Cloning and characterization of a murine macrophage lipoxygenase

We have isolated a murine macrophage cDNA encoding a 12-lipoxygenase, that represents the homolog of the human 15-lipoxygenase. The predicted amino acid sequence of this lipoxygenase is highly similar to the rat 12-lipoxygenase isolated from brain and human 15-lipoxygenase. The recombinant enzyme expressed in Cos-7 cells oxidizes arachidonic acid to 12- and 15-HETE with a profile similar to that obtained from peritoneal macrophages. A polyclonal antibody generated against a putative peptide recognizes a 75 kDa protein in cell extracts from mouse peritoneal macrophages and transfected Cos-7 cells. The lipoxygenase cDNA hybridizes to a 2.5 kb mRNA present in peritoneal macrophages, lung, spleen, heart and liver. RT-PCR analysis indicates that the same lipoxygenase is expressed in mouse reticulocytes. A partial genomic clone for this lipoxygenase has also been characterized. Southern blot analysis of mouse genomic DNA indicates that this is a single copy gene.     

Development and characterization of monoclonal antibodies to murine macrophage colony-stimulating factor

The macrophage-specific CSF (CSF-1), purified from murine L cell-conditioned medium, supports the in vitro proliferation and survival of various murine mononuclear phagocyte colony-forming cells. In this report we describe the production and functional characterization of two monoclonal antibodies (mAb) to CSF-1 obtained from rat X rat hybridomas. These two mAb are functionally distinct and recognize different epitopes on CSF-1. The mAb 5A1 binds to and inhibits the biologic function of CSF-1, and the second mAb (D24) binds CSF-1 but does not neutralize its biologic activity. The mAb 5A1 inhibits colony formation of tissue mononuclear phagocyte colony-forming cells as well as the committed bone marrow stem cells for both granulocytes and monocytes. The extent of colony inhibition by mAb 5A1 is dependent on the tissue origin of colony-forming cells. CSF-1 complexed with mAb 5A1 does not bind to its cell surface receptor of peritoneal exudate macrophages, and mAb 5A1 does not complex with cell-bound CSF-1. Although both bone marrow cell-derived macrophages and J774.1 macrophages bind CSF-1, mAb 5A1 inhibits the proliferation of only bone marrow cell-derived macrophages. The non-neutralizing mAb D24 does not block binding of CSF-1 to its cellular receptor, and it recognizes cell-bound CSF-1.     

Establishment and characterization of a novel murine model for pollen allergy

Although there have been many studies revealing the mechanism and establishing the therapeutical method for allergic rhinitis, no suitable animal models for allergic rhinitis, especially for pollen allergy, are currently available. We therefore aimed in this study to develop a murine model producing IgE in response to an inhaled antigen without using any adjuvants. Ovalbumin (OVA)-specific T cell receptor transgenic mice (DO11.10) inhaled an OVA solution for one h, twice a week, for six weeks. The resulting increase of OVA-specific IgE in the serum was observed depending on the times of inhalation. Spleen cells from mice that had inhaled the antigen produced more IL-4 and less IFN-γ than those from the control mice in vitro. These results indicate that inhaled antigen enhanced the Th2-type responses and induced IgE production in a T cell-mediated manner. Our findings would contribute to studies on prevention and treatment of pollen allergy.     

Establishment and characterization of osteogenic cell lines from a spontaneous murine osteosarcoma

Five clonal cell lines were established from a spontaneous BALB/c mouse osteosarcoma, and characterized. Four of these lines showed some similarities in morphology, in vitro growth properties, production of collagenous and noncollagenous extracellular matrix proteins and osteogenic differentiation. The cells formed colonies with characteristic differences in size and morphology in soft agar, and osteogenic sarcomas and metastases in syngeneic mice after transplantation. Ultrastructurally, cells in the transplant tumours showed marked osteogenic features. There were no osteoclast-like cells. The fifth cell line had somewhat different characteristics. All five lines expressed infectious endogenous murine leukemia viruses. Increased c-myc protoon-cogene expression was found in one cell line and c-fos expression at different levels in all lines. There was only very low expression of c-Ha-ras and no expression of c-Ki-ras and c-sis. DNA analysis showed the presence of newly acquired proviral genomes integrated at different sites in the cellular DNA. The results show that distinct osteogenic neoplastic subclones can be obtained from a primary mouse osteosarcoma. Although the clones exhibited an appreciable morphological, functional, and molecular diversity they retained the basic pathogenic properties of the tumour from which they were derived.     

