Transformed Macrophage Cell Line Research Articles

Production of Murine Macrophages from Hoxb8-Immortalized Myeloblasts: Utility and Use in the Context of Salmonella Infection.

Salmonella enterica is a Gram-negative intracellular pathogen that causes a range of life-threatening diseases in humans and animals worldwide. In a systemic infection, the ability of Salmonella to survive/replicate in macrophages, particularly in the liver and spleen, is crucial for virulence. Transformed macrophage cell lines and primary macrophages prepared from mouse bone marrow are commonly used models for the study of Salmonella infection. However, these models raise technical or ethical issues that highlight the need for alternative methods. This chapter describes a technique for immortalizing early hematopoietic progenitor cells derived from wild-type or transgenic mice and using them to produce macrophages. It validates, through a specific example, the interest of this cellular approach for the study of Salmonella infection.

Evaluation of Antioxidant, Anti-Inflammatory and Cytoprotective Properties of Ethanolic Mint Extracts from Algeria on 7-Ketocholesterol-Treated Murine RAW 264.7 Macrophages.

The present study consisted in evaluating the antioxidant, anti-inflammatory and cytoprotective properties of ethanolic extracts from three mint species (Mentha spicata L. (MS), Mentha pulegium L. (MP) and Mentha rotundifolia (L.) Huds (MR)) with biochemical methods on murine RAW 264.7 macrophages (a transformed macrophage cell line isolated from ascites of BALB/c mice infected by the Abelson leukemia virus). The total phenolic, flavonoid and carotenoid contents were determined with spectrophotometric methods. The antioxidant activities were quantified with the Kit Radicaux Libres (KRLTM), the ferric reducing antioxidant power (FRAP) and the 2,2-diphenyl-1-picrylhydrazyl (DPPH) assays. The MS extract showed the highest total phenolic content, and the highest antioxidant capacity, while the MR extract showed the lowest total phenolic content and the lowest antioxidant capacity. The cytoprotective and anti-inflammatory activities of the extracts were quantified on murine RAW 264.7 macrophages treated with 7-ketocholesterol (7KC; 20 µg/mL: 50 µM) associated or not for 24 h and 48 h with ethanolic mint extracts used at different concentrations (25, 50, 100, 200 and 400 µg/mL). Under treatment with 7KC, an important inhibition of cell growth was revealed with the crystal violet test. This side effect was strongly attenuated in a dose dependent manner with the different ethanolic mint extracts, mainly at 48 h. The most important cytoprotective effect was observed with the MS extract. In addition, the effects of ethanolic mint extracts on cytokine secretion (Interleukin (IL)-6, IL-10, Monocyte Chemoattractant Protein (MCP)-1, Interferon (IFN)-ϒ, Tumor necrosis factor (TNF)-α) were determined at 24 h on lipopolysaccharide (LPS, 0.2 µg/mL)-, 7KC (20 µg/mL)- and (7KC + LPS)-treated RAW 264.7 cells. Complex effects of mint extracts were observed on cytokine secretion. However, comparatively to LPS-treated cells, all the extracts strongly reduce IL-6 secretion and two of them (MP and MR) also decrease MCP-1 and TNF-α secretion. However, no anti-inflammatory effects were observed on 7KC- and (7KC + LPS)-treated cells. Altogether, these data bring new evidences on the potential benefits (especially antioxidant and cytoprotective properties) of Algerian mint on human health.

Antioxidant and Anti-Inflammatory Effects of Defatted Rice Bran (Oryza SativaL.) Protein Hydrolysates on Raw 264.7 Macrophage Cells

