Bone Marrow-derived Macrophage Cell Line Research Articles

Generation and characterization of bovine bone marrow-derived macrophage cell line.

Macrophages, as the forefront of innate immune defense, have an important role in the host responses to mycobacterial infection. Therefore, a stable macrophage cell line is needed for future bovine immune system research on the bacterial infection. In this study, we established a bovine macrophage cell line by introducing the human telomerase reverse transcriptase (hTERT) gene into bovine bone marrow-derived macrophages (bBMMs). The TERT-bBMMs cells expressed macrophage surface antigen (CD11b, CD282) and upregulated expression of the cytokines IL-1β, IL-6, IL-10, IL-12, TNF-α in response to bacterial invasion. These results demonstrate that this cell line provide reliable cell model system for future studies on interactions between the bovine macrophages and Mycobacterium tuberculosis.

Anti-WASP intrabodies inhibit inflammatory responses induced by Toll-like receptors 3, 7, and 9, in macrophages

Wiskott-Aldrich syndrome protein (WASP) is an adaptor molecule in immune cells. Recently, we showed that the WASP N-terminal domain interacted with the SH3 domain of Bruton's tyrosine kinase (Btk), and that the complex formed by WASP and Btk was important for TLR2 and TLR4 signaling in macrophages. Several other studies have shown that Btk played important roles in modulating innate immune responses through TLRs in immune cells. Here, we evaluated the significance of the interaction between WASP and Btk in TLR3, TLR7, and TLR9 signaling. We established bone marrow–derived macrophage cell lines from transgenic (Tg) mice that expressed intracellular antibodies (intrabodies) that specifically targeted the WASP N-terminal domain. One intrabody comprised the single-chain variable fragment and the other comprised the light-chain variable region single domain of an anti-WASP N-terminal monoclonal antibody. Both intrabodies inhibited the specific interaction between WASP and Btk, which impaired the expression of TNF-α, IL-6, and IL-1β in response to TLR3, TLR7, or TLR9 stimulation. Furthermore, the intrabodies inhibited the phosphorylation of both nuclear factor (NF)-κB and WASP in response to TLR3, TLR7, or TLR9 stimulation, in the Tg bone marrow-derived macrophages. These results suggested that WASP plays important roles in TLR3, TLR7, and TLR9 signaling by associating with Btk in macrophages.

Specific binding of the WASP N-terminal domain to Btk is critical for TLR2 signaling in macrophages

Wiskott–Aldrich syndrome protein (WASP) is an adaptor molecule in immune cells. Recently, we revealed that WASP is involved in lipopolysaccharide-TLR4 signaling in macrophages by association of Bruton's tyrosine kinase (Btk) with the WASP N-terminal domain. Btk has been shown to play important roles in the signaling of several TLRs and to modulate the inflammatory response in macrophages. In this study, we evaluated the importance of the interaction between Btk and WASP in TLR2 signaling by using bone marrow-derived macrophage cell lines from transgenic (Tg) mice expressing anti-WASP N-terminal domain single-chain variable fragment (scFv) or VL single-domain intrabodies. In this Tg bone marrow-derived macrophages, specific interaction between WASP and Btk were strongly inhibited by masking of the binding site in the WASP N-terminal domain. There was impairment of gene expression of TNF-α, IL-6, and IL-1β and phosphorylation of inhibitor of κB α/β (IKKα/β) and nuclear factor (NF)-κB upon stimulation with TLR2 ligands. Furthermore, tyrosine phosphorylation of WASP following TLR2-ligand stimulation was severely inhibited in the Tg bone marrow-derived macrophages, as shown by the impairment in WASP tyrosine phosphorylation following lipopolysaccharide stimulation. These results strongly suggest that the association between the WASP N-terminal domain and Btk plays an important role in the TLR2-signaling pathway in macrophages.

