Failure to obtain hybridomas between human macrophages and human tumoral U-937 cells is probably due to parental macrophages

Summary Despite more than 50 attempts and the use of various methods, it has been impossible to establish homologous hybridomas between human mature macrophages and 8-azaguanine-resistant U-937 clones prepared in the laboratory. To rule out the possibility that these clones were unsuitable for the selection of hybrids, a study of their properties was done. It was shown that U-937 wild type cells were able to produce HPRT, whereas 8-azaguanine (8-aza)-resistant clones did not. Curiously, exonic and intronic HPRT sequences were undetectable both in wild type and in 8-aza-resistant cell genomes, under conditions where they were detected in control cells. Chromosome analysis of the clone UM9, one of the most frequently used in fusion experiments, revealed many qualitative and quantitative differences with the U-937 wild type cells. 8-aza-resistant U-937 cells were capable of fusion with human macrophages and gave rise to heterokaryons and probably to synkaryons, which survived for weeks without dividing in hypoxanthine-aminopterin-thymidine medium. The results could be interpreted in terms of the existence of a transacting negative regulatory mechanism of the macrophage genome on the proliferative capacity of homospecific hybridomas.     

The study of agglutination of trypsin-treated sheep red cells by Corynebacterium diphtheriae strains

Abstract 620 Corynebacterium diphtheriae strains from 472 sick and healthy persons were studied for their adhesive activity (AA) in direct agglutination of trypsin-treated sheep erythrocytes. Toxigenic strains had more active AA than non-toxigenic ones which was not dependent on the presence of toxin in the culture. Neither biotype nor serotype of the strains correlated with their AA. Several lysotypes among toxigenic and non-toxigenic strains were more active than others. Toxigenic strains from patients had higher AA than those from carriers. Both toxigenic and non-toxigenic strains isolated from the prolonged carriers possessed the highest AA. It was concluded that AA measured in this way was an important colonization factor for all diphtheria strains and a pathogenicity factor for toxigenic strains.     

Stimulation of cell growth in the U-937 human myeloid cell line by honey royal jelly protein

Royal jelly was fractionated by ion-exchange chromatography and a protein (DIII protein) that had growth stimulating activity to the U-937 human myeloid cell line was obtained. The molecular weight of the DIII protein was 58 kDa on SDS-PAGE. The growth stimulating activity of the DIII protein was shown to be relatively heat and pH stable.     

Characteristics of U-937 and HL-60 leukemia cells differentiated by spinach extract

Some characteristics of U-937 and HL-60 leukemia cell lines treated with a fraction of non-dialyzable extract of spinach are reported. The absorbed fraction separated by a DEAE-Tyopearl 650 column chromatography of the non-dialyzable extract induced NBT reducing activity of U-937 and HL-60 cells. This fraction also induced substrate adhesion of U-937 cells, and the non-specific esterase activity of HL-60 cells. The expression of CD11b, CD11c and CD36 antigens on the U-937 cell surface was enhanced by the treatment with the fraction, whereas CD24 antigen was not. The treatment of HL-60 cells with the fraction also induced the expression of CD11b and CD11c antigens, but CD24 and CD36 were not expressed. These results indicated that the non-dialyzable extract of spinach induced immature differentiation of U-937 and HL-60 cells into monocyte/macrophages.Abbreviations NBT nitroblue tetrazolium - TPA 12-O-tetradecanoyl-phorbol-13-acerate - PBS phosphate buffered saline - FITC fluorescein isothiocyanate     

Dynamic process of phagocytosis and forms of macrophage cell death induced by ingestion of apoptotic neutrophils

Clearance of apoptotic neutrophils by macrophages is important for both the successful resolution of acute inflammation and homeostasis.However,the dynamic process of phagocytosis of apoptotic neutrophils by macrophages and the fate of macrophages after the ingestion of apoptotic neutrophils has not been well documented.In the present study,we staged the recognition and tethering,internalization,digestion and exocytosis steps of phagocytosis of apoptotic neutrophils.Furthermore,we found that after the ingestion of apoptotic cells,a subset of macrophages underwent cell death by autophagy,apoptosis or oncosis as revealed by transmission electron microscopy and confocal microscopy combined with specific dyes.The percentage of autophagic,apoptotic and oncotic macrophages were 8.00%±2.00%,12.33%±2.08%,and 3.66%±1.50%,respectively.These results indicated that after ingestion of apoptotic neutrophils,a subset of macrophages undergoes autophagy and apoptosis.We propose that autophagy of macrophages after the ingestion of apoptotic cells may be a new mechanism present in the resolution of inflammation.     