Defatted rice bran (DRB) is a by-product of oil extraction from rice bran (Oryza sativa L.), and has health benefits. This in vitro study investigated the antioxidant and anti-inflammatory effects of four crude DRB protein hydrolysate fractions prepared by pepsin-pancreatin digestion and fractionated by ultrafiltration on a transformed macrophage cell line, RAW264.7. Protein hydrolysates with MW <3 kDa (F4 fraction) had the lowest IC50 for NO-, DPPH˙ and ABTS radical scavenging activities (0.38 ± 0.01, 1.24 ± 0.03 and 0.17 ± 0.01 µg/mL, respectively). The pooled fraction formed after Reverse phase-High-performance liquid chromatography (HPLC) (F4-5) had the highest percentage inhibition of radical scavenging for DPPH˙, and NO- (25.04 ± 0.88, and 6.50 ± 0.39%, respectively). LC-MS of F4-5 revealed peptides exhibiting antioxidant activity were composed of His (H), Trp (W) and Tyr (Y) that known to enhance the scavenging activities of peptides. The F4 fraction suppressed lipopolysaccharide and rmIFN-γ mediated increase in iNOS, IL-6 and TNF-α mRNA levels in RAW264.7 cells, suggesting the interference was mediated at the transcriptional level. Practical Application Defatted rice bran (DRB) is a good source of nutritional proteins. In this study, DRB protein was hydrolyzed using proteases and further fractionated by ultrafiltration. The results demonstrated that DRB hydrolysates and their fractions exhibited antioxidant and anti-inflammatory activities indicating these as a potential source of functional food and medicines.

Cartilage proteoglycan degradation by a mouse transformed macrophage cell line is mediated by macrophage metalloelastase.

Identify and characterize the matrix metalloproteinase responsible for cartilage proteoglycan degradation mediated by a macrophage cell line in a cell culture model that resembles some aspects of rheumatoid pannus. Supernatants from the transformed mouse macrophage cell line J774A.1 were used to purify the proteoglycan degrading activity. J774A.1 macrophage culture supernatants were purified by sequential column chromatography and proteins were identified by zymography, western blotting and amino acid sequence analysis. Cartilage degradation was measured using 35S labeled bovine nasal cartilage. The cartilage degrading proteolytic activity in the mouse macrophage supernatants proved to be due to two major proteins with approximate molecular masses of 48 kDa and 22 kDa that were identified as macrophage metalloelastase (MME). Incubation of purified MME at 37 degrees C for up to 16 h resulted in the processing of the 48 kDa protein to several novel bands including a previously undescribed protein of approximately 25 kDa without accumulation of fully processed 22 kDa protein. A number of proteinases increased the rate of this processing. J774A.1 macrophage metalloelastase degraded cartilage proteoglycan with an efficiency approximately equal to human macrophage metalloelastase (MMP-12) and matrilysin (MMP-7) and twice that of stromelysin-1 (MMP-3). These data identify the cartilage proteoglycan degrading metalloproteinase secreted by J774A.1 macrophages in this cell culture model as MME, and describes mechanisms of activation and processing of this enzyme that may play an important role in cartilage degradation.

Mouse Macrophages Carrying Both Subunits of the Human Interferon-γ (IFN-γ) Receptor Respond to Human IFN-γ but Do Not Acquire Full Protection against Viral Cytopathic Effect

Studies of hamster-human and mouse-human somatic fibroblast hybrids and transfected mouse fibroblasts have demonstrated that signaling through the human interferon-gamma receptor (hu-IFN-gammaR) requires the formation of a complex consisting of ligand (IFN-gamma), a ligand binding receptor chain (IFN-gammaR1), and a signal transducing receptor chain (IFN-gammaR2). To date, the ability of this receptor complex to transduce the full repertoire of biological signals has been difficult to assess due to the limited number of activities that IFN-gamma can exert on fibroblasts. The current report assesses the ability of hu-IFN-gammaR chains to transduce signals in the absence of background human gene products by expressing hu-IFN-gammaR2 in a transformed macrophage cell line (F10/96) derived from a hu-IFN-gammaR1 transgenic mouse. Our results indicate that F10/96 clones expressing both human receptor proteins bind hu-IFN-gamma with an affinity comparable to that of human cells. Binding of either human or mouse IFN-gamma to its respective receptor elicits classic IFN-gamma responses such as up-regulation of major histocompatibility complex antigens, enhanced expression of IRF-1, and increased production of NO2- radicals, interleukin-6, tumor necrosis factor-alpha, and granulocyte macrophage-colony stimulating factor. However, hu-IFN-gamma could not fully protect the clones from cytopathic effects of encephalomyocarditis virus and vesicular stomatitis virus while mo-IFN-gamma could. These results demonstrate that while co-expression of hu-IFN-gammaR1 and hu-IFN-gammaR2 is necessary and sufficient for most IFN-gamma-induced responses, it is not sufficient to confer a generalized antiviral state. These findings further suggest that additional species-specific accessory factor(s) are necessary for full signaling potential through the IFN-gamma receptor complex. The nature and potential role of such factors in IFN-gammaR signaling is discussed.

