and currently, there is no vaccine commercially available against this form of mastitis. In the current study, cell-free extracts (CFE) were prepared from each of three different S. uberis strains, designated as #3, #24 and #363 representative of the three main sequence types of S. uberis that cause mastitis in New Zealand. These proteins were formulated into vaccines with Emulsigen-D and the immunogenicity of the vaccines was determined in both calves and dairy cows. Two groups of calves (n=5/group) were vaccinated subcutaneously with CFE from strain #24 or strains #3, #24 and #363 formulated with Emulsigen-D, respectively. A third group (n=5) was vaccinated with CFE from the three strains formulated with Emulsigen-D and also containing recombinant bovine granulocyte macrophage colony-stimulating factor while, a control group (n=5) was not vaccinated. Vaccinated animals produced strong antibody responses to the S. uberis antigens and an antigen-specific cytotoxic effect against blood monocytes/macrophages that had phagocytosed S. uberis, with no significant differences in responses observed between the three vaccinated groups. In a second trial, the safety and immunogenicity of the vaccine containing CFE from all three strains of S. uberis and Emulsigen-D was determined in dairy cows. A group of six cows were vaccinated subcutaneously at 3 and 1 week prior to dry off and revaccinated 2–3 weeks before calving. Immune responses in blood and mammary gland secretions (MGS) were monitored during the dry period and in the subsequent lactation. The vaccine was well tolerated with no adverse effect from vaccination observed in any of the cows. Vaccination induced an antigen-specific cytotoxic effect against blood monocytes/macrophages that had phagocytosed S. uberis, moderate antigen-specific IFN-γ responses in blood and strong antibody responses in both blood and MGS. In conclusion, the results suggest vaccination of cattle with S. uberis CFE by the subcutaneous route can induce both cellular and humoral responses.

Isolation and characterization of macrophages from a mixed primary culture of bovine liver cells

Previously, we developed a simple and efficient method to isolate liver macrophages from a mixed primary culture of adult rat liver cells. To extend the applicability of this method, we isolated macrophages from mixed primary cultures of bovine liver cells. Macrophage cells proliferated on the cell sheet of mixed bovine liver cells after 8–16d of culture. These cells were detached by shaking of the culture flasks. Subsequent transfer and brief incubation in plastic dishes resulted in selective adhesion of macrophages. After rinses with PBS, attached macrophages were harvested. More than 106 cells could be harvested from the culture flask at intervals of 2–3d for more than three weeks. The isolated cells were strongly positive for bovine macrophage markers, such as CD68, CD172a and Iba-1. These cells exhibited functional properties of macrophages, including active phagocytosis of polystyrene microbeads, proliferative response to recombinant bovine granulocyte-macrophage colony-stimulating factor, upregulation of specific inflammatory cytokine genes upon stimulation with lipopolysaccharide, and formation of multinucleated giant cells. The shaking and attachment method provides a simple and efficient alternative to obtain bovine liver macrophages without requiring complex equipment or specialized technical skills.

Conservation and variation of the parapoxvirus GM-CSF-inhibitory factor (GIF) proteins

The GIF protein of orf virus (ORFV) binds and inhibits the ovine cytokines granulocyte-macrophage colony-stimulating factor (GM-CSF) and interleukin-2 (IL-2). An equivalent protein has so far not been found in any of the other poxvirus genera and we therefore investigated whether it was conserved in the parapoxviruses. The corresponding genes from both the bovine-specific pseudocowpox virus (PCPV) and bovine papular stomatitis virus (BPSV) were cloned and sequenced. The predicted amino acid sequences of the PCPV and BPSV proteins shared 88 and 37 % identity, respectively, with the ORFV protein. Both retained the six cysteine residues and the WSXWS-like motif that are required for biological activity of the ORFV protein. However, an analysis of the biological activity of the two recombinant proteins revealed that, whilst the PCPV GIF protein bound to both ovine and bovine GM-CSF and IL-2 with very similar binding affinities to the ORFV GIF protein, no GM-CSF- or IL-2-binding activity was found for the BPSV protein.

Development of efficient method for purified recombinant bovine granulocyte-macrophage colony-stimulating factor production with baculovirus-silkworm gene expression system

Recombinant bovine granulocyte-macrophage colony-stimulating factor (rboGM-CSF) was produced by the baculovirus-silkworm expression system. It was purified to 98% by (NH(4))(2)SO(4), followed by a three-step column chromatography with silica gel, ion exchange resin and a metal chelate column. The specific activity of purified rboGM-CSF was 1.6-6.3 x 10(6) ED(50) mg(-1). By this method, the specific activity was raised 160-fold and 21% of the expressed rboGM-CSF was recovered.

