Canine Granulocyte-macrophage Colony-stimulating Factor Research Articles

Functional Granulocyte-Macrophage Colony-Stimulating Factor (GM-CSF) Delivered by Canine Histiocytic Sarcoma Cells Persistently Infected with Engineered Attenuated Canine Distemper Virus.

The immune response plays a key role in the treatment of malignant tumors. One important molecule promoting humoral and cellular immunity is granulocyte-macrophage colony-stimulating factor (GM-CSF). Numerous successful trials have led to the approval of this immune-stimulating molecule for cancer therapy. However, besides immune stimulation, GM-CSF may also accelerate tumor cell proliferation, rendering this molecule a double-edged sword in cancer treatment. Therefore, detailed knowledge about the in vitro function of GM-CSF produced by infected tumor cells is urgently needed prior to investigations in an in vivo model. The aim of the present study was to functionally characterize a persistent infection of canine histiocytic sarcoma cells (DH82 cells) with the canine distemper virus strain Onderstepoort genetically engineered to express canine GM-CSF (CDV-Ondneon-GM-CSF). The investigations aimed (1) to prove the overall functionality of the virally induced production of GM-CSF and (2) to determine the effect of GM-CSF on the proliferation and motility of canine HS cells. Infected cells consistently produced high amounts of active, pH-stable GM-CSF, as demonstrated by increased proliferation of HeLa cells. By contrast, DH82 cells lacked increased proliferation and motility. The significantly increased secretion of GM-CSF by persistently CDV-Ondneon-GM-CSF-infected DH82 cells, the pH stability of this protein, and the lack of detrimental effects on DH82 cells renders this virus strain an interesting candidate for future studies aiming to enhance the oncolytic properties of CDV for the treatment of canine histiocytic sarcomas.

Up-Regulation of Cluster of Differentiation (CD) 11b Expression on the Surface of Canine Granulocytes with Human Granulocyte-Macrophage Colony-Stimulating Factor (GM-CSF)

ABSTRACTGranulocyte-macrophage colony-stimulating factor (GM-CSF) is a pleiotropic cytokine, sharing a common beta subunit (CDw131) with interleukins 3 and 5. GM-CSF is important for its direct and indirect involvement in host defense. In veterinary medicine, human (h) GM-CSF has been used as a substitute for canine GM-CSF to stimulate canine granulocytes and macrophages. In this study, we compared the effects of three distinct hGM-CSFs produced by bacteria, yeasts and Chinese hamster ovary (CHO) cells with those of Escherichia (E) coli-produced canine GM-CSF on the cluster of differentiation 11b (CD11b) expression in canine granulocytes. The median effective dose (ED50) of hGM-CSFs from bacteria, yeasts and CHO cells was 3.09, 4.09 and 4.27 ng/ml, respectively, with no significant difference among three. In contrast, a significant difference was observed between ED50 of canine GM-CSF (0.56 ng/ml) and three hGM-CSFs according to the paired t-test (P<0.05). We conclude that hGM-CSF can activate canine granulocytes, but the average activity of the three rhGM-CSFs was approximately 15% of that of canine GM-CSF.

Priming Effects of Lipopolysaccharide and Inflammatory Cytokines on Canine Granulocytes

Granulocytes play a pivotal role in natural immunity. Under inflammatory conditions, granulocytes are universally primed by several agents, such as endotoxins and inflammatory cytokines. Primed granulocytes exert potent adhesiveness, chemotaxis, phagocytosis and reactive oxygen species (ROS) production, effectively eliminating invading agents. Reactivity against priming agents is known to vary with species; however, there have been few reports on the effects of priming agents on canine granulocytes. In the present study, we assayed the priming effects of lipopolysaccharide (LPS), recombinant canine tumor necrosis factor-alpha (rcTNF-alpha) and recombinant canine granulocyte macrophage colony-stimulating factor (rcGM-CSF) on canine granulocyte function in vitro. Isolated recombinant canine were primed with various concentrations of LPS, rcTNF-alpha and rcGM-CSF, and CD11b expression was assayed. Furthermore, actin polymerization, phagocytosis and ROS production were then assayed at primer concentrations where enhancement of CD11b expression was observed. LPS did not enhance canine granulocyte function. Phagocytosis and actin polymerization were not enhanced by priming agents; however, rcTNF-alpha and rcGM-CSF enhanced CD11b expression and ROS production in canine granulocytes. These results suggest that priming effects are mainly reflected in CD11b expression and ROS production, with rcGM-CSF and rcTNF-alpha having a priming effect similar to that observed in humans.