Specific inhibition of PI3K p110δ inhibits CSF‐1‐induced macrophage spreading and invasive capacity

Colony stimulating factor-1 (CSF-1) stimulates mononuclear phagocytic cell survival, growth and differentiation into macrophages through activation and autophosphorylation of the CSF-1 receptor (CSF-1R). We have previously demonstrated that CSF-1-induced phosphorylation of Y721 (pY721) in the receptor kinase insert triggers its association with the p85 regulatory subunit of phosphoinositide 3'-kinase (PI3K). Binding of p85 PI3K to the CSF-1R pY721 motif activates the associated p110 PI3K catalytic subunit and stimulates spreading and motility in macrophages and enhancement of tumor cell invasion. Here we show that pY721-based signaling is necessary for CSF-1-stimulated PtdIns(3,4,5)P production. While primary bone marrow-derived macrophages and the immortalized bone marrow-derived macrophage cell line M-/-.WT express all three class IA PI3K isoforms, p110δ predominates in the cell line. Treatment with p110δ-specific inhibitors demonstrates that the hematopoietically enriched isoform, p110δ, mediates CSF-1-regulated spreading and invasion in macrophages. Thus GS-1101, a potent and selective p110δ inhibitor, may have therapeutic potential by targeting the infiltrative capacity of tumor-associated macrophages that is critical for their enhancement of tumor invasion and metastasis.

Pegylated Granulocyte Colony Stimulating Factor (Peg-G-CSF) Enhances Mapatumumab-Mediated Antibody Dependent Cellular Cytotoxicity (ADCC) Against Non-Hodgkin's Lymphoma (NHL) Cell Lines Independent of NADPH Oxidase-Derived Reactive Oxidant Intermediates (ROIs).

ADCC is an important mechanism of action of rituximab and other monoclonal antibodies (mAb). In an attempt to improve the efficacy of rituximab while minimizing its toxicity, our group have explored the use of cytokines that target neutrophil maturation and activation in combination with rituximab in pre-clinical and clinical models. In response to a growing need to develop alternative biological therapies, a new generation of humanized antibodies are under development. Mapatumumab is a humanized mAb targeting the death receptor-4 (DR4), active against NHL. A variable degree of ADCC has been observed in mapatumumab-treated NHL cells, raising the possibility that its biological activity may be augmented by cytokines. To this end, we studied the effects of cytokine-priming of neutrophils in the activity of mapatumumab and the role of free radical species in mAb-mediated ADCC. We used Raji cells, a CD20 and DR4 expressing Burkitt's lymphoma cell line, as target cells in a standardized 51Cr release assay. Effector cells consisted of peripheral blood mononuclear cells (PBMC) and neutrophils collected from healthy volunteers or lymphoma patients who had received peg-G-CSF for the prevention of chemotherapy-induced neutropenia. In addition, thiolglycollate-elicted peritoneal neutrophils and a bone marrow-derived macrophage cell line from C57BL/6 and genetically engineered chronic granulomatous disease (CGD) mice were used to evaluate the role of NADPH oxidase-derived ROIs in mAb-mediated ADCC. For ex vivo priming, healthy volunteer PBMC/neutrophils were exposed to peg-G-CSF (1mcg/ml) or placebo for 24hrs at 37oC,5%CO2. Subsequently, effector cells were co-cultured with 51Cr-labeled Raji cells in the presence of mapatumumab or isotype control at an effector:target ratio of 40:1. Following a period of 6 hrs of incubation, the supernatant was collected and the percentage of lysis was calculated. For in vivo cytokine priming, PBMC/neutrophils were collected from lymphoma patients who had received peg-G-CSF (6mg sq) at the time of white cell count recovery and used as effector cells in ADCC assays as described above. Finally, to establish the role of ROIs in mapatumumab-associated ADCC, we conducted standard 51Cr release assays with mapatumumab plus C57BL/6 or CGD murine neutrophils or macrophages as effector cells. Ex vivo cytokine-primed killing (34.54% +/− 1.69) and in vivo cytokine-primed killing (26.17% +/−0.28) resulted in an improvement in mapatumumab-associated ADCC as compared to controls (10.26% +/−0.79; P = 0.002, and P = 0.003, respectively). In murine studies, mapatumumab resulted in significant neutrophil and macrophage-mediated ADCC against Raji cells (20% and 15%, respectively). No differences were observed in mapatumumab anti-tumor activity between C57BL/6 versus CGD effector cells suggesting that NADPH oxidase-derived ROIs do not influence mapatumumab-induced ADCC. In summary, our data strongly suggest that peg-G-CSF can potentiate the anti-tumor activity of mapatumumab and support additional translational and clinical research of the combination of cytokine-primed effector cells plus mAbs targeting the death receptor pathway.