Nitric oxide inducing factor as a measure of antigen and mitogen-specific T cell responses in chickens

We describe here an assay to measure responses of T cells to in vitro stimulation with antigens and a T cell mitogen (ConA). Spleen cells from chickens immunized with live viruses and an inactivated antigen produced macrophage activating factors (MAF) in response to in vitro stimulation with homologous antigens. The production of MAF, quantitated by the induction of NO in a retrovirus transformed macrophage cell line, HD11 (Beug et al., 1979, Cell 18, 375) was antigen-specific and correlated well with T cell proliferation. Further studies showed that production of MAF was abrogated by cyclosporin A, anti-CD4 and anti-CD8 monoclonal antibodies. These data suggested that production of MAF required T cell activation and can be used as measure of antigen and mitogen-specific T cell responses in chickens.

Interferon-gamma inhibits macrophage insulin-like growth factor-I synthesis at the transcriptional level.

Spontaneous production of insulin-like growth factor-I (IGF-I) by inflammatory macrophages contributes to aberrant wound healing, but little is known about regulation of IGF-I synthesis in myeloid cells. The T cell-derived cytokine interferon-gamma (IFN gamma) inhibits several fibrogenic and angiogenic components of the wound-healing response. We have used metabolic labeling of primary colony stimulating factor-1 (CSF-1)-derived macrophages and a transformed macrophage cell line (PU5-1R) followed by immunoprecipitation to demonstrate that synthesis of the 17 kilodalton (kDa) prepro-IGF-I protein by these cells is substantially inhibited by IFN gamma. An exon 4 IGF-I/beta-actin riboprobe expression cassette was used in RNase protection assays to show that IFN gamma also reduces steady state levels of IGF-I mRNA in three different populations of macrophages in a time- and dose-dependent manner. This effect is specific for IFN gamma because neither the IFNs-alpha/beta nor lipopolysaccharide (LPS) affects expression of steady state IGF-I transcripts. Down-regulation of IGF-I mRNA by IFN gamma is dependent on de novo protein synthesis and is abrogated by coculture with cycloheximide. Nuclear run-on assays revealed that elongation of IGF-I transcripts is absent in fresh bone marrow cells but is induced several-fold after cells are cultured for 6 days with CSF-1. Treatment of these CSF-1-derived macrophages with IFN gamma for 6 h substantially inhibits synthesis of IGF-I mRNA. Studies on the decay of IGF-I mRNA in PU5-1R macrophages treated with an RNA polymerase inhibitor confirmed that the decline in IGF-I steady state mRNA in IFN gamma-treated cultures arises from an inhibition of transcription rather than from a reduction in mRNA stability. Since a variety of inflammatory mediators can induce expression of IGF-I in macrophages, inhibition of macrophage IGF-I synthesis by IFN gamma provides a mechanism by which leukocytes regulate levels of this growth factor in their microenvironment.

Monoclonal c-myc transformed macrophage cell lines. I. Heterogeneity in ability to process and present antigen.

Processing of proteins into immunogenic forms and their subsequent presentation to T cells are mediated by APC. Monocytes and macrophages have long been recognized as one of the APC types. However, little is known about whether functional heterogeneity in processing and presentation exist within the monocyte/macrophage population. Past difficulties in obtaining clonal representatives of these populations have limited investigations in this regard. The c-myc-containing retrovirus MRV, previously shown to immortalize murine macrophages, was used to generate a large panel of macrophage cell clones. Differences observed in cell surface antigen expression and morphology demonstrated phenotypic heterogeneity among these clones. Functional heterogeneity was also observed both before and after IFN-gamma and IL-4 stimulation. The clones differ in their capacity to present several nominal antigens to T cell hybridomas. When parallel variation in ability to present both a nominal antigen and a peptide representing the epitope for which a T cell hybridoma was specific was observed among the clones, this variation correlated with the levels of surface MHC class II antigen the clones expressed. In contrast, diversity in the ability to process and present certain nominal antigens among clones that all presented the corresponding antigenic peptide with similar efficiency did not appear to be due to differences in levels of surface MHC class II molecules. Our results suggest that the macrophage clones are heterogeneous in their ability to both process and present several antigens. The ability to obtain macrophage tissue culture cell lines displaying phenotypic and functional heterogeneity should allow insight into the impact of normal macrophage heterogeneity on the outcome of immune responses in vivo.

