Chicken Myelomonocytic Growth Factor Research Articles

Integration of gene expression profile data to screen and verify immune-related genes of chicken erythrocytes involved in Marek's disease virus

Chicken erythrocytes participated in immunity, but the role of erythrocytes in the immunity of Marek's disease virus (MDV) has not been reported related to the immunity genes. The purpose of this study was to screen and verify the immune-related genes of chicken erythrocytes which could be proven as a biomarker in MDV. The datasets (GPL8764-Chicken Gene Expression Microarray) were downloaded from the GEO profile database for control and MDV infected chickens to obtain differentially expressed genes (DEGs) through bioinformatics methods. Kyoto Encyclopedia of Genes and Genomes (KEGG) was performed to find enriched pathways, including Gene Ontology (GO). Based on enriched pathways, the top 19 immune-related genes were screened-out and process further to construct the protein-protein interaction (PPI) networks. The screened genes were validated on RT-PCR and qPCR. Results suggested that the mRNA transcription of Toll-like receptors 2, 3, 4, 6 (TLR2, TLR3, TLR4, TLR6), major histocompatibility complex-II (MHCII), interleukin-7 (IL-7), interferon-βeta (IFN-β), chicken myelomonocytic growth factor (cMGF) and myeloid differentiation primary response 88 (MyD88) were significantly up-regulated. The expression of toll-like receptor 5, 7 (TLR5, TLR7) interleukin-12 (IL-12 p40), interleukin-13 (IL-13), and interferon-αlpha (IFN-α) were significantly down-regulated in the erythrocytes of the infected group (P < 0.05). In contrast, the expression of toll-like receptor-1, 15, 21 (TLR1, TLR15, TLR21), major histocompatibility complex I (MHCI) and Tumor necrosis factor receptor (TNFR)-associated factor 6 (TRAF6) were not significant. In conclusion, it has been verified on qRT-PCR results that 19 immune-related genes, which included TLRs, cytokines and MHC have immune functions in MDV infected chickens.

Marek's Disease Virus Induces Th-2 Activity During Cytolytic Infection

Marek's disease (MD) is a lymphoproliferative disease of chickens that is caused by a highly cell-associated oncogenic alpha-herpesvirus, Marek's disease virus (MDV). The role of cytokines and other related proteins in MD pathogenesis and immunity is poorly understood. The aim of this study was to examine the transcriptional profiling of a panel of cytokines and other immune-related genes in the splenic tissues of chickens infected with a highly oncogenic strain of MDV during cytolytic infection and latency. Real-time polymerase chain reaction analysis revealed significant upregulation in the expression levels of interleukins (IL)-1beta, IL-4, IL-6, IL-10, IL-12p35, and IL-13, interferons (IFN)-1alpha, IFN-1beta, and IFN-gamma, chicken myelomonocytic growth factor (cMGF), granulocyte-macrophage colony-stimulating factor (GM-CSF), and inducible nitric oxide synthase (iNOS) in the infected chickens at 5 d post-inoculation (lytic infection). The changes in the mRNA levels of IL-18 and MHC I were minimal in comparison to those of the control birds. There was no significant difference in the expression levels of IL-2, IL-8, MHC II, Bcl-2, Bcl-x, and Nr-13 between the two groups. With the exception of IL-10, which showed high transcriptional activity beyond the lytic phase, the expression patterns of all the tested genes were similar between the infected and age-matched control birds at 15 d post-inoculation (latency infection). Of the genes examined, in addition to the high transcriptional activities of IL-1beta, IL-6, IL-12, iNOS, and type 1 and 2 IFNs, the relative expression levels of IL-4, IL-10, and IL-13 were significantly upregulated in the infected chickens during the lytic phase of infection compared to uninfected controls (a 9- to 50-fold difference). This observation suggests that (1) an immune response with a Th-2 characteristic is induced by a very virulent plus MDV strain during the lytic phase of infection; and (2) there is no significant MDV-specific immune response in the latent phase of infection.