In vitro macrophage activation by organic waste materials

The EU-wide ban on antibiotic growth promoters in animal production has stimulated research on functional feed additives that improve animal health and performance in the related industries. Organic wastes from industries like pharmaceutical or food production often have to be disposed at high costs and sometimes also cause environmental problems. The potential further use of such plant residues or by-products as value-added products in animal or human nutrition is currently evaluated in the course of the EU-funded project SAFEWASTES. Here we report on possible immunomodulating activity of several organic wastes by in vitro activation of a chicken macrophage cell line. Macrophages are important elements of the innate, non-specific and specific immune reaction of vertebrates. They represent a first-line defence against invading pathogens by phagocytosis, cytokine and nitric monoxide (NO) production and antigen presentation. In vitro activation of a chicken bone marrow-derived macrophage cell line (HD-11) was measured by NO production in a cell culture assay. Active substances were checked for lipopolysaccharide (LPS) contaminations caused by gram-negative bacteria that could lead to false-positive results. 52 extracts prepared from 18 different organic waste materials were investigated. Nine water extracts showed high macrophage activation comparable to the positive control, a bacterial cell wall preparation. Among them, willow bark water extract had no detectable amounts of LPS while they probably accounted for some activity of the other extracts. Bioassay-guided fractionation was done to further identify the active principles of the materials.

Abrupt Expression of TLR4 in TLR4-Deficient Macrophages Imposes a Selective Disadvantage: Genetic Evidence for TLR4-Dependent Responses to Endogenous, Nonmicrobial Stimuli

TLR4 is crucial for macrophage responses to LPS. It is less clear whether TLR4 may also transduce signals from host factors, and if so, with what consequences. Immortalized bone marrow-derived macrophage cell lines, termed T4Cr and T4ko, were established from TLR4null strains, C57BL/10ScNCr and TLR4 knockout mice, respectively. Multiple transfections and selections were conducted to stably introduce TLR4 into these cell lines. Among 196 individual clones isolated, 48 expressed TLR4 on the cell surface but did not respond to LPS due to a deletion in the MyD88 gene. The remaining clones integrated TLR4 DNA into the genome but expressed neither detectable TLR4 mRNA nor TLR4 protein. To test the possibility that TLR4null cells lack modulating factors to protect against a harmful effect of TLR4, 15 stably transfected clones were generated in the presence of conditioned media from wild-type macrophages. Some of these cells expressed a small amount of TLR4 and regained responsiveness to LPS. Because no microbial ligands were available to the cell lines during their generation, signaling via endogenous ligands is likely to have occurred in TLR4-expressing, signal-competent macrophages and imposed a proliferative or other selective disadvantage. These studies support the existence of constitutive signaling via TLR4 during in vitro culture of macrophages without microbial products, and help account for the lack of reports of restoration of TLR4 expression in normally TLR4-expressing types of cells in vitro whose TLR4 genes are deleted or disrupted.