The role of intracellular oxidation in death induction (apoptosis and necrosis) in human promonocytic cells treated with stress inducers (cadmium, heat, X-rays)

Treatment of U-937 human promonocytic cells with the stress inducers cadmium chloride (2 h at 200 microM), heat (2 h at 42.5 C) or X-rays (20 Gy), followed by recovery, caused death by apoptosis and stimulated caspase-3 activity. In addition, all stress agents caused intracellular oxidation, as measured by peroxide and/or anion superoxide accumulation. However, while pre-incubation with antioxidants (N-acetyl-L-cysteine or butylated hydroxyanisole) inhibited the induction of apoptosis by cadmium and X-rays, it did not affect the induction by heat-shock. Pre-incubation for 24 h with the GSH-depleting agent L-buthionine-[S,R]-sulfoximine (BSO) switched the mode of death from apoptosis to necrosis in cadmium-treated cells. By contrast, BSO only caused minor modifacions in the rate of apoptosis without affecting the mode of death in heat- and X-rays-treated cells. BSO potentiated peroxide accumulation in cells treated with both cadmium and X-rays. However, while the accumulation of peroxides was stable in the case of cadmium, it was transient in the case of X-rays. Moreover, the administration of antioxidants during the recovery period sufficed to prevent necrosis and restore apoptosis in BSO plus cadmium-treated cells. Cadmium and X-rays caused a decrease in intracellular ATP levels, but the decrease was similar in both apoptotic and necrotic cells. Taken together, these results demonstrate that (i) stress inducers cause intracellular oxidation, but oxidation is not a general requirement for apoptosis; and (ii) the duration of the oxidant state seems to be critical in determining the mode of death.     

Corynebacterium diphtheriae putative tellurite-resistance protein (CDCE8392_0813) contributes to the intracellular survival in human epithelial cells and lethality of Caenorhabditis elegans

Corynebacterium diphtheriae, the aetiologic agent of diphtheria, also represents a global medical challenge because of the existence of invasive strains as causative agents of systemic infections. Although tellurite (TeO3^2-) is toxic to most microorganisms, TeO3^2--resistant bacteria, including C. diphtheriae, exist in nature. The presence of TeO3^2--resistance (Te^R) determinants in pathogenic bacteria might provide selective advantages in the natural environment. In the present study, we investigated the role of the putative Te^R determinant (CDCE8392_813gene) in the virulence attributes of diphtheria bacilli. The disruption of CDCE8392_0813 gene expression in the LDCIC-L1 mutant increased susceptibility to TeO3^2- and reactive oxygen species (hydrogen peroxide), but not to other antimicrobial agents. The LDCIC-L1 mutant also showed a decrease in both the lethality of Caenorhabditis elegans and the survival inside of human epithelial cells compared to wild-type strain. Conversely, the haemagglutinating activity and adherence to and formation of biofilms on different abiotic surfaces were not regulated through the CDCE8392_0813 gene. In conclusion, the CDCE8392_813 gene contributes to the Te^R and pathogenic potential of C. diphtheriae.     

Priming and activation of mouse macrophages by trehalose 6,6'-dicorynomycolate vesicles from Corynebacterium glutamicum

Vesicles consisting of pure trehalose dicorynomycolate (TDCM), the corynebacterial analog of the most studied mycobacterial glycolipid 'cord factor', were isolated from Corynebacterium glutamicum cells by mild detergent treatment; these induced in vivo a macrophage priming similar to that obtained with mycobacterial-derived trehalose dimycolate. In vitro, both TDCM and bacterial lipopolysaccharide (LPS) induced in macrophages the production of nitric oxide (NO) and tumor necrosis factor-alpha (TNF-alpha), endotoxin tolerance, and were primed for an enhanced secondary NO response to LPS. Interferon-gamma pretreatment did not influence the LPS-induced TNF-alpha response, but considerably increased the TDCM-induced response.     