Effect of recombinant cytokines on leucocytes and physiological changes in bovine mammary glands during early involution.

We examined the effects of administering recombinant bovine cytokines to non-lactating dairy cows and measured mammary gland leucocytes and the involution process. After the final milking, groups of cows were given an intramammary infusion of cytokine in two quarters. These cytokines were recombinant bovine interleukin-2 (rbolL-2) (2 x 10(5) units, n = 6), recombinant bovine granulocyte-macrophage colony stimulating factor (rboGM-CSF) (500 microg, n = 4) and recombinant bovine interleukin-1beta (rbolL-1beta) (10 microg, n = 10). Each animal also received an infusion of phosphate-buffered saline (PBS) in the other two quarters as controls. The rbolL-2 and rboGM-CSF were produced in a yeast expression system, while rbolL-1beta was produced in Escherichia coli. Leucocyte numbers, bactericidal activity of leucocytes, and concentrations of citrate and lactoferrin in quarter secretion samples were monitored after infusion of cytokine or PBS. Infusion of rbolL-2 had minimal effect on leucocyte numbers and concentrations of citrate and lactoferrin. Both rboGM-CSF and rbolL-1beta induced a rapid increase in the number of neutrophils and macrophages compared with control PBS quarters. Concentrations of lactoferrin in secretions were increased by rboGM-CSF and rbolL-1beta compared with control PBS quarters. In addition, infusion of glands with rbolL-1beta lowered the citrate:lactoferrin molar ratio compared with PBS control quarters. The results indicate that intramammary infusion of either rboGM-CSF or rbolL-1beta at cessation of milking immediately increased the number of phagocytic cells in the gland. These cytokines, in particular rbolL-1beta, also increased the rate of mammary gland involution during the early dry period.

Molecular cloning and expression of woodchuck granulocyte‐macrophage colony stimulating factor*

Granulocyte-macrophage colony stimulating factor (GM-CSF) has immunoregulatory and antiviral effects, and may thus be promising for the treatment of chronic hepatitis B. Using woodchuck hepatitis virus (WHV)-infected woodchuck as an animal model to test the efficacy and safety of GM-CSF on the therapy of chronic hepatitis B, woodchuck GM-CSF will be required due to the apparent species-specific activity of GM-CSF. The cDNA of woodchuck GM-CSF was cloned using reverse transcription-polymerase chain reaction (RT-PCR) with primers deriving from highly conserved regions of GM-CSF genes from other species. The deduced amino acids, including the signal peptide, is 138 in length and its identities to human, murine, canine and bovine GM-CSFs are 63, 49, 63, and 63% respectively. The genomic DNA of woodchuck GM-CSF was also cloned by PCR. Its organization is highly homologous to that of human and murine GM-CSF genes, consisting of four exons and three introns. Cloned woodchuck GM-CSF was expressed transiently in 293T cells. The recombinant protein expressed was found to stimulate the growth and differentiation of woodchuck bone marrow cells, indicating the protein expressed by the cloned gene is functional. These results pave the way for future studies on the potential role of GM-CSF for the treatment of chronic hepatitis B by using this animal model.

Flt-3 ligand, in combination with bovine granulocyte-macrophage colony-stimulating factor and interleukin-4, promotes the growth of bovine bone marrow derived dendritic cells.

Culture of bone marrow precursor cells with cytokines, including granulocyte-macrophage colony-stimulating factor (GM-CSF), interleukin-4 (IL-4) and the tyrosine kinase receptor binding proteins Flt-3 ligand (Flt-3L) and stem-cell factor (SCF), has previously been shown, in both mouse and human, to result in the generation of large numbers of dendritic cells. We extend these findings to bovine dendritic cells. Culture of bovine bone marrow cells with GM-CSF, IL-4 and either Flt-3L or SCF enhanced the generation of low buoyant-density dendritic cells. However, only the addition of Flt-3L to cells cultured with GM-CSF and IL-4 was shown to increase the number of dendritic cells and induce the differentiation of dendritic cells with potent capacity to stimulate allogeneic T cells and resting CD4+ memory T cells. The effective ability to stimulate T cells was associated with the expression of major histocompatibility complex (MHC) class II molecules and CD80/86 by dendritic cells. Bovine bone marrow derived dendritic cells appeared to be exclusively of myeloid origin because they expressed the myeloid-related antigens CD14, MyD-1 and CD11b.