Proteasome-mediated Degradation of STAT1α following Infection of Macrophages with Leishmania donovani

Activation of the Janus-activated kinase 2 (JAK2)/STAT1alpha signaling pathway is repressed in Leishmania-infected macrophages. This represents an important mechanism by which this parasite subverts the microbicidal functions of the cell to promote its own survival and propagation. We recently provided evidence that the protein tyrosine phosphatase (PTP) SHP-1 was responsible for JAK2 inactivation. However, STAT1 translocation to the nucleus was not restored in the absence of SHP-1. In the present study, we have used B10R macrophages to study the mechanism by which this Leishmania-induced STAT1 inactivation occurs. STAT1alpha nuclear localization was shown to be rapidly reduced by the infection. Western blot analysis revealed that cellular STAT1alpha, but not STAT3, was degraded. Using PTP inhibitors and an immortalized bone marrow-derived macrophage cell line from SHP-1-deficient mice, we showed that STAT1 inactivation was independent of PTP activity. However, inhibition of macrophage proteasome activity significantly rescued Leishmania-induced STAT1alpha degradation. We further demonstrated that degradation was receptor-mediated and involved protein kinase C alpha. All Leishmania species tested (L. major, L. donovani, L. mexicana, L. braziliensis), but not the related parasite Trypanosoma cruzi, caused STAT1alpha degradation. Collectively, results from this study revealed a new mechanism for STAT1 regulation by a microbial pathogen, which favors its establishment and propagation within the host.

Trypanosoma congolense infections: antibody-mediated phagocytosis by Kupffer cells.

Immunohistochemical double-label technique was used to detect trypanosomal antigen in macrophages. Immunoglobulin (Ig)M as well as IgG2a monoclonal antibodies (mAb) specific for the variant surface glycoprotein (VSG) mediated phagocytosis of Trypanosoma congolense variant antigenic type (VAT) TC13 by macrophages [bone marrow-derived macrophage cell line from BALB/c (BALB.BM)] in vitro. Administration of these IgM or IgG2a antibodies to BALB/c mice 30 min after injection of 3 x 10(8) T. congolense mediated phagocytosis of trypanosomes by Kupffer cells of the liver within 1 h. Plasma levels of the monokines interleukin (IL)-1beta, IL-10, and IL-12p40 were significantly increased 6-48 h after phagocytosis. In BALB/c mice infected with 10(3) T. congolense, a small degree of phagocytosis of trypanosomes by Kupffer cells, mediated by actively synthesized antibodies, was detected as early as 5 days after infection. Phagocytosis of trypanosomes was dramatically enhanced on day 6. Concomitantly, the Kupffer cells trippled in size. In BALB/c mice infected for 6 days, treatment with IgM or IgG2a mAb specific for T. congolense VSG led to clearance of VAT TC13 parasitemia but did not prevent death at the second parasitemia of a different VAT. We conclude that IgM as well as IgG antibody mediate phagocytosis of trypanosomes by Kupffer cells.

Toll-like receptor 4 (TLR4)-deficient murine macrophage cell line as an in vitro assay system to show TLR4-independent signaling of Bacteroides fragilis lipopolysaccharide.

Bacterial lipopolysaccharides (LPS) activate cells of innate immunity, such as macrophages, by stimulating signaling through toll-like receptor 4 (TLR4). We and others have hypothesized that LPS derived from different bacterial species may function through TLR4-independent mechanisms. To test this hypothesis, we have generated using a nonviral transformation procedure a bone marrow-derived macrophage cell line called 10ScNCr/23 from mouse strain C57BL/10ScNCr. This mouse strain has a deletion of the TLR4 locus, causing the mouse strain to be nonresponsive to stimulation by LPS from Escherichia coli while responding normally to other bacterial substrates, such as lipoteichoic acid (LTA) from Staphylococcus aureus, which signal TLR4 independently. Stimulation with LTA induces five- and sixfold increases in 10ScNCr/23 cell line tumor necrosis factor alpha and macrophage inflammatory protein-2 (MIP-2) secretion, but no increases in either cytokine were found when cells were stimulated with E. coli LPS. Bacteroides fragilis-derived LPS, however, can effectively stimulate MIP-2 expression in the absence of functional TLR4 in the 10ScNCr/23 cell line. This gives rise to the notion that LPS from some bacterial species will utilize alternative receptors to stimulate the innate immune response.