Fate of ecto-NAD+ glycohydrolase during phagocytosis of normal and mannosylated latex beads by murine macrophages

In order to gain a better understanding of the role of ecto-NAD+ glycohydrolase, an enzyme predominantly associated with phagocytic cells, we have studied its fate in murine macrophages (splenic, resident peritoneal and Kupffer cells) during phagocytosis of opsonized on mannosylated latex beads. In parallel, we have also monitored nucleotide pyrophosphatase, another ecto-enzyme of macrophages. Phagosomes were isolated by flotation in a discontinuous sucrose gradient and the enzyme activities were determined with fluorometric methods. Low levels of NAD+ glycohydrolase and nucleotide pyrophosphatase could be measured associated with the phagosomal fractions, eg, respectively less than 4.5% and 10% in spleen macrophages. The phagosomal activities originate from the plasma membrane, ie they were latent and inactivation of ecto-NAD+ glycohydrolase with the diazonium salt of sulfanilic acid resulted in a marked decrease of this enzyme activity in the phagosomal fractions. Pre-labelling of the cell surface by [3H]-galactosylation indicated that NAD+ glycohydrolase is internalized to a lesser extent than an average surface-membrane unit. These results indicate that if ecto-NAD+ glycohydrolase of macrophages can be internalized to a limited extent during phagocytosis of opsonized or mannosylated latex beads, this enzyme appears to be predominantly excluded from the surface area involved in the uptake of such particles.     

Comparison of the phagocytosis of two types of cyclosporin (SDZ OXL 400 and SDZ IMM 125) by alveolar macrophages from hamsters

The aim of this study was to compare two types of cyclosporin (Cs) particles, SDZ OXL 400 and SDZ IMM 125, the latter being more hydrophilic, to understand their uptake by airway macrophages. Alveolar macrophages (AM), harvested by bronchoalveolar lavage (BAL) of hamster lungs, were cultured with two different doses (0.1 mg and 0.5 mg) for 1 h, 6 h, and 24 h. Control incubations without Cs particles or with latex particles were carried out simultaneously. Cell viability, cell activation (i.e., respiratory burst, interleukin-6 (IL-6) synthesis) and mean volume of particles phagocytosed per macrophage were measured. Both types of Cs particles did not modify the AM viability, and failed to induce IL-6 synthesis during phagocytosis but slightly decreased the cell oxidative respiratory burst. The comparison between SDZ OXL 400 and SDZ IMM 125 showed that for the lower dose the mean volume of both Cs types phagocytosed was similar at 1 h and 6 h. At 24 h an increase of the mean volume phagocytosed was seen for SDZ IMM 125 but not for SDZ OXL 400. For the higher dose the mean volume of SDZ IMM 125 phagocytosed was higher than SDZ OXL 400 at 1 h and 6 h and comparable for both types at 24 h. SDZ IMM 125 particles were phagocytosed more rapidly than SDZ OXL 400. The mean volume of phagocytosed latex particles increased with time and dose and was higher than for both Cs particle types. In conclusion, AM were seen to phagocytose particles of different physical properties (i.e., form, size, and shape), chemical properties (i.e., inert or peptidic) and degrees of hydrophilicity in a different manner.     

Spontaneous rejection of intradermally transplanted non-engineered tumor cells by neutrophils and macrophages from syngeneic strains of mice

It is not surprising that tumors arising spontaneously are rarely rejected by T cells, because in general they lack molecules to elicit a primary T-cell response. In fact, cytokine-engineered tumors can induce granulocyte infiltration leading to tumor rejection. In the present study, we i.d. injected seven kinds of non-engineered tumor cells into syngeneic strains of mice. Three of them (i.e. B16, KLN205, and 3LL cells) continued to grow, whereas four of them (i.e. Meth A, I-10, CL-S1, and FM3A cells) were spontaneously rejected after transient growth or without growth. In contrast to the i.d. injection of B16 cells into C57BL/6 mice, which induces infiltration of TAMs into the tumors, the i.d. injection of Meth A cells into BALB/c mice induced the invasion of cytotoxic inflammatory cells, but not of TAMs, into or around the tumors leading to an IFN-γ-dependent rejection. On day 5, the cytotoxic activity against the tumor cells reached a peak; and the effector cells were found to be neutrophils and macrophages. The i.d. Meth A or I-10 cell-immunized, but not non-immunized, mice rejected i.p.- or i.m.-transplanted Meth A or I-10 cells without growth, respectively. The main effector cells were CTLs; and there was no cross-sensitization between these two kinds of tumor cells, suggesting specific rejection of tumor cells by CTLs from i.d. immunized mice. These results indicate that infiltration of cytotoxic myeloid cells (i.e. neutrophils and macrophages, but not TAMs) into or around tumors is essential for their IFN-γ-dependent spontaneous rejection.     