Expression of granulocyte-macrophage colony-stimulating factor (GM-CSF) receptor on B-1a cell from persistent lymphocytosis (PL) cows and lymphoma cell induced by bovine leukemia virus

The effect of granulocyte-macrophage colony-stimulating factor (GM-CSF) on B lymphocytes from persistent lymphocytosis (PL) cattle and lymphoma cells induced by bovine leukemia virus (BLV) was studied in vitro. Flow cytometric analysis showed that high levels of receptors to GM-CSF were expressed on these cell types. Proliferation of these B cells was induced in response to bovine GM-CSF. In tumor cell lines, the rate of cell proliferation was correlated with expression of GM-CSF receptors. A monoclonal antibody to GM-CSF inhibited lymphocyte proliferation and blocked the GM-CSF binding of lymphocytes. Cells expressing GM-CSF receptor were Ig positive and both CD5 and CD11 positive (B-1a cell). These results suggest that an abnormal expression of GM-CSF receptors on B lymphocytes from PL and lymphoma cells induced by BLV plays important roles in the PL and proliferation of lymphoma.

Effects of recombinant bovine granulocyte-macrophage colony-stimulating factor on bovine peripheral blood neutrophil functions in vitro and in vivo.

Effects of recombinant bovine granulocyte-macrophage colony-stimulating factor (rboGM-CSF) on bactericidal activity of bovine peripheral blood neutrophils in vitro and in vivo were studied. In in vitro experiment, bovine blood neutrophils were cultured for 9 hr in media containing 0.005, 0.05 or 0.5 microg/ml of rboGM-CSF. Neutrophils treated with rboGM-CSF showed significantly higher luminol-dependent chemiluminescence (LDCL) than control cells. In in vivo experiment, neutrophils isolated from cows injected 5.0 microg/kg of rboGM-CSF showed significantly higher Nitrobluetetrazolium (NBT) reduction value than that from control cows 24 hr post injection. Total leukocyte counts of cows injected rboGM-CSF sharply decreased 6 hr post injection and recovered to normal level 2 days post injection. Body temperature of these cows rose 6 hr post injection and back to normal level at 24 hr post injection. It was suggested that rboGM-CSF enhanced bactericidal activity of bovine neutrophils both in vitro and in vivo.

Interleukin-1 receptor mRNA expression in activated bovine leukocytes in vitro.

Interleukin-1 (IL-1) is a key player in inflammation and the immune response. To better understand the complex interactions of IL-1 and its receptors in inflammation, we need to investigate how type I and type II IL-1 receptors (IL-1RI and IL-1RII) are regulated by cytokines and other mediators. Using semiquantitative reverse transcriptase PCR and Northern analysis, we examined the regulation of IL-1RI and IL-1RII mRNA levels in bovine polymorphonuclear leukocytes (PMNs) (i.e., neutrophils) and peripheral blood mononuclear cells (PBMCs) in vitro. IL-1RI mRNA levels were up-regulated in PBMCs by recombinant bovine IL-1beta (rBoIL-1beta), recombinant bovine granulocyte-macrophage colony-stimulating factor (rBoGM-CSF), rBoIL-4, recombinant bovine gamma interferon (rBoIFN-gamma), and dexamethasone. IL-1RI mRNA was increased in bovine PMNs exposed to rBoGM-CSF, rBoIL-4, and dexamethasone but was down-regulated by rBoIL-1beta and rBoIFN-gamma. IL-1RII mRNA was increased in bovine PBMCs and PMNs after exposure to rBoIL-1beta, rBoGM-CSF, rBoIL-4, and dexamethasone. In contrast, rBoIFN-gamma down-regulated the expression of bovine IL-1RII mRNA in PBMCs. These findings suggest that the expression of bovine IL-1RI and IL-1RII mRNAs is regulated differently by certain soluble stimuli (e.g., IFN-gamma) in PMNs and PBMCs.

Granulocyte-macrophage colony-stimulating factor promotes development of in vitro produced bovine embryos.

The objective was to determine whether granulocyte-macrophage colony-stimulating factor (GM-CSF) stimulates development of bovine embryos. In each experiment, oocytes were fertilized in vitro, GM-CSF was added to embryo culture medium at 8-10 h or 5 days after insemination, and development was monitored as the proportion of oocytes that formed blastocysts. Addition of recombinant bovine GM-CSF to serum-free medium at 8-10 h after insemination increased the percentage of oocytes that formed blastocysts (7.2% and 15.2% for control and GM-CSF, respectively). GM-CSF did not affect cleavage rate. Rather, the effect of GM-CSF seems to be exerted after Day 5 after insemination, as indicated by the following findings: 1) GM-CSF did not alter embryo cell number at Day 5 after insemination; 2) administration of GM-CSF at Day 5 increased the proportion of oocytes that developed to the blastocyst stage (6.7%, 13.0%, and 22.4% for control and 1 and 10 ng/ml GM-CSF, respectively); and 3) addition of serum at Day 5 increased development but prevented a further increase due to addition of GM-CSF at 10 h after insemination. Blastocysts from GM-CSF-treated cultures tended to be at earlier stages of morphological development (i.e., fewer blastocysts expanded at Day 7 and fewer were hatching or hatched at Day 9 after insemination). GM-CSF may play a role in the early development of bovine embryos and might be a useful molecule for increasing blastocyst production rates in serum-free culture systems.