Preventive and therapeutic effects of gene therapy using silica nanoparticles–binding of GM-CSF gene on white blood cell production in dogs with leukopenia

Our previous study has shown that granulocyte-macrophage colony-stimulating factor (GM-CSF) gene/silica nanoparticles have a leukocytosis effect in normal dogs. Therefore, this study was conducted to determine whether treatment of canine GM-CSF gene/silica nanoparticles has preventive or therapeutic effects in dogs with leukopenia. To induce leukopenia, vinblastine was administered intravenously at a dose of 2 mg/m(2) of body surface area on day 0. Then 7.5 microg GM-CSF/nanoparticles (1:100, w/w) were administered intravenously to each of four dogs in the prevention group on day 2 and an equivalent amount of GM-CSF/nanoparticles was administered to the post-nadir group on day 4 (other groups were administered phosphate-buffered saline intravenously). Therapeutic GM-CSF gene was expressed in peripheral blood mononuclear cells for 10 days and both the prevention and post-nadir groups showed significant increases in white blood cell counts when compared with the control group, as confirmed by complete blood count, differential count, and flow cytometry. GM-CSF/nanoparticles can be useful for correction of acute leukopenia, such as chemotherapy-induced myelosuppression, without developing neutralizing antibodies.

Generation of Blood-derived Dendritic Cells in Dogs with Oral Malignant Melanoma

Advances in treatment of human melanoma indicate that immunotherapy, particularly dendritic cell (DC) immunization, may prove useful. The aim of this study was to investigate whether blood-derived DCs could be generated from canine melanoma patients. Peripheral blood mononuclear cells were isolated from three such dogs and cultured with recombinant canine granulocyte-macrophage colony stimulating factor (GM-CSF), canine interleukin 4 and human Flt3-ligand for 7 days. The resulting cells demonstrated a typical dendritic morphology, and were enriched for cells expressing CD1a, CD11c and MHC II by flow cytometric analysis. Thus, canine blood-derived DCs can be generated in vitro and DC immunization should be feasible in dogs.

Cloning of canine GM-CSF and SCF genes

Cytokines have pleiotropic regulatory effects on hematopoietic cells and many other cell types that participate in host defence and repair processes. Granulocyte-macrophage colony-stimulating factor (GM-CSF) mediates the growth and differentiation of granulocytes and macrophages and regulates the biological functions expressed by mature cells of these lineages. Stem cell factor (SCF) is a multifunctional cytokine involved in hematopoiesis, melanogenesis and gametogenesis. In order to determine the complementary DNA (cDNA) of canine GM-CSF and canine SCF, cDNA clones were generated from lipopolysaccharide (LPS) stimulated peripheral blood mononuclear cells (PBMCs) and bone marrow cells by reverse transcription PCR amplification. The canine GM-CSF cDNA obtained in this study contains an open reading frame encoding 144 amino acid residues and has 53-75% homology with those of human, cat, sheep, pig, cow and mouse, Canine SCF cDNA consist of an open reading frame encoding 274 amino acid residues and shares 81-92% homology with those of human, cat, pig, cow and mouse.

Intramarrow cytokine gene transfer by adenoviral vectors in dogs.