Thrombospondin-1 differentially regulates release of IL-6 and IL-10 by human monocytic cell line U937

We investigated possible regulatory effects of thrombospondin-1 (TSP-1), a multifunctional extracellular matrix protein, on cytokine release from macrophages. Immobilized TSP-1 enhanced IL-6 release from the human monocytic U937 cells stimulated with phorbol myristate acetate and LPS, whereas it inhibited IL-10 release. The 70-kDa fragment of TSP-1 containing the type 1 repeats showed the same regulatory effects. The enhanced IL-6 release by TSP-1 was inhibited by anti-CD36 antibody or antibody against the sequence of the binding site to CD36 in the type 1 repeats of TSP-1. Conversely, the decrease in IL-10 release by TSP-1 was strengthened by the blocking of the interaction between CD36 and TSP-1. Furthermore, the involvement of TGF-beta1 in the inhibition of IL-10 release by TSP-1 was indicated by the facts that (i) TSP-1 induced activation of TGF-beta1 produced by the U937 cells, (ii) exogenously added TGF-beta1 inhibited IL-10 release, and (iii) antibody against TGF-beta1 blocked the inhibition of IL-10 release by TSP-1. Together, the present findings suggest that TSP-1 enhances IL-6 release from macrophages by interaction with CD36, whereas IL-10 release is regulated by the balance between the enhancing effect of TSP-1 via CD36 and the suppressive effect by TSP-1-activated TGF-beta1.     

Regulation of lymphocyte blastogenesis and antibody production by soluble factor released by a human B-lymphoblastoid cell line

Yam 1B, a human B lymphoblastoid cell line, spontaneously produced an immunoregulatory factor, which suppresses blastogenesis and antibody formation by human lymphocytes. The Yam 1B cells, which were derived from the peripheral blood of an adult T-cell leukemia patient, have been established and maintained in our laboratory since 1985. This cell line expressed mature B-cell surface antigens including surface immunoglobulin M (IgM), CD23, and HLA-DR; had cytoplasmic IgM; and secreted small amounts of IgM in the culture supernatants. Yam 1B was positive for Epstein-Barr virus-associated antigen (EBNA) but negative for adult T-cell-associated antigen (ATLA). The serum-free Yam 1B culture supernatants (SN) inhibited the expression of transferrin R, but neither the expression of interleukin 2 (IL-2) R(CD25) nor the production of IL-2 in the lymphocytes stimulated with phytohemagglutin. Yam 1B SN also inhibited DNA synthesis by human T and B lymphocytes and immunoglobulin generation by normal B cells as well as by Epstein-Barr virus-transformed human B lymphoblastoid cell lines. The inhibitory activity of Yam 1B SN was inactivated at 56 degrees C and at pH 10 but was relatively stable at pH 2. It was abrogated by digestion with pronase and was partially stable by digestion with trypsin. Fractions collected from a Sephacryl S-300 gel filtration column (Pharmacia Fine Chemicals, Uppsala, Sweden) were found to have a peak of inhibitory activity of cell proliferation associated with molecules of apparent MWr of 43,000 to 67,000. The inhibitory activity of Yam 1B SN was not blocked by the anti-transforming growth factor beta antibody.(ABSTRACT TRUNCATED AT 250 WORDS)     

Cytotoxicity of carboplatin on human glioblastoma cells is reduced by the concomitant exposure to an extremely low-frequency electromagnetic field (50 Hz, 70 G)

Glioblastoma multiforme (GBM) is a malignant brain cancer that causes high mortality in patients. GBM responds weakly to the common cancer treatments such as chemotherapy and radiotherapy and even surgery. Carboplatin is an alkylating agent widely used to treat cancer. However, resistance to this drug is a common problem in its use in cancer treatment. Concomitant exposure to extremely low-frequency electromagnetic fields (ELF-EMFs) and carboplatin is one unexplored possibility for overcoming this resistance. Indeed, many lines of evidence show that EMF affects cancer cells and drug action. In this study, we evaluated the effect of concomitant administration of carboplatin and EMF (50 Hz, 70 G) and also concomitant administration of carboplatin and static magnetic field (SMF) (70 G) on human glioma cell line (U-87). The results showed that cotreatment reduced the efficiency of carboplatin in U-87 cells, by decreasing caspase-3 in comparison to drug groups. Overall, EMF reduced the apoptotic effect of carboplatin, possibly through a redox regulation mechanism. Therefore, we have to avoid coadministration of magnetic field (MF) and carboplatin in tumor area, because the MF decreased the toxicity of the drug. However, further studies are needed to reveal the action mechanism of this combination therapeutic method.     