Lack of effect of granulocyte-macrophage colony-stimulating factor on secretion of interferon-τ, other proteins, and prostaglandin E 2 by the bovine and ovine conceptus

Three experiments tested the effects of recombinant bovine granulocyte-macrophage colony-stimulating factor (rbGM-CSF) on the preimplantation bovine and ovine conceptus. There was no effect of rbGM-CSF on the secretion of total radiolabeled protein in conditioned medium, immunoreactive interferon-τ (IFNτ), antiviral activity, or prostaglandin E 2 from Day 16–18 bovine conceptuses cultured for 24 hr with [ 3H]leucine and ±10 ng/ml rbGM-CSF. Similarly, there was no effect of 1 ng/ml rbGM-CSF on the secretion of total radiolabeled protein, IFNτ, or antiviral activity from Day 17 ovine conceptuses. There was also no beneficial effect of 1 or 10 ng/ml rbGM-CSF on the presence of immunoreactive IFNτ in conditioned medium from in vitro-produced bovine blastocysts at Day 7–8 after fertilization. Results indicate that IFNτ secretion from bovine and ovine conceptuses are unresponsive to rbGM-CSF at the concentrations tested.

Monomeric bovine IgG2 is a potent stimulus for bovine neutrophils

Bovine plasma contains factors that can stimulate bovine neutrophils. Bovine plasma at 1:1,000,000-1:1,000 dilution induced a dose-dependent superoxide production in bovine neutrophils. When bovine plasma was fractionated with a protein G column, only the IgG fraction contained induction activity. At similar concentrations purified IgG induced a much stronger response than that of plasma IgG. Purified monomeric bovine IgG induced a dose-dependent increase in superoxide production. The maximum induction can be achieved at 100 micrograms/ml of bovine IgG. When subclasses of bovine IgG were examined, monomeric bovine IgG2 potently stimulated bovine polymorphonuclear leukocytes. In contrast, bovine IgG 1 failed to induce a response at similar concentrations, and neither bovine IgG F(ab')2 and Fc were effective. Both recombinant bovine granulocyte-macrophage colony-stimulating factor (r-BoGM-CSF) and recombinant bovine granulocyte colony-stimulating factor (r-BoG-CSF) primed bovine neutrophils for superoxide production induced by bovine IgG. The above results suggest that: (1) bovine plasma contain factors that can activate bovine neutrophils; (2) bovine plasma IgG is the major component that is responsible for bovine neutrophil activation; (3) bovine plasma contains factors that can inhibit the effect of bovine IgG; (4) monomeric bovine IgG2, but not IgG1, can activate bovine neutrophils directly without Fc receptor cross-linkage; (5) the integrity of bovine IgG is important in bovine polymorphonuclear leukocyte activation; and (6) bovine neutrophil activation induced by bovine IgG can be primed by r-BoGM-CSF or r-BoG-CSF.

Culture of ovine bone marrow-derived macrophages and evidence for serum factors distinct from M-CSF contributing to their propagation in vitro.