The granulocyte colony-stimulating factors (CSF3s) of fish and chicken

Granulocyte colony-stimulating factor (CSF3) is a glycoprotein cytokine, which influences the hematopoiesis of the phagocytic neutrophils and its precursors and was used extensively in cancer therapy and for the treatment of neutropenia in mammals. However, CSF3 is yet to be identified in nonmammalian species mainly because of its rapid mutation. Here, we report the first CSF3 genes from three teleost fishes: Japanese flounder (Paralichthys olivaceus), fugu (Takifugu rubripes), and green-spotted pufferfish (Tetraodon nigroviridis) and present evidence that the chicken (Gallus gallus) myelomonocytic growth factor is in fact the chicken CSF3 orthologue. We support this by showing significant conservation of the CSF3 genes' structure, domains, regulatory motifs, and synteny across species and by phylogenetic analysis. CSF3 orthologues are indeed evolving rapidly and appears to be undergoing purifying selection in mammals but positive selection in fish and chicken. Furthermore, the paralogous fugu and pufferfish CSF3-1s and CSF3-2s are shown to be the ancestral and duplicate genes, respectively. Finally, we demonstrate that the Japanese flounder CSF3 gene is at least involved in immunity based on its basal expression in immune-related tissues and its upregulation in kidney and peripheral blood leukocytes after in vitro stimulation with lipopolysaccharide and a combination of concanavalin A/phorbol myristate acetate.

Oral delivery of novel therapeutics: development of a fowl adenovirus vector expressing chicken IL-2 and MGF

Delivery methods for commercial scale applications must be simple, inexpensive and cost-effective. The use of live vectors such as fowl adenoviruses (FAV) has previously been proven successful for the effective oral delivery of chicken interferon gamma to broilers under commercial growing conditions. FAV can be delivered via food or water or can be used as a spray. The ability of cytokines to modulate immune responses to vaccines and their therapeutic and growth promoting capability makes them an important component in future health management strategies for poultry. In this report, we describe the development of recombinant FAV vectors expressing chicken interleukin-2 (ChIL-2) and chicken myelomonocytic growth factor (cMGF). The ChIL-2 and cMGF genes were cloned into the right hand terminal region of FAV serotype 8, under the control of the adenovirus major late promoter. The in vitro biological activity of FAV-ChIL-2 and FAV-cMGF was comparable to that of the corresponding recombinant proteins expressed in prokaryotic (E. coli) and eukaryotic (COS cell) systems. These constructs may be particularly useful as therapeutics for mucosal infections.

Antiviral and antitumoral effects of recombinant chicken myelomonocytic growth factor in virally induced lymphoma

Chicken myelomonocytic growth factor (cMGF) is a 27-kDa glycoprotein that stimulates the growth and activation of cells from the monocyte/macrophage lineage. Recombinant cMGF was produced in a prokaryotic ( Escherichia coli) expression system and purified via a C-terminal His-tag. Treatment of 2-week-old histocompatible B 13/B 13 chickens highly susceptible to Marek's disease (MD) with rcMGF (two daily injections of 50 μg rcMGF per chicken) enhanced background and LPS-inducible systemic NO (NO 3 −+NO 2 −) responses 3 days later. NO has antiviral activity on Marek's disease virus (MDV), a herpesvirus specifically inducing T cell-lymphomas in chickens. When the very virulent strain of MDV RB1-B was inoculated 3 days after treatment with rcMGF, MDV viremia was significantly inhibited and development of visceral tumors was drastically reduced. Combination of rcMGF with partially protective vaccination using a herpesvirus of turkey (HVT) further reduced tumor burden and significantly delayed mortality, but only in very young birds. In conclusion, rcMGF might be worth considering as a stimulator of innate immune response in chickens, such as activation of macrophages and NO production, and thus be beneficial for its antiviral and antitumoral effects in vaccination against MD lymphoma.

Protective effect of avian myelomonocytic growth factor in infection with Marek's disease virus.