Clearance of infection with Mycobacterium bovis BCG in mice is enhanced by treatment with S28463 (R-848), and its efficiency depends on expression of wild-type Nramp1 (resistance allele).

The mouse bcg host resistance gene is known to control the activation of host macrophages for killing of intracellular parasites like Leishmania donovani as well as intracellular bacteria, including Mycobacterium bovis BCG and Salmonella enterica serovar Typhimurium. The Nramp1 gene has been mapped to this locus and affects the efficiency of macrophage activation. It has been shown that imidazoquinoline compounds, including S28463, are able to improve the clearance of a number of intracellular pathogens such as herpes simplex virus 2, human papillomavirus, and Leishmania. The goal of this study was to determine whether S28463 is efficient against infection with another intracellular pathogen, M. bovis BCG, and to determine the molecular basis underlying this effect. To achieve this, B10A.Nramp1(r) and B10A.Nramp1(-/-) mice were infected with M. bovis BCG and treated with S28463. The bacterial content in the spleen from these mice was assayed by a colony-forming assay. In addition, in vitro experiments were performed using bone marrow-derived macrophage cell lines from these mice. These cells were treated with S28463 and/or gamma interferon (IFN-gamma), and nitric oxide (NO) production was measured. Our study was able to show that S28463 acts in synergy with IFN-gamma to increase the production of NO in vitro. We were also able to demonstrate that mice that carried the resistant allele of the Nramp1 gene and were infected with M. bovis BCG responded to treatment with S28463, resulting in a decreased bacterial load after 2 weeks of treatment. Mice that do not express the Nramp1 gene responded only to a very large dose of S28463, and the response was not as efficient as that observed in mice carrying a wild-type Nramp1 allele. Our data provide evidence for the potential of S28463 as an immunomodulator that may be helpful in designing efficient strategies to improve host defense against mycobacterial infection.

Ship is a positive regulator of nitric oxide production

Studies to date with the hemopoietic-specific inositol 5-phosphatase, SHIP, have established that it is a negative regulator of hemopoietic cell proliferation, survival, and end cell activation. It appears to carry out this inhibitory function, at least in part, by hydrolysing the PI-3 kinase-generated second messenger, PI-3,4,5-P3, to PI-3,4-P2. However, recent studies in our laboratory with SHIP +/+ and −/− macrophages have established a hitherto unexpected role for SHIP as a positive regulator of nitric oxide (NO) synthesis. Both resident and thioglycollate-elicited peritoneal macrophages as well as retrovirally-immortalized bone marrow-derived macrophage cell lines (J2M) from SHIP −/− mice are impaired in their induction of the inducible isoform of NO synthase (iNOS) in response to LPS/IFN-γ co-stimulation, and produce 5 to 10-fold less NO than wild-type cells. Furthermore, upon LPS stimulation alone, wild-type J2M macrophages induce significantly more iNOS mRNA and protein and 5 to 10-fold more NO than SHIP −/− J2M cells. Consistent with these results, we found that the luciferase activity of a wild type iNOS promoter/luciferase construct in SHIP+/+ J2M cells, following 18 h with LPS, was substantially higher than that in SHIP−/− cells. We are currently testing various mutant iNOS promoter/luciferase constructs to hone in on the transcription factors that are regulated by SHIP and these results will be presented.

Macrophages derived from C3H/HeJ (Lpsd) mice respond to bacterial lipopolysaccharide by activating NF-kappa B.