An in vitro system allowing the culture of ovine bone marrow-derived macrophages (BMMs) is described. Bone marrow (BM) cells from the sternum of 4- to 9-month-old sheep were cultured in liquid suspension in hydrophobic bags with medium containing 20% autologous serum and 20% fetal calf serum (FCS). Cells with macrophage characteristics were positively selected and increased four- to five-fold between day (d) 0 and d18. Granulocytes and cells of lymphoid appearance including progenitor cells were negatively selected and were diminished 50-fold during this 18-d culture. The addition of macrophage colony-stimulating factor (M-CSF)-containing supernatants to liquid cultures did not significantly improve the yield of BMM in 18-d cultures. In contrast, cell survival at d6 and macrophage cell yield at d18 depended on the concentration and source of serum in the culture medium. FCS and 1:1 mixtures of FCS and autologous serum were superior to autologous serum alone. Analysis of growth requirements of ovine BMMs suggested that they are under more complex growth control than their murine counterparts. In an [3H]thymidine incorporation assay with BM cells collected at different times of culture, d3 or d4 BM cells responded to human recombinant M-CSF, human recombinant granulocyte-macrophage colony-stimulating factor (GM-CSF), bovine GM-CSF, murine M-CSF or murine M-CSF-containing supernatants, and bovine interleukin 1 beta (IL-1 beta) in decreasing order of magnitude. Likewise, pure murine BMM populations harvested at d6 responded to homologous GM-CSF, IL-3, and human or murine M-CSF. FCS did not stimulate the proliferation of murine BMMs (d6) and of ovine BM cells (d3 or d4). In contrast, ovine BM cells harvested at d12 responded to FCS by proliferation in a dose-dependent manner but failed to proliferate in the presence of human or murine M-CSF or M-CSF-containing supernatants of mouse and sheep fibroblasts containing mouse macrophage growth-promoting activity. Likewise, various cytokine-containing supernatants and recombinant cytokines (murine IL-3, murine and human GM-CSF, murine and bovine IL-1 beta) did not promote proliferation of ovine d12 BM cells to an extent greater than that achieved with 15% FCS alone. Thus, ovine BMM proliferation is under the control of at least two factors acting in sequence, M-CSF and an unidentified factor contained in FCS. The ovine BMM culture system may provide a model for the analysis of myelomonocytopoiesis in vitro.

Cloning and sequencing of the cDNA for ovine granulocyte-macrophage colony-stimulating factor (GM-CSF)

Colony-stimulating factors (CSFs) are not only regulators of haemopoiesis but can also enhance the function of mature myeloid cells, and are therefore potential immune adjuvants. By use of the polymerase chain reaction (PCR) with primers based on the bovine granulocyte-macrophage CSF (GM-CSF) sequence, we have amplified the cDNA for ovine GM-CSF, produced from crude mRNA extracted from alveolar macrophages. The PCR product was cloned into pUC119, and electroporated into Escherichia coli. The complete nucleotide sequence of two clones, and the partial sequence of eight others, was determined. At the nucleotide and amino acid levels, the ovine and bovine GM-CSF sequences are 91% and 81% homologous, respectively.

Cloning and expression of the cDNA for bovine granulocyte-macrophage colony-stimulating factor

A sequence encoding bovine granulocyte-macrophage colony-stimulating factor (GM-CSF) has been identified from a concanavalin A-stimulated bovine lymphocyte cDNA library. This sequence was isolated by hybridization with synthetic oligonucleotide probes based upon the human GM-CSF sequence. This bovine cDNA was engineered for expression and secretion of activity into the periplasmic space of E. coli. Periplasmic extracts contain a 14 500-dalton protein and stimulate colony formation of bovine bone marrow progenitor cells. The predicted protein is 70% homologous with human GM-CSF and 55% homologous with murine GM-CSF. Numerous structural features are conserved among these three proteins, such as location of cysteine residues, glycosylation sites, and overall charge. The biological activity of bovine GM-CSF is species specific, since recombinant preparations do not cause proliferation of human or murine bone marrow cells. Similarly, murine GM-CSF does not exhibit activity on cells of bovine or human origin. However, human GM-CSF does stimulate colony formation of bovine bone marrow cells, although the specific activity appears reduced when compared to assays on human cells.

Bovine GM-CSF: Molecular cloning and biological activity of the recombinant protein

The lymphokine granulocyte-macrophage colony-stimulating factor (GM-CSF) mediates the growth and differentiation of granulocytes and macrophages from bone marrow progenitors, and regulates biological functions expressed by mature cells of these lineages. In order to isolate a bovine GM-CSF cDNA, a cDNA library, generated from the BT2 bovine T cell line, was screened with a human GM-CSF cDNA probe. A cDNA clone was isolated with an insert of 783 bp, that would encode a protein of 143 amino acids, with a predicted mol. wt of 16,160. Bovine GM-CSF exhibits a high degree of sequence homology with mouse and human GM-CSF at both the nucleotide and amino acid levels. Comparison of GM-CSF amino acid sequences from the three species indicates that the bovine GM-CSF precursor contains a putative 17 amino acid signal sequence, cleavage of which would yield a 14,250 mol. wt protein. The cDNA was inserted into a mammalian expression vector and transfected into COS-7 monkey kidney cells. Biologically active bovine GM-CSF was secreted as judged by a bovine bone marrow proliferation assay. Bovine GM-CSF was weakly active in both human and mouse bone marrow proliferation assays. In contrast, human GM-CSF was weakly active on bovine but not murine mouse bone marrow cells and mouse GM-CSF was only active on murine bone marrow cells.