Marek's disease virus (MDV) is a herpesvirus that induces T lymphomas in chickens. The aim of this study was to assess the role of the macrophage activator chicken myelomonocytic growth factor (cMGF) in controlling MDV infection. B13/B13 chickens, which are highly susceptible to MD, were either treated with cMGF delivered via a live fowlpox virus (fp/cMGF) or treated with the parent vector (fp/M3) or were left as untreated controls. Seven days later, when challenged with the very virulent RB-1B strain of MDV, the spleens of chickens treated with fp/cMGF showed increased expression of the inducible nitric oxide synthase (iNOS) gene compared to those of control chickens and fp/M3-treated chickens. Increased iNOS gene expression was also accompanied by greater induction of gamma interferon and macrophage inflammatory protein (K203) gene expression, both possible activators of iNOS. fp/cMGF treatment also increased the number of monocytes and systemic NO production in contrast to fp/M3 treatment. Even though cMGF treatment was unable to prevent death for the chickens, it did prolong their survival time, and viremia and tumor incidence were greatly reduced. In addition, cMGF treatment improved the partial protection induced by vaccination with HVT (herpesvirus isolated from turkeys) against RB-1B, preventing 100% mortality (versus 66% with vaccination alone) and greatly reducing tumor development. Treatment with fp/M3 did not have such effects. These results suggest that cMGF may play multiple roles in protection against MD. First, it may enhance the innate immune response by increasing the number and activity of monocytes and macrophages, resulting in increased NO production. Second, it may enhance the acquired immune response, indicated by its ability to enhance vaccine efficacy.

Potential use of cytokine therapy in poultry

Newly hatched chickens are highly susceptible to infection during the first 2 weeks of life. The utilisation of cytokines as therapeutic agents in livestock animals, in particular poultry, has become more feasible with the recent cloning of cytokine genes and the progression of new technologies such as live vectors. We have constructed a live recombinant fowlpox virus (FPV) that expresses chicken myelomonocytic growth factor (fp/cMGF). Administration of fp/cMGF to chicks resulted in a marked and sustained increase in the number of circulating blood monocytes as well as an increase in their state of activation, as measured by enhanced phagocytic activity and elevated production of nitric oxide. We have recently cloned the gene for chicken interferon- γ (ChIFN- γ). Recombinant ChIFN- γ was capable of protecting chick fibroblasts from undergoing virus-mediated lysis and induced nitrite secretion from chicken macrophages in vitro. Preliminary vaccination trials have indicated that co-administration of ChIFN- γ with antigen (sheep red blood cells) resulted in enhanced secondary (IgG) antibody responses and allowed a 10-fold lower dose of antigen to be used. Furthermore, administration of ChIFN- γ resulted in enhanced weight gain in chicks and improved their resistance to disease challenge. The ability of cytokines to combat infection and enhance vaccine efficacy makes them excellent candidates as a therapeutic agents and adjuvants.

Enhanced expression of cytokine genes in spleen macrophages during acute infection with infectious bursal disease virus in chickens

We examined the effects of infectious bursal disease virus (IBDV) on splenic T cells and macrophages. In acute IBDV infection, splenocytes responded poorly to Con A stimulation. However, when T cells were isolated from whole spleen cells, purified T cells responded normally to Con A. This result indicated that functional T cells were present in the spleen but mitogen-induced proliferation of T cells was being suppressed by other cells. Previous studies indicated that soluble factors from suppressor cells may mediate this inhibition of T cell mitogenesis. We thus examined the effects of IBDV on spleen adherent cells. Reverse transcriptase–polymerase chain reaction (RT–PCR) was used to quantitate the expression of several cytokine genes in splenic macrophages. In acute IBDV infection, splenic macrophages exhibited enhanced gene expression of type I interferon (IFN), chicken myelomonocytic growth factor (cMGF), an avian homolog of mammalian IL-6, and 9E3/CEF4, an avian homolog of mammalian IL-8. Mitogen-stimulated spleen cell cultures also produced elevated levels of nitric oxide. The elevation of cytokine gene expression by macrophages occurred transiently during the acute phase of viral infection and coincided with in vitro inhibition of T cell mitogenic response of spleen cells.