Cantharidin decreased viable cell number in human osteosarcoma U-2 OS cells through G2/M phase arrest and induction of cell apoptosis

ABSTRACT

Cantharidin (CTD), a sesquiterpenoid bioactive substance, has been reported to exhibit anticancer activity against various types of cancer cells. The aim of the present study was to investigate the apoptosis effects and the underlying mechanisms of CTD on osteosarcoma U-2 OS cells. Results showed that CTD induced cell morphologic changes, reduced total viable cells, induced DNA damage, and G₂/M phase arrest. CTD increased the production of reactive oxygen species and Ca²⁺, and elevated the activities of caspase-3 and ?9, but decreased the level of mitochondrial membrane potential. Furthermore, CTD increased the ROS- and ER stress-associated protein expressions and increased the levels of pro-apoptosis-associated proteins, but decreased that of anti-apoptosis-associated proteins. Based on these observations, we suggested that CTD decreased cell number through G₂/M phase arrest and the induction of cell apoptosis in U-2 OS cells and CTD could be a potential candidate for osteosarcoma treatments.     

Regulation of phospholipase D from human hepatocarcinoma cell line by purine nucleotides and protein kinase A

The regulation of phosphatidylcholine-specific phospholipase D by purine nucleotides and protein kinase A were studied in vitro using an enzyme preparation partially purified from the membranous fraction of 7721 hepatocarcinoma cells. It was found that the enzyme activity was elevated by low concentrations of some purine nucleotides, but the activating effects were decreased when the concentrations of the nucleotides were higher. The optimal concentrations of GTP, GTP[S] , GDP and ATP for maximal activation were 0.1mM, 5M,1 mM and 1 mM respectively. The activation caused by 1mM ADP was lower. The enzyme was not activated by 1mM AMP, but significant activation was observed by the addition of 1mM cAMP. The latter was mediated by protein kinase A, as a specific inhibitor of protein kinase A ablished the activation. There were synergic effects between ATP and GTP, ATP and PIP₂, but not between ATP and GTP[S] , or PIP₂ and GTP[S]. The activating effects of GTP and ATP were abolished by neomycin, a PIP₂ scavenger. These results suggest that phospholipase D is regulated by GTP-binding protein and the presence of PIP₂ is required for the activation induced by GTP. Protein kinase A may be another protein kinase in addition to protein kinase C and protein tyrosine kinase which regulate the activity of phospholipase D, when the intracellular concentration of cAMP is increased.     

Establishment of a human hepatocyte line (OUMS-29) having CYP 1A1 and 1A2 activities from fetal liver tissue by transfection of SV40 LT

Summary Immortalized human hepatocytes that can retain functions of drug-metabolizing enzymes would be useful for medical and pharmacological studies and for constructing an artificial liver. The aim of this study was to establish immortalized human hepatocyte lines having differentiated liver-specific functions. pSVneo deoxyribonucleic acid, which contains large and small T genes in the early region of simian virus 40, was introduced into hepatocytes that had been obtained from the liver of a 21-wk-old fetus. Neomycin-resistant immortalized colonies were cloned and expanded to mass cultures to examine hepatic functions. Cells were cultured in a chemically defined serum-free medium, ASF104, which contains no peptides other than recombinant human transferrin and insulin. As a result, an immortal human hepatocyte cell line (OUMS-29) having liver-specific functions was established from one of the 13 clones. Expression of CYP 1A1 and 1A2 messenger ribonucleic acid by the cells was induced by treatment with benz[a]pyrene, 3-methylcholanthrene, and benz[a]anthracene. OUMS-29 cells had both the polycyclic aromatic hydrocarbon receptor (AhR) and AhR nuclear translocator. Consequently, 7-ethoxyresorufin deethylase activity of the cells was induced time- and dose-dependently by these polycyclic aromatic hydrocarbons. This cell line is expected to be instrumental as an alternative method in animal experiments for studying hepatocarcinogenesis, drug metabolisms of liver cells, and hepatic toxicology.