The effects of bacterial lipopolysaccharide (LPS) on macrophage gene expression are mediated in part by its ability to induce activation of transcription factor NF-kappa B. We compared the ability of LPS-treated macrophages from Lpsn (LPS-responsive) C3H/HeN and Lpsd (LPS-hyporesponsive) C3H/HeJ mice to mobilize NF-kappa B by electrophoretic mobility shift assays with oligonucleotide probes containing a unique NF-kappa B sequence from the promoter of inducible nitric oxide synthase (iNOS). In response to ng/ml concentrations of LPS, this probe bound proteins that appeared rapidly in the nuclei of thioglycollate-elicited macrophages and bone marrow-derived macrophage cell lines from both Lpsn and Lpsd mice. Only in macrophages from Lpsn mice, however, was LPS able to induce iNOS or tumor necrosis factor alpha. NF-kappa B-containing DNA-protein complexes from Lpsd macrophages were formed in lesser amounts than from Lpsn macrophages but shared the same composition, insofar as they displayed the same electrophoretic mobilities and content of heterodimers of p50/RelA (p65) and p50/c-rel. Two conclusions emerge from these findings: (1) NF-kappa B activity alone is not sufficient for induction of certain LPS-responsive genes and (2) An LPS-response pathway involving activation of NF-kappa B is preserved in Lpsd mice. The inability of cells from Lpsd mice to induce gene expression in response to LPS thus cannot be attributed to inability to activate NF-kappa B.

Opposite Effects of Bacterial Lipopolysaccharide on Fc-Receptor-Mediated Phagocytosis of Two Bone Marrow-Derived Macrophage Cell Lines, BDM-1 and BDM-1W3.

We have reported the isolation and characterization of three factor-dependent macrophage cell lines from bone marrow cells of C3H/HeN mice. We have since isolated a subclone, BDM-1W3, from one of these cell lines. We found previously that BDM-1W3 has a different sensitivity to bacterial lipopolysaccharide (LPS) for growth than its parental cell line, BDM-1. In this report, we show that LPS inhibits BDM-1W3 phagocytosis of antibody-coated sheep erythrocytes (Fc-mediated phagocytosis), whereas it enhanced Fc-mediated phagocytosis by BDM-1. It was observed that a loss of Fc-receptor capacity parallels a loss of phagocytic activity in LPS-treated BDM-1W3 cells. LPS stimulated phagocytosis of latex beads by BDM-1 and BDM-1W3, suggesting that Fc-mediated phagocytosis and phagocytosis of latex beads differ in their regulatory mechanisms. When BDM-1 cells were cultured with LPS, they underwent drastic morphological changes, whereas LPS-treated BDM-1W3 cells did not change significantly. Gamma interferon enhanced FC-mediated phagocytosis by BDM-1, while it has no significant effect on that by BDM-1W3. These cell lines should be useful for studying signal transduction mechanisms in LPS-mediated macrophage activation.

Evidence for effects of interleukin 4 (B cell stimulatory factor 1) on macrophages: enhancement of antigen presenting ability of bone marrow-derived macrophages.

We have studied the effects of recombinant mouse interleukin 4 (IL 4) (previously known as B cell stimulatory factor 1) on the antigen-presenting ability of murine splenic B cells and bone marrow macrophages. Our assay is based on the induction of antigen-presenting ability in these cells after incubation with IL 4 for 24 hr. The presenting cells were then used to stimulate IL 2 production by antigen-specific, I-Ad-restricted T cell hybridomas, a response mainly dependent on the induction of Ia antigens. Consistent with our previously published data using partially purified natural IL 4, we show here that recombinant IL 4 (but not interferon-gamma (IFN-gamma) or IL 1) induces antigen-presenting ability in B cells. Recombinant IL 4 was also found to induce antigen-presenting ability in a cloned, bone marrow derived-macrophage cell line (14M1.4), and in normal bone marrow-derived macrophages. These macrophage populations also respond to IFN-gamma showing enhanced antigen-presenting ability (mediated by increased Ia antigen expression). A small but significant increase in Ia antigen expression was also detected in 14M1.4 macrophages induced with IL 4. However, additional analysis suggested that the effect of IL 4 on 14M1.4 is different from that of IFN-gamma, because IL 4 (but not IFN-gamma) is able to maintain the viability and increase the size of and metabolic activity of bone marrow macrophages. However, IL 4 may not affect all macrophages because the macrophage cell line P388D1, which responds to IFN-gamma, failed to show enhanced antigen-presenting function after stimulation with IL 4. These observations indicate that IL 4, a lymphokine previously considered to be B cell lineage specific, has effects on macrophages and may be involved in their activation.