STAT5 involvement in the differentiation response of primary chicken myeloid progenitor cells to chicken myelomonocytic growth factor.

We have investigated the activation of the STAT5 protein during the differentiation of myelomonocytic cells. In the human U937 promonocytic cell line, STAT5 activation occurred in response to several inducers of monocytic differentiation (phorbol ester, 1alpha,25-dihydroxyvitamin D3, and retinoic acid). In the promyelocytic HL60 cell line, STAT5 was activated in the course of either phorbol ester-induced monocytic differentiation or DMSO-induced granulocytic differentiation. To test for involvement of STAT5 in the differentiation of primary nonimmortalized cells, chicken myeloid progenitor cells transfected with the ts21-E26 avian retrovirus were studied. At 37 degrees C the temperature-sensitive oncoprotein of the ts21-E26 virus (p135(gag-myb-ets)) installs a differentiation block that is released by a shift to the nonpermissive temperature (42 degrees C). Both proliferation and differentiation of ts21-E26-transfected myeloblasts require the continuous presence of chicken myelomonocytic growth factor (cMGF). Addition of cMGF to growth factor-starved cells rapidly caused STAT5 tyrosine phosphorylation, DNA binding, and transcriptional activity at both permissive and nonpermissive temperatures. Moreover, the temperature shift-induced onset of myelomonocytic differentiation strictly correlated with the appearance of activated STAT5 in ts21-E26-infected myeloblasts, but not in cells infected with wtE26 retrovirus. These data position STAT5 in a nuclear signaling cascade induced by the cMGF receptor and suggest a contribution of STAT5 to the process of myelomonocytic differentiation or to the functional changes that accompany the maturation of myeloid progenitor cells to a terminally differentiated stage.

Applications in in ovo technology

By mid-August 1995, 55% of broiler embryos in North America were vaccinated for Marek's disease using the INOVOJECT system, with 201 INOVOJECT machines placed with 16 of the top 25 poultry producers, providing the industry with the capacity to inject in excess of 400 million eggs per month or about 5 billion eggs per annum. In ovo administration of a bursal disease antibody-infectious bursal disease virus (BDA-IBDV) complexed vaccine to specific-pathogen-free (SPF) embryos was safer and more potent than conventional IBDV vaccine alone because it delayed the appearance of bursal lesions, produced no early mortality, produced higher geometric mean antibody titers against IBDV, and generated protective immunity against challenge. In ovo administration of a BDA-IBDV complexed vaccine to broiler embryos generated antibody titers against IBDV sooner than conventional virus vaccinates, and generated protective immunity against challenge Direct DNA injection of plasmid DNA encoding beta-galactosidase into breast muscle in ovo and posthatch was an effective means to achieve both gene transfer and expression, with potential for the development of gene vaccines using plasmids encoding protective antigens from poultry pathogens. In ovo administration of 800 U chicken myelomonocytic growth factor (cMGF), a chicken hematopoietic cytokine for cells of the monocytic-granulocytic lineages, significantly reduced mortality associated with Escherichia coli exposure within the hatcher when compared to PBS controls (6.1 vs 12.4, P < or = 0.05), but not when compared to a yeast expression control. A procedure was developed enabling injection prior to the onset of incubation without compromising embryo viability. This in ovo injection process has opened up the window of embryo development during incubation for intervention, as illustrated by the 100% male phenotype produced in chicks hatching from eggs injected with aromatase inhibitor prior to incubation. These data illustrate some of the in ovo applications presently in use by the poultry industry, and under development or in research at EMBREX.

In vivo effects of chicken myelomonocytic growth factor: delivery via a viral vector.