Daily systemic administration of hematopoietic growth factors can be associated with dose-limiting systemic side effects. To overcome this, we have investigated hematopoietic cytokine gene transfer to the marrow cavity of dogs by direct intramarrow injection of adenoviral vectors. In marrow culture, replication-deficient (E1-deleted) adenoviral vectors were able to transduce marrow stromal cells, demonstrating 30-fold greater expression than from other marrow cell types. High-level (ng/ml) cytokine production from transduced stromal cells persisted for 14 days in culture. Because adenovectors could efficiently transduce marrow stromal cells in culture, we investigated if stromal cells could also be transduced in vivo following direct intramarrow vector injection. Adenovectors with genes for interleukin 6 (IL-6) and Lac Z (beta-galactosidase) were injected directly into the marrow cavity of dogs resulting in protein expression localized to within the treated marrow. To evaluate this approach further in dogs, we constructed a vector expressing biologically active canine granulocyte-macrophage colony stimulating factor (GM-CSF). 293 cells infected with ADGM-CSF demonstrated prevalent GM-CSF mRNA by Northern blot and 135 +/- 30 ng/ml of protein as measured by enzyme-linked immunosorbent assay (ELISA). In vitro bioactivity of protein expressed was confirmed by canine GM colony-forming assay (CFU-GM). In vivo high-level protein production was noted in supernatants of marrow aspirates 72 hr following direct intramarrow administration of ADGM-CSF (baseline mean +/- SEM, 27 +/- 22 ng/ml, 72-hr sample 921 +/- 461 ng/ml). A localized myeloid expansion of marrow and significant peripheral leukocytosis (neutrophilia) were noted in all ADGM-CSF-treated dogs. Peripheral blood changes lasted for up to 3 weeks in dogs following single intramarrow injection. Thus, adenoviral cytokine expression from the marrow of a single large bone (ilium) led to compartmentalized expression of growth factor and an increase of hematopoiesis sufficient to cause peripheral blood changes in a large animal model.

Thrombocytopenia in Dogs Induced by Granulocyte-Macrophage Colony-Stimulating Factor: Increased Destruction of Circulating Platelets

Administration of recombinant canine granulocyte-macrophage colony-stimulating factor (rcGM-CSF) to normal dogs in previous studies induced an increase in peripheral blood neutrophils and a dose-dependent decrease in platelet counts. In six dogs that received the highest tested dose of rcGM-CSF (50 micrograms/kg/d) for a minimum of 12 days, the mean nadir of the platelet count was 46,000/microL (range, 4,000 to 91,000/microL) on day 9 +/- 1.1 after starting therapy, compared with a mean baseline platelet count of 398,000/microL (range, 240,000 to 555,000/microL). In three dogs, survival of autologous 111In-labeled platelets was reduced from a mean of 4.9 days to 1.3 days during the administration of rcGM-CSF. Biodistribution studies with gamma camera imaging indicated that there was an increase in mean hepatic uptake during the administration of rcGM-CSF, from 15% to 44% of the total injected 111In-labeled platelets at 2 hours, whereas splenic uptake was not significantly changed. In contrast, in two evaluable dogs who were recipients of 111In-labeled platelets from matched allogeneic donors receiving rcGM-CSF, platelet survival was not reduced and no increased hepatic uptake was noted. A third dog became alloimmunized to the matched donor platelets and was not evaluable. Immunohistologic studies of liver and spleen were performed with monoclonal antibodies specific for canine gpIIb/IIIa and P-selectin in dogs treated with rcGM-CSF and compared with untreated controls. On treatment, a marked reduction of platelets in the red pulp of the spleen was evident, and in general, the presence of platelet antigen in the liver was unchanged. Therefore, platelets were not being sequestered, but destroyed in the liver and spleen. The platelet antigens, P-selectin and gpIIb/IIIa, were identified in association with Kupffer cells in the liver, but no difference in the number of distribution of these Kupffer cells was found between controls and rcGM-CSF-treated dogs. In the spleen during rcGM-CSF treatment, most platelet antigens were associated with large mononuclear cells in the marginal zone. During administration of rcGM-CSF, CD1c and CD11c expression was increased on Kupffer cells. Platelet P-selectin expression and binding of leukocytes to circulating platelets were unchanged from baseline studies with rcGM-CSF treatment. In conclusion, during the administration of rcGM-CSF to dogs, a local process in the liver and spleen is induced resulting in thrombocytopenia.(ABSTRACT TRUNCATED AT 400 WORDS)