The inhibition of cholesterol esterification by cyclandelate in transformed mouse macrophages

Cyclandelate (trimethylcyclohexanyl mandelate) inhibited cholesterol esterification in a transformed mouse macrophage cell line (J774) with a concentration of approximately 20 μM being required for half-maximal inhibition. The intact drug was required for its inhibitory action since neither of its hydrolysis products, trimethylcyclohexanol and mandelic acid, caused any inhibition even at high concentrations. The drug entered the cells very rapidly with inhibition being apparent within the shortest time possible to measure esterification (15 min after drug addition). The rate of cholesterol esterification returned to control values when drug-inhibited cells were incubated in drug-free medium indicating a rapid loss of drug from the cells. Loading of cells with cholesterol had no effect on the inhibitory action of cyclandelate, and the inhibition of esterification of cholesterol appeared to be specific, since the syntheses of phospholipid and triacylglycerol (which also involve the action of acyltransferases) were not affected by the drug. Similar inhibitions of cholesterol esterification were seen in four other cell lines, a human osteosarcoma, Chinese hamster ovary cells, a human transformed macrophage cell line (U937) and human umbilical cord vein endothelial cells, as well as in slices of pig aorta, indicating a general action in extra-hepatic tissues where the drug is not hydrolysed.

Cartilage degradation by cocultures of transformed macrophage and fibroblast cell lines. A model of metalloproteinase-mediated connective tissue degradation.

A number of human and mouse macrophage and fibroblast cell lines were examined for their ability to degrade cartilage proteoglycan in an attempt to establish a cell culture model of cartilage degradation. The mouse transformed macrophage cell line J774A.1 alone or in combination with the mouse transformed fibroblast cell line 10ME HD A.5R.1 were the only cell lines capable of extensively degradating cartilage proteoglycan. Incubation of the macrophage cell line J774A.1 on heat-killed cartilage disks resulted in the release of 36% +/- 8 (mean +/- SEM, n = 5) of the radiolabeled cartilage proteoglycan. The fibroblast cell line 10ME HD A.5R.1 alone did not degrade cartilage. However, cocultures of J774A.1 macrophages and 10ME HD A.5R.1 fibroblasts incubated on cartilage discs resulted in the release of 69% +/- 6 (mean +/- SEM, n = 5) of radiolabeled proteoglycan. There was little degradation of cartilage by macrophage/fibroblast cocultures during the first 3 days of culture. Cartilage degradation increased with each subsequent day in culture from 7% +/- 2 on day 4 to 68% +/- 3 (n = 3) by day 7. Supernatants from the macrophage/fibroblast cocultures were incubated with cartilage discs in the presence of general class-specific proteinase inhibitors. The metalloproteinase inhibitors 1,10 phenanthroline, EDTA, and recombinant tissue inhibitor of metalloproteinase were the only inhibitors that significantly blocked cartilage degradation by coculture supernatant. The cartilage degrading metalloproteinase in the macrophage/fibroblast coculture supernatant eluted as a broad peak on Sephacryl S-200HR with an estimated molecular mass between 22 and 55 kDa. These studies suggest that the macrophage/fibroblast coculture model of cartilage degradation may be a useful experimental system for the study of metalloproteinase-mediated connective tissue degradation.

Expression and complex assembly of calcium-binding proteins MRP8 and MRP14 during differentiation of murine myelomonocytic cells.

In the present study we analyzed expression and biochemical properties of the two recently cloned calcium-binding proteins MRP8 and MRP14, both members of the S-100 family, in murine myelomonocytic cells. Expression of MRPs was found to be restricted to granulocytes and distinct stages of macrophage differentiation when compared to the expression of other markers (Mac-1, F4/80) in transformed macrophage cell lines (M1, RMB.TG, WEHI.TG, J774A, P388D) and resident and exudate peritoneal and tissue macrophages. Similarly, mRNA and protein levels of MRP8/MRP14 in murine bone marrow cells decreased during culture in L cell-conditioned medium. Applying a cross-linking technique, the formation of noncovalently associated heteromeric MRP8/MRP14 complexes of 25, 35, and 48 kd was demonstrated. Our data suggest that MRP8/MRP14 expression and complexation are characteristic for granulocytes and distinct stages of macrophage differentiation in mice. Analysis of the MRP8/MRP14+ phenotype of macrophages may provide further insights into the mechanisms underlying macrophage differentiation.