We have constructed a recombinant fowlpox virus (FPV) that expresses chicken myelomonocytic growth factor (cMGF). Administration of this construct (fp/cMGF) to 1-day-old chicks resulted in a marked and sustained increase in the number of circulating blood monocytes compared with chicks infected with the parental FPV strain (fp/M3). Blood monocyte numbers were elevated within 4 days of fp/cMGF infection, reached maximal levels at day 9, and returned to normal levels by day 16. During the peak response, approximately 35% of blood leukocytes were monocytes, compared with 4 to 7% in uninfected control birds. Infection with fp/M3 also resulted in a detectable increase in monocyte numbers; however, the effect was less dramatic. Compared with fp/M3, fp/cMGF consistently induced two- to threefold higher monocyte numbers, and the period of monocytosis was longer (10 vs 5 days). No other specific changes in white blood cell populations were observed. Associated with the increase in the number of monocytes was an increase in their state of activation, as measured by the ability to produce nitric oxide (NO) and to phagocytose latex beads. Blood monocytes from birds infected with fp/cMGF produced about 6 times as much NO per cell compared with monocytes from fp/M3-infected birds. Monocytes from normal birds failed to produce detectable levels of NO. Furthermore, cMGF treatment specifically induced enhanced phagocytic activity in blood monocytes. Overall, these results indicate that viral vectors are suitable for the delivery of biologically active cytokines and that they allow an assessment of cytokine activities in vivo.

Avian Hematopoiesis in Response to Avian Cytokines

The objective of this study was to examine the hematopoietic cell proliferation and differentiation potential of growth factors produced by chicken macrophages. Bone marrow (BM) cells (25 × 103) from newly hatched B15B15K-strain Leghorn chicks were seeded in .5 mL serum-free semi-solid culture supplemented with 10% (vol/vol) of a conditioned medium (CM) from a chicken macrophage cell line, MQ-NCSU. The conditioned medium was obtained by culturing MQ-NCSU cells either in LM-HAHN (CMI) or RPMI-1640 (CMII) growth medium. The control cultures contained only LM-HAHN or RPMI medium. Bone marrow cells in the presence of CMI differentiated predominately into granulocyte colonies (Experiment 1 = 84±9.2; Experiment 2 = 105 ± 5). No colonies were observed in, the control cultures. Stimulation of MQ-NCSU cells with lipopolysaccharide (LPS) produced a CM that differentiated BM cells predominantly into macrophage colonies (122±16.3 in CMI and 92±5.6 in CMII). These data suggest that MQ-NCSU cells spontaneously secrete a factor with the potential to promote granulocyte differentiation. However, upon stimulation with LPS, the factor secreted had macrophage colony stimulation potential (M-CSF), which was similar in activity when compared with the activity of recombinant chicken myelomonocytic growth factor (r-cMGF). Another CM from chicken fibroblasts (FCM) was tested on BM cells from K-strain Leghorns and Arbor Acres × Arbor Acres broiler chicks. Data from three experiments showed that 25 × 103 BM cells from K-strain chicken yielded more macrophage and granulocytes colonies (82 ± 14) than those from broilers (56 ± 12). This study suggests that avian cytokines exhibit progenitor cell differentiation potential and that this activity is dependent upon the source of cytokines and their targets.

In vivo cooperation of two nuclear oncogenic proteins, P135gag-myb-ets and p61/63myc, leads to transformation and immortalization of chicken myelomonocytic cells.

To investigate a possible in vivo cooperation between the p61/63myc and P135gag-myb-ets proteins, we used a previously constructed retrovirus, named MHE226, which contains the fused v-myb and v-ets oncogenes of the E26 retrovirus and the v-myc oncogene of MH2. For that purpose, chicken neuroretina cells producing MHE226 and pseudotyped with the Rous associated virus-1 (RAV-1) helper virus were injected in 1-day-old chickens. In control experiments, we also injected chicken neuroretina cells producing E26 (RAV-1), RAV-1 alone, or constructs lacking one of the oncogenes of MHE226. The average life span of MHE226-infected chickens is half that of E26-infected chickens. MHE226-infected chickens harbor tumors scattered in many organs, but compared with E26, MHE226 induced a weak leukemia. Study of integration sites suggests that the majority of the tumors results from clonal or oligoclonal events. Cell cultures were derived from the tumors of MHE226-infected chickens and grown in standard medium without addition of exogenous chicken myelomonocytic growth factor. These cells still divide at high rate after more than 100 passages and can thus be considered immortalized. By using several criteria, these cells were characterized as precursors of the myelomonocytic lineages.