Effect of Recombinant Canine Granulocyte-Macrophage Colony-Stimulating Factor on Hematopoietic Recovery After Otherwise Lethal Total Body Irradiation

AbstractRecombinant canine granulocyte-macrophage colony-stimulating factor (rcGM-CSF) was studied in normal dogs and in dogs receiving otherwise lethal total body irradiation (TBI) without marrow transplant. Five normal dogs receiving 25 μg/kg of rcGM-CSF by subcutaneous (SO injection twice daily (BID) for 14 days showed increases in peripheral blood neutrophil counts of three to five times the baseline. Platelet counts decreased during administration of rcGM-CSF to a mean nadir of 52,800. Ten dogs received 400 cGy TBI at 10 cGy/min from two opposing 60Co sources and no marrow graft. Within 2 hours of TBI, rcGM-CSF was begun at a dose of 50 μg/kg SC BID for 5 doses and then continued at 25 μg/kg SC BID for 21 days. Only 1 of the 10 dogs receiving rcGM-CSF survived with complete and sustained recovery of hematopoiesis. One of 13 historical control dogs survived after 400 cGy with no hematopoietic growth factor or marrow infusion. Results with rcGM-CSF were compared with previous and concurrent data with G-CSF studied in the same model. Of 10 dogs receiving G-CSF, 8 survived with complete and sustained hematopoietic recovery, a significantly better survival than that seen with rcGM-CSF (P = .006). Neutrophil counts were sustained at higher levels after TBI for the first 18 days in the G-CSF group (P < .016) and the neutrophil nadirs were higher. No differences in neutrophil nadirs were noted between the rcGM-CSF and control groups. Dogs treated with rcGM-CSF experienced a more rapid decline of platelet counts than G-CSF-treated or control dogs over the first 18 days (P < .001). The nadir of the platelet count was higher in the control group than in either the G-CSF or rcGM-CSF group and no significant difference was observed between the G-CSF and rcGM-CSF groups. After otherwise lethal TBI (400 cGy) in dogs, rcGM-CSF was not effective in promoting hematopoietic recovery or improving survival.

Effect of recombinant canine granulocyte-macrophage colony-stimulating factor on hematopoietic recovery after otherwise lethal total body irradiation

Recombinant canine granulocyte-macrophage colony-stimulating factor (rcGM-CSF) was studied in normal dogs and in dogs receiving otherwise lethal total body irradiation (TBI) without marrow transplant. Five normal dogs receiving 25 micrograms/kg of rcGM-CSF by subcutaneous (SC) injection twice daily (BID) for 14 days showed increases in peripheral blood neutrophil counts of three to five times the baseline. Platelet counts decreased during administration of rcGM-CSF to a mean nadir of 52,800. Ten dogs received 400 cGy TBI at 10 cGy/min from two opposing 60Co sources and no marrow graft. Within 2 hours of TBI, rcGM-CSF was begun at a dose of 50 micrograms/kg SC BID for 5 doses and then continued at 25 micrograms/kg SC BID for 21 days. Only 1 of the 10 dogs receiving rcGM-CSF survived with complete and sustained recovery of hematopoiesis. One of 13 historical control dogs survived after 400 cGy with no hematopoietic growth factor or marrow infusion. Results with rcGM-CSF were compared with previous and concurrent data with G-CSF studied in the same model. Of 10 dogs receiving G-CSF, 8 survived with complete and sustained hematopoietic recovery, a significantly better survival than that seen with rcGM-CSF (P = .006). Neutrophil counts were sustained at higher levels after TBI for the first 18 days in the G-CSF group (P &lt; .016) and the neutrophil nadirs were higher. No differences in neutrophil nadirs were noted between the rcGM-CSF and control groups. Dogs treated with rcGM-CSF experienced a more rapid decline of platelet counts than G-CSF-treated or control dogs over the first 18 days (P &lt; .001). The nadir of the platelet count was higher in the control group than in either the G-CSF or rcGM-CSF group and no significant difference was observed between the G-CSF and rcGM-CSF groups. After otherwise lethal TBI (400 cGy) in dogs, rcGM-CSF was not effective in promoting hematopoietic recovery or improving survival.