Isolation and characterization of three lysophospholipases from the murine macrophage cell line WEHI 265.1.

Anion exchange chromatography of WEHI 265.1 cell homogenates resolved the lysophospholipase activity into three peaks, when assayed using lysophosphatidylcholine as a substrate. Peaks 1 and 2 were purified by sequential hydrophobic interaction and gel filtration chromatography. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of the purified peaks 1 and 2 indicated homogeneous proteins with apparent masses of 28 and 27 kDa, respectively. Peak 3 lysophospholipases was partially purified by hydrophobic, hydroxyapatite and gel filtration chromatography. Peak 3 lysophospholipase also had calcium-dependent phospholipase A2 activity, which further co-purified with the lysophospholipase activity. The three lysophospholipases were characterized with respect to substrate specificity, additional enzymatic activities and the effects of lipids, metal ions and other compounds on enzymatic activity. Peaks 1, 2 and 3 hydrolyzed lysophosphatidylcholine most readily, but lysophosphatidylethanolamine also served as substrate for each enzyme. Furthermore, all three enzymes hydrolyzed platelet activating factor and acetylated lysophosphatidylcholine. Each lysophospholipase was inhibited by free fatty acids and by palmitoyl carnitine, although the relative sensitivities to these agents differed among the enzymes. The lysophospholipase activities of peaks 1 and 2, but not peak 3, were inhibited by phenylmethylsulfonyl fluoride, diisopropyl fluorophosphate and N-ethylmaleimide. Although they had similar masses, the amino acid compositions of peaks 1 and 2 differed, indicating that these are distinct proteins rather than posttranslational modifications of the same gene product.     

Establishment and characterization of somatic hybrids between human differentiated macrophages and mouse myeloma NS1 cells

Human macrophages obtained from circulating monocytes matured in vitro by culture for seven days in hydrophobic flexible teflon bags were successfully fused with murine myeloma NS1 cells. Six of 20 clones, selected for their adherence properties, were further studied. All possessed human chromosomes (mean number ranging from 4 to 14 depending on the clones studied), exhibited non-specific esterases (but no peroxidase activity) and expressed CD14 antigen and C3 receptors (but no MAX-1 antigen). Moreover, the hybridomas retained phagocytic activity and high interferon plus lipopolysaccharide-activable cytolytic activity against tumor cells.     

Isolation and characterization of cell cycle-enriched subpopulations of a murine macrophage cell line by centrifugal elutriation

The cell cycle of the P388D 1 murine macrophage line was delineated and suspensions of exponentially growing cells were separated by centrifugal elutriation into subpopulations enriched in the various phases of the cycle. Analysis of both growth and labelled mitoses curves disclosed that the doubling and cell-cycle times were essentially identical (18.4 and 18.3 h), indicating that all cells were in cycle. In addition, G1 + 1/2M was 4.3 h, whereas S phase and G2 + 1/2M lasted about 12 and 1.5 h. The most homogeneous subpopulations of phase-enriched cells obtained by elutriation were cells in G1 and S, where purities (estimated by both labelling indices and analyses of DNA histograms obtained by flow cytometry) exceeded 80%. Isolation of G2 + M-phase cells was not as efficient, although the purity of these subpopulations was consistently greater than of 50%, an approx. 10-fold enrichment over unseparated suspensions of cells. Comparison of IgG2a-Fc-receptor-mediated phagocytic activities among the phase-enriched subpopulations showed that cells in G2 had appreciably enhanced activity.     