Constitutive monocyte-restricted activity of NF-M, a nuclear factor that binds to a C/EBP motif.

In a search for monocyte-specific nuclear factors, we analyzed in human cells the promoter of the chicken myelomonocytic growth factor, a gene that, in the chicken, is expressed in myeloid and myelomonocytic cells. Reporter gene constructs were active in monocytic Mono Mac 6 cells and in monoblastic THP-1 cells but not in the hematopoietic stem cell line K562. When a region with homology to the sequence recognized by CAAT enhancer-binding proteins (C/EBP) was inactivated by site-directed mutagenesis, the reporter activity was reduced by a factor of 10. Multimers of this region, termed F, in front of a heterologous promoter were active in Mono Mac 6 and THP-1 cells but not in K562 cells, WIL2 B cells, BT20 mammary carcinoma cells, MelJuso melanoma cells, or SK-Hep-1 hepatoma cells. Gel shift analysis with the F oligonucleotide identified DNA-binding activity in monocytic Mono Mac 6, monoblastic THP-1, and myelomonocytic HL60 cells. No binding was detected in myelomonocytic RC2A cells, in myeloid KG-1 cells, or in the hematopoietic stem cell line K562. Furthermore, a panel of solid tumor cell lines, representing various tissues, were also negative. Stimulation by PMA could not induce this binding factor in any of the negative cell lines. Analysis of primary cells (granulocytes, T cells, monocytes, and alveolar macrophages) revealed binding activity only in monocytes and macrophages. This DNA-binding factor, termed NF-M, was found to consist of two molecules, of 50 and 72 kDa, as determined by affinity cross-linking. Binding of NF-M was competed by the region F oligonucleotide and by the C/EBP motif from the albumin enhancer but not by an AP-2 motif. These data suggest that NF-M is a member of the C/EBP family of nuclear factors. The monocyte-restricted activity of NF-M suggests that this nuclear factor may be involved in regulation of monocyte-specific genes.

Structure of the chicken myelomonocytic growth factor gene and specific activation of its promoter in avian myelomonocytic cells by protein kinases.

In chicken myeloid cells but not in erythroid cells, kinase-type oncogenes activate expression of the chicken myelomonocytic growth factor (cMGF). The autocrine loop established this way plays a key role in lineage-specific cooperation of nuclear and kinase-type oncogenes in retrovirally induced myeloid leukemia. In this report, we describe the cloning of the cMGF gene, including its promoter. The structure of the cMGF gene is homologous to those of the granulocyte colony-stimulating factor and interleukin-6 genes. Expression from reporter constructs containing the cMGF promoter is specific to myelomonocytic cells. Kinases activate cMGF at the transcriptional level in macrophages and strongly induce reporter expression in myelomonocytic cells.

Structure of the chicken myelomonocytic growth factor gene and specific activation of its promoter in avian myelomonocytic cells by protein kinases.

In chicken myeloid cells but not in erythroid cells, kinase-type oncogenes activate expression of the chicken myelomonocytic growth factor (cMGF). The autocrine loop established this way plays a key role in lineage-specific cooperation of nuclear and kinase-type oncogenes in retrovirally induced myeloid leukemia. In this report, we describe the cloning of the cMGF gene, including its promoter. The structure of the cMGF gene is homologous to those of the granulocyte colony-stimulating factor and interleukin-6 genes. Expression from reporter constructs containing the cMGF promoter is specific to myelomonocytic cells. Kinases activate cMGF at the transcriptional level in macrophages and strongly induce reporter expression in myelomonocytic cells.

Autocrine growth induced by kinase type oncogenes in myeloid cells requires AP-1 and NF-M, a myeloid specific, C/EBP-like factor.