Effect of endotoxin on serum granulocyte and granulocyte-macrophage colony-stimulating factor levels in dogs.

The biologic effects of endotoxin are attributed to the release of several cytokines, including interleukin-1, interleukin-6, tumor necrosis factor, and the colony-stimulating factors. To investigate the mechanism of endotoxin-induced neutrophilia in dogs, several cell lines known to proliferate selectively in response to recombinant human colony-stimulating factors were examined to determine their responses to recombinant canine granulocyte colony-stimulating factor (rcG-CSF) or recombinant canine granulocyte-macrophage colony-stimulating factor (rcGM-CSF). The murine cell line NFS-60 was found to respond well to rcG-CSF and the human cell line TALL-101 to rcGM-CSF, and these responses were neutralized by antibodies to these recombinant proteins. These bioassays were then used to determine G-CSF and GM-CSF levels in dogs after intravenous endotoxin administration. G-CSF levels increased by 2 h, peaked at 4 h, and had not returned to normal by 24 h after endotoxin. In contrast, GM-CSF was not detectible before or after endotoxin administration.

Molecular cloning and in vivo evaluation of canine granulocyte- macrophage colony-stimulating factor

Canine granulocyte-macrophage colony-stimulating factor (caGM-CSF) was cloned and expressed to allow further investigation of GM-CSF in a large animal model. The cDNA is 850 base pairs (bp) long and encodes a peptide of 144 amino acids. The nucleotide and amino acid sequence homology between caGM-CSF and human GM-CSF (hGM-CSF) is 80% and 70%, respectively. A mammalian expression vector pCMV/CAGM was constructed and used to transfect COS cells for expression of caGM-CSF. Supernatant from transfected COS cells enriched with caGM-CSF was shown to have significant stimulating activity in granulocyte-macrophage colony forming unit (CFU-GM) assays of canine marrow. caGM-CSF, expressed from bacteria, was used to treat seven dogs at varying doses twice daily subcutaneously (sc) for 14 to 16 days. Circulating blood neutrophils and monocytes increased significantly. The increase in circulating eosinophils was variable. Thrombocytopenia developed during administration of caGM-CSF but corrected rapidly after cessation of treatment. Evaluation of survival times of 51Cr-labeled autologous platelets suggested increased consumption as the primary reason for thrombocytopenia. A species-specific GM-CSF will be a useful tool for hematologic or immunologic studies in dogs.

Molecular Cloning and In Vivo Evaluation of Canine Granulocyte-Macrophage Colony-Stimulating Factor

Abstract Canine granulocyte-macrophage colony-stimulating factor (caGM-CSF) was cloned and expressed to allow further investigation of GM-CSF in a large animal model. The cDNA is 850 base pairs (bp) long and encodes a peptide of 144 amino acids. The nucleotide and amino acid sequence homology between caGM-CSF and human GM-CSF (hGM-CSF) is 80% and 70%, respectively. A mammalian expression vector pCMV/CAGM was constructed and used to transfect COS cells for expression of caGM-CSF. Supernatant from transfected COS cells enriched with caGM-CSF was shown to have significant stimulating activity in granulocyte-macrophage colony forming unit (CFU-GM) assays of canine marrow. caGM-CSF, expressed from bacteria, was used to treat seven dogs at varying doses twice daily subcutaneously (sc) for 14 to 16 days. Circulating blood neutrophils and monocytes increased significantly. The increase in circulating eosinophils was variable. Thrombocytopenia developed during administration of caGM-CSF but corrected rapidly after cessation of treatment. Evaluation of survival times of 51Cr-labeled autologous platelets suggested increased consumption as the primary reason for thrombocytopenia. A species-specific GM-CSF will be a useful tool for hematologic or immunologic studies in dogs.