Establishment and characterization of a new murine mammary tumor cell line,balb/c-MC

Summary A new murine mammary tumor cell line (BALB/c-MC) was established from a spontaneous mammary tumor in a 17-mo.-old female mouse of the low mammary cancer strain BALB/cHe. The cell line was derived from a papillary adenocarcinoma. In monolayer culture the line exhibits a pavementlike arrangement of cells and forms “domes” or “hemicysts” as the cells become confluent. The cell line rapidly forms tumors when transplanted into young syngeneic BALB/cHe mice. The subcutaneous injection of 10⁶ cells resulted in the development of mammary tumors (typical papillary adenocarcinomas) in 33 of 37 (87% recipients within 2 to 3 mo. after injection. These mammary tumors also metastasize to lung [14 of 33 (42%) of recipients] during this time. The number of chromosomes in this cell line is hyperdiploid (average of 43, range 39 to 44).     

In situ accessibility of murine macrophage gangliosides

Gangliosides are implicated in cell signal transduction. Priorto investigating this phenomenon in macrophages, the in situaccessibility of gangliosides to macromolecules was assessedfor peritoneal macrophages isolated from normal C3H/HeN andendotoxin-hyporesponsive C3H/HeJ mice. C3H/HeJ resident andthioglycolate-elicited macrophage gangiioside patterns are thesame as normal strains, and no strain differences in galactoseoxidase accessibility for resident or thioglycolate-elicitedmacrophage gangliosides were found. The only gangliosides accessibleto galadose oxidase in resident macrophages are G_M1a structures.In thioglycolate-elicited macrophages, an additional ganglieosideis accessible. For Escherichia coli-activated macrophages, whereganglioside distribution differs between strains, a differencein galactose oxidase-accessible gangliosides also exists. Escherichiacoli-activated C3H/HeN patterns show three triplets absent inC3H/HeJ patterns. There were no differences in ganglioside accessibilityto Vibrio cholenre sialidase between the thioglycolate-elicitedC3H/HeJ and C3H/HeN macrophages. However, despite differencesin sialidase-sensitive ganglioside content between E.coli-activatedmacrophages of these strains, sialidase accessibility for E.coli-activatedmacrophages was also similar. Sialidase-susceptible G_M3. wascryptic in either strain under all conditions examined. Theaccessibility of murine macrophage gangliosides to galactoseoxidase or sialidase was independent of their sialic acid speciesand chain length of the ceramide fatty acid. With the exceptionof G_M3, major murine macrophage gangliosides are accsdble insitu to macromolecules, especially to exogenous pathogenic bacterialsialidase which can alter macrophage cell surface characteristics.Altered macrophage ganglioside accessibility appears sometimesas a consequence, but not a cause, of C3H/HeJ endotoxin hyporesponsiveness. accessibility galactose oxide gangliosides macrophages sialidase     

Insertional activation of N-myc by endogenous Moloney-like murine retrovirus sequences in macrophage cell lines derived from myeloma cell line-macrophage hybrids.

Hybrids formed from a myeloma cell line, NS1, and macrophages initially show myeloma properties but later, after loss of the parental macrophage genome and consequent loss of myeloma characteristics, express macrophage properties. Molecular studies demonstrated that macrophage properties in the hybridomas originate from the NS1 parental cells (M. Setoguchi, S. Yoshida, Y. Higuchi, S. Akizuki, and S. Yamamoto, Somatic Cell Mol. Genet. 14:427-438, 1988). In such hybrids, N-myc was activated by insertion of endogenous Moloney-like retrovirus sequences into mouse N-myc exon 3 when the hybrids gained macrophage properties. Interestingly, expression of N-myc took place in all aged hybrids. These results suggest that such unique insertional mutagenesis occurs in a regionally specific manner and that expression of N-myc may play a role in hematopoietic lineage conversion.     

Altered N-glycosylation in macrophage x melanoma fusion hybrids.