The nuclear oncogenes v-myc or v-myb specifically transform avian myeloid cells. In both cases, the transformed cells remain dependent on chicken myelomonocytic growth factor (cMGF). This factor dependence can be relieved by expression of kinase-type oncogenes such as v-mil or v-erbB, leading to expression of cMGF and autocrine growth stimulation. In erythroid cells the same kinase-type oncogenes cause transformation but do not induce cMGF expression. Here we investigated the molecular mechanisms of the observed lineage specific oncogene collaboration. We found that kinase-type oncogenes and TPA activate the cMGF promoter via AP-1 like transcription factors. The activation of the cMGF promoter is, however, strictly dependent on the binding of nuclear proteins to both halves of an inverted repeat adjacent to the AP-1 binding site. These proteins are related to C/EBP. They are expressed exclusively in myeloid cells and were therefore termed NF-M. Our results indicate that the lineage specific cooperation of kinase type oncogenes with v-myb or v-myc in leukemia formation is based on the concerted action of AP-1 and NF-M on the cMGF promoter.

Activation of cMGF expression is a critical step in avian myeloid leukemogenesis.

A non-leukemogenic version of the v-myb oncogene causes in vitro transformation of avian myeloblasts, which are dependent on chicken myelomonocytic growth factor (cMGF). We have shown that this version of v-myb, when combined with the erythroleukemia-inducing v-erbB oncogene, is capable of causing a mixed myeloid and erythroid leukemia. Myeloid leukemic cells transformed by this construct produce cMGF. To test whether autocrine growth stimulation via cMGF is the essential contribution of the tyrosine kinase oncogene v-erbB in avian myeloid leukemogenesis we constructed another retrovirus containing both the non-leukemogenic v-myb and the cMGF cDNA. This virus induced myeloid leukemia at high efficiency. In a third construct we combined v-myb with the human EGF-receptor gene. Myeloid cells transformed by this construct could be stimulated to grow by the addition of cMGF or EGF. Growth stimulation with EGF was blocked by a cMGF antiserum indicating that activation of a normal tyrosine kinase-type receptor induces cMGF expression but does not bypass the cMGF requirement. We conclude that cMGF plays a key role in the growth regulation of normal and transformed avian myeloid cells.

Rapid and transient expression of Ets2 in mature macrophages following stimulation with cMGF, LPS, and PKC activators.

We reported previously that Ets2 is expressed in normal and transformed macrophages. We show here that the expression of both c-ets-2 mRNA proteins is induced rapidly and transiently in chicken nondividing bone marrow-derived macrophages but not in E26-transformed myeloblasts in response to chicken myelomonocytic growth factor (cMGF), an avian hematopoietic growth factor required for survival, proliferation, and colony formation of avian myeloid cells. c-ets-2 expression is also rapidly induced in chicken bone marrow-derived macrophages, human monocytes, and mouse peritoneal macrophages in response to LPS and/or PKC activators. The rapid induction of Ets2 after treatment of chicken bone marrow-derived macrophages by cMGF is blunted after down-regulation or inactivation of PKC, suggesting a role of PKC in the cMGF-induced signal transduction pathway. Because Ets2 is localized in the nucleus of macrophages and binds to DNA in vitro, the kinetics of its expression suggest a role for Ets2 in the transduction within the nucleus of specific signals received at the cell membrane and involved in securing the survival and/or the development of functional competence of these cells.

The mutated, myeloid cell-specific growth factor receptor v-fms transforms avian erythroid but not myeloid cells.

In avian hematopoietic cells, transformation by tyrosine kinase oncogenes is restricted to the erythroid lineage. To study the mechanism of this striking target cell specificity, we constructed an avian retrovirus correctly expressing the feline v-fms protein, an oncogenic version of the myeloid-specific CSF-1 receptor. Similar to other tyrosine kinase oncogenes (erbB, src, fps, sea), the v-fms oncogene induced progenitor cells to self-renew in an erythropoietin-independent manner. Spontaneous differentiation of the transformed cells was arrested by v-erbA. v-fms failed to induce transformation of myeloid cells but caused myeloid cells transformed by the v-myb oncogene, as well as normal macrophages, to proliferate independent of cMGF (chicken myelomonocytic growth factor). Unlike the other tyrosine kinases, v-fms did not induce cMGF secretion in myb-myeloblasts, suggesting a nonautocrine mechanism of growth factor independence.