Growth inhibition of Cryptococcus neoformans by cloned cultured murine macrophages

Cloned and unselected bone marrow-derived macrophage cell lines were obtained from A/J, AKR/J, BIO.A(5R), CBA/J, DBA/2, HPC, NZW, and [NZB X NZW]F 1 mice, and their interactions were studied in vitro with a lightly encapsulated natural serotype A isolate of Cryptococcus neoformans. Growth inhibition of C. neoformans was seen with all of the cell lines, as determined by enumeration of colony-forming units. Inhibition was enhanced by a high concentration (8%) of fresh mouse serum and was the same for serum obtained from AKR/J (C5 deficient) and BIO.A (C5 normal) mice. Macrophage incubation with fresh AKR/J serum which had been absorbed with heat-killed Cryptococcus cells also inhibited C. neoformans growth. Heat-inactivation, EDTA addition or anti-C3 antibody treatment of fresh serum abolished the opsonic activity for C. neoformans, while EGTA addition to fresh serum was without effect on opsonization. In addition, neither IgM nor IgG 1 murine monoclonal antibodies specific for C. neoformans enhanced phagocytosis or killing of the yeast by macrophages. These findings are consistent with the interpretation that C3b is an important modulator of interactions between macrophages and C. neoformans.

Avian acute leukemia virus OK10 has an 8.2-kilobase genome and modified glycoprotein gp 78.

We have analyzed the structure of OK10-BM virus, an avian acute leukemia virus produced by a bone marrow-derived cell line of macrophage origin, and compared it with that of OK10 AV, an associated virus originally present in the OK10 virus stock. The RNAs of OK10-BM virus and OK10 AV had the same mobility in agarose gels, corresponding to 8.0 to 8.5 kilobases, a size considerably larger than that of the transforming component (5 to 6 kb) of most other avian acute leukemia viruses. Fingerprint analysis showed a close relationship between OK10-BM virus and OK10 AV RNAs. The polypeptide compositions of OK10-BM and OK10 AV viruses were similar except for the envelope glycoproteins. In analysis by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, the large envelope glycoprotein of OK10-BM virus migrated at M(r) = 78,000 (gp78), whereas OK10 AV had the characteristic 85,000-dalton glycoprotein (gp85) of nondefective avian leukemia viruses. gp78 was weakly labeled with methionine, glycine, proline, or mannose, suggesting that purified OK10-BM virus had reduced amounts of the modified envelope glycoprotein. In cell-free rabbit reticulocyte lysates, OK10-BM virion RNA directed the synthesis of a 200,000-dalton polypeptide (p200), a 180,000-dalton polypeptide (pr180), and a 76,000-dalton polypeptide (pr76), whereas OK10 AV RNA gave rise only to pr180 and pr76, suggesting that p200 may represent an OK10-BM-encoded transforming protein. No biochemical evidence for the presence of an associated helper virus was found in the OK10-BM virus population produced by the macrophage cell line. However, when OK10-BM virus was serially passaged in chicken embryo fibroblasts, a virus having structural properties similar to those of OK10 AV (OK10 AV-specific oligonucleotides and gp85) appeared after three passages. Moreover, nonproducer clones of transformed cells could be readily obtained in OK10-BM virus-infected quail cell cultures. It is thus likely that the bone marrow-derived macrophage cell line produces a transforming virus defective in its env gene and low amounts of an associated helper virus, which upon transfer to fibroblasts is preferentially replicated.