It was recently reported that a majority of hybrids generated in vitro between weakly metastatic mouse Cloudman S91 melanoma cells and human or mouse macrophages showed enhanced metastatic potential (Rachkovsky et al., 1998). With few exceptions, hybrids with enhanced metastatic potential also had elevated basal melanin content, enhanced chemotactic responses to fibroblast-conditioned media, and stronger responsiveness to MSH compared to parental cells. Analyses revealed that altered N-glycosylation in metastatic hybrids could explain the multiple phenotypic changes. Tyrosinase, TRP-2 and LAMP-1 from hybrids migrated more slowly on gels compared to the same proteins from parental melanoma cells, consistent with increased glycosylation. Migration of LAMP-1 from hybrids was similar to that from peritoneal macrophages which also appeared to be more heavily glycosylated than LAMP-1 from Cloudman cells. The incorporation of 3H-glucosamine, as a marker of N-glycosylation, into tyrosinase and LAMP-1 was found to be elevated in hybrids, suppressed by N-glycosylation inhibitors and stimulated by MSH to a greater degree in hybrids compared to parental cells. These results indicate N-glycosylation as an important regulatory pathway for MSH-induced melanogenesis, and further suggest that altered N-linked glycosylation may be an underlying mechanism for regulation of both melanogenesis and metastasis in macrophage x melanoma hybrids.     

Replication-competent hybrids between murine leukemia virus and foamy virus

Replication-competent chimeric retroviruses constructed of members of the two subfamilies of Retroviridae, orthoretroviruses and spumaretroviruses, specifically murine leukemia viruses (MuLV) bearing hybrid MuLV-foamy virus (FV) envelope (env) genes, were characterized. All viruses had the cytoplasmic tail of the MuLV transmembrane protein. In ESL-1, a truncated MuLV leader peptide (LP) was fused to the complete extracellular portion of FV Env, and ESL-2 to -4 contained the complete MuLV-LP followed by N-terminally truncated FV Env decreasing in size. ESL-1 to -4 had an extended host cell range compared to MuLV, induced a cytopathology reminiscent of FVs, and exhibited an ultrastructure that combined the features of the condensed core of MuLV with the prominent surface knobs of FVs. Replication of ESL-2 to -4 resulted in the acquisition of a stop codon at the N terminus of the chimeric Env proteins. This mutation rendered the MuLV-LP nonfunctional and indicated that the truncated FV-LP was sufficient to direct Env synthesis into the secretory pathway. Compared to the parental viruses, the chimeras replicated with only moderate cell-free titers.     

Isolation and partial characterization of messenger RNA, from murine T cell hybrids, coding for suppressive immunoglobulin G-binding factor

Poly A RNA has been isolated from a murine T cell hybridoma ( T2D4 ) that spontaneously secretes suppressive immunoglobulin G-binding factor ( IgGBF ). Translation products, obtained from a rabbit reticulocyte lysate translation system and after injection into Xenopus laevis oocytes, contain material with the biologic activity, the affinity, and the m.w. of murine IgGBF ; it suppresses secondary in vitro IgG antibody production in a dose-dependent fashion. The suppressive factor binds to IgG but not to IgM immunoadsorbents and, after mild NaDodSO4 treatment, dissociates in NaDodSO4 polyacrylamide gels into two peaks at 78 and 40 kD. Translation products from two non- IgGBF -secreting cell lines (BW-5147, a T lymphoma line, and A9, a fibroblast cell line) fail to exert any suppressive activity. On sucrose gradients, the RNA responsible for the biologic activity was found in one major peak located at 11S. IgGBF synthesized in a cellfree translation system by using poly A RNA and sucrose gradient fractions was also characterized by immunoprecipitation with Fc fragments of [35S]methionine-labeled proteins. On NaDodSO4 polyacrylamide gels, it migrates in one peak located at 37 kD. We conclude that IgGBF is coded for by 11S poly A RNA and that no post-translational modifications (other than proteolytic cleavage) are necessary to obtain a biologically active factor with Ig-binding properties.