Properties of colony-stimulating factors produced by macrophage cell lines and hybrid cells.

Colony-stimulating factors (CSFs) produced by two simian virus 40(SV40) transformed macrophage cell lines (BAM1 and BAM3), and three hybrids (HM3-11, HM3-12, and HM3-14) derived from fusion between BAM3 and a Chinese hamster cell line (hs222-16) were examined. HM3-11 and HM3-14 produce two molecular species of CSF, which are not found in the conditioned media from cultures of BAM1 and BAM3 or lipopolysaccharide (LPS), phorbolmyristate-acetate (PMA), and zymosan-stimulated BAM3. HM3-12, which is classified into another group in terms of CSF secretion, does not produce these two CSFs. On the basis of various criteria, one of these CSF species (peak 1-CSF) was characterized as a macrophage-colony-stimulating factor (M-CSF). The other CSF (peak 2-CSF) induced a group of bone marrow cells in granulocytes and macrophages as well as growth of a mast cell line, IC2. This CSF has an apparent molecular weight of 18,000, estimated by SDS-polyacrylamide gel electrophoresis. Unlike interleukin 3 (IL3) from WEHI-3 cells, the growth factor activity of peak 2-CSF binds to DEAE-Sephacel. Thus, peak 2-CSF is similar to a granulocyte-macrophage colony-stimulating factor (GM-CSF) rather than to IL3. The anti L cell CSF serum does not inhibit the CSF activity in Chinese hamster fibroblast conditioned medium, and the IC2 cells do not respond to Chinese hamster lung conditioned medium (CHLCM), suggesting that peak 1- and peak 2-CSF are of mouse origin.

Murine macrophage cell lines can be ordered in a linear differentiation sequence

The present study investigated whether transformed macrophage cell lines represent certain stages in macrophage differentiation. Cell surface markers linked to macrophage differentiation were characterized in 11 murine macrophage cell lines and compared with markers on isolated resident and exudate peritoneal macrophages. Furthermore, the capacity of the cell lines to phagocytose latex microspheres was analyzed. This analysis indicated that cell lines arrested at an early differentiation stage were characterized by the expression of the ‘immature’ markers Thy-1 and MIV 113, and the lack of expression of ‘mature’ macrophage markers such as Mac-1, Mac-2, and F4/80. More mature cell lines, which do not express ‘immature’ markers, show an increase in the expression of ‘mature’ macrophage markers. Furthermore, the expression of the ‘mature’ markers was found to be correlated with the phagocytic capacity of the cells. We have ordered the cell lines in a linear differentiation sequence based on these data. We propose that this sequence represents various stages in the differentiation of macrophages. This panel of cell lines provides a new model of early macrophage differentiation.

Colony-stimulating factor (CSF-1): its enhancement of plasminogen activator production and inhibition of cell growth in a mouse macrophage cell line.

CSF-1 is a hemopoietic growth factor that specifically causes the proliferation and differentiation of mononuclear phagocytic cells. J774 cells are a monocyte precursor-like macrophage cell line. This transformed macrophage cell line exhibits specific 125I-CSF-I-binding activity similar to that of normal murine macrophages, although its survival and growth is independent of CSF-1. At 0 degrees C, saturation of binding sites was achieved at 240 pM 125I-CSF-1. At 37 degrees C, the bound 125I-CSF-1 was rapidly internalized and degraded by the target cells with a T1/2 of approximately 30 min; degradation was inhibited by the addition of NH4Cl. The addition of CSF-1 to cultures caused dose-dependent inhibition rather than stimulation of [3H]thymidine uptake by J774 cells. Whereas CSF-1 stimulated the clonal growth of normal mouse peritoneal exudate macrophages, it inhibited the clonal growth of J774 cells in agar cultures. Furthermore, CSF-1 exhibited a concentration-dependent enhancement of the production of plasminogen activator (PA) by J774 cells. The enhanced production of PA was detected 6 hr after the addition of CSF-1 and was inhibited by the simultaneous addition of the anti-inflammatory drug. It appears that the effects of CSF-1 on cell proliferation and PA production by CSF-1 receptor-bearing cells are mediated by distinct intracellular pathways albeit through the same receptor.