Effects of selected herbicides on cytokine production in vitro.

To evaluate possible deleterious effects of commonly used herbicides on leukocytes, cytokine production was selected as a sensitive indicator. After in vitro exposure of human peripheral blood mononuclear cells from normal donors, the production of all 3 cytokines tested--interferon-gamma (a type 1 cytokine), interleukin-5 (a type 2 cytokine) and tumor necrosis factor-alpha (an inflammatory cytokine)--was impaired by up to 70, 50 and 70% respectively in a concentration-dependent manner in cultures exposed to atrazine (0.03-3 microM in 1% dimethylsulfoxide, DMSO). The effect paralleled that seen with dexamethasone, a known immunosuppressive agent. Other pesticides also dissolved in DMSO--mecoprop, simazine or MCPA (each up to 1 microM)--or dissolved in phosphate-buffered saline--diuron (up to 1 microM), isoproturon (up to 3 microM), metoxuron (up to 8 microM) or metamitron (up to 80 microM)--showed no concentration-related effects on cytokine production. There was, however, an inhibition of IFN-gamma and TNF-alpha production by simazine, metoxuron and mecoprop and of all three cytokines tested by diuron. MCPA (0.01 and 0.1 microM) stimulated the production of TNF-alpha. Thus, exposure to herbicides leading to plasma levels in the micromolar range induces imbalance in cytokine production.

Use of the fluorescence-activated cell sorter (FACS) for in vitro assays of developmental toxicity.

Our objective is to predict embryotoxicity with reliable in vitro techniques. In several experimental systems, differentiation is accompanied by changes in the glycosylation pattern of cell-surface glycoconjugates. This is also the case with embryonal carcinoma cells. We have monitored the expression of receptors for wheat germ agglutinin (WGA). Murine embryonal carcinoma cells (P19 and F9) were exposed in vitro to xenobiotics for 1-3 days, then incubated successively with WGA-biotin (15 mug/ml) and streptavidin-phycoerythrin (SA-PE) (20 mug/ml), each for 30 min at room temperature. Cell-surface fluorescence was then analysed using a fluorescence-activated cell sorter (FACS). Exposure to 1 mum retinoic acid, a known inducer of differentiation, altered glycosylation as indicated by changes in WGA binding. Clear-cut effects were also observed after exposure to salts of arsenic (20 muM), or nickel (50 mum), and to methotrexate (1 mug/ml), fluorouracil (1.3 mug/ml) or actinomycin D (0.04 mug/ml). These compounds affected the percentage of positive cells the intensity of labelling, or both. Two non-teratogenic compounds (metronidazole and sulfonilamide) have also been tested and had no effect. Lectin histochemistry of embryonal carcinoma cells exposed to potentially toxic agents holds promise as a method for predicting embryotoxicity. FACS analysis allows rapid quantification.

Expression of IL-6 mRNA in normal rat and human pituitaries and in human pituitary adenomas.

Cells expressing IL-6 mRNA were detected by in situ hybridization in normal pituitaries. In normal untreated rat pituitary the expression was very low. Within hours after IP administration of liposaccharide, IL-6 mRNA accumulated in the anterior lobe of the pituitary. Production of IL-6 was monitored after dissociation and culture of pituitary cells. High levels (8000 pg/ml) were recovered after 72 hr in culture. In normal human pituitaries, less than 1% of cells expressed IL-6 mRNA or IL-6 receptor mRNA (IL-6-R mRNA). In gonadotropinomas, prolactinomas, and non-functioning adenomas, only rare, scattered positive cells were found for either IL-6 or IL-6-R. In contrast, both genes were highly expressed in ACTH- and GH-secreting tumors at the junction of adenoma and infiltrating fibrous tissue and around blood vessels. The combined expression of IL-6 and IL-6-R suggests that IL-6 acts in an autocrine or in a paracrine way in ACTH and GH adenomas.

The transcription factor Pit-1/GHF-1 is expressed in hemopoietic and lymphoid tissues.

The expression of the Pit-1/GHF-1 transcription factor (hereafter Pit-1), which controls the expression of growth hormone (GH) and prolactin (PRL) in the pituitary gland, has been documented in human and rat hemopoietic and lymphoid tissues and cell lines. Pit-1 mRNA was detected by in situ hybridization in about 1% of rat bone marrow cells and in the spleen red pulp and marginal zone. Pit-1 was also expressed in human tonsils (mantle zone), in the thymus (rat and human, non-lymphoid cells), in lipopolysaccharide-stimulated rat peritoneal cells and in non-hepatocyte cells in the liver (rat and human). A detailed investigation of the rat spleen showed a very similar distribution for Pit-1, GH and PRL mRNA and Pit-1, GH and PRL proteins (detected by immunocytochemistry). Using polymerase chain reaction followed by Southern hybridization, the expression of Pit-1 could be confirmed in human and rat spleen, bone marrow and thymus. HL60 and RAJI leukemic cells were also positive. The sequence of fragments amplified from rat spleen and from human bone marrow completely matched published sequences of rat and human pituitary Pit-1, respectively. Expression of GH and PRL in lymphoid tissues has been documented. The straightforward hypothesis would therefore be that Pit-1's main function in lymphoid tissues is controlling GH and PRL expression, as in the pituitary gland. GH and PRL may be hemopoietic and lymphoid growth and differentiation factors.

Effect of glycosylation inhibitors on N-glycosylpeptides and on invasion of malignant mouse MO4 cells in vitro.

Cell surface glycans are believed to play a role in tumour invasion and metastasis. Yet, we have previously shown that the inhibitors of N-linked glycan processing swainsonine (SW) and 1-deoxynojirimycin (dNM) did not prevent invasion of chick heart fragments by MO4 murine fibrosarcoma cells in organ culture. We now present biochemical evidence that these and other inhibitors of processing were indeed effective in remodeling glycans, including those expressed at the cell surface. After metabolic labeling with tritiated mannose or fucose, glycosylpeptides were obtained by Pronase treatment of material released from intact cells by trypsin. Glycosylpeptides were separated by Biogel P-10 chromatography. With all drugs tested, there was a shift towards lower molecular weight of the glycan chains. There were, however, major quantitative differences between the different drugs and also, for monensin (MON; 0.1 microgram ml-1), between fucose-labeled and mannose-labeled chains. The shift in apparent molecular weight affected mainly fucose-labeled peptides after treatment of MO4 cells with SW (0.4 microgram ml-1). The shift induced by dNM (10 mM) + SW (0.4 microgram ml-1) in both fucosylated and mannosylated chains was much larger than that induced by SW given alone. 1-Deoxymannojirimycin (dMM; 1 mM) had major effects on both mannose and fucose-labeled structures and so did N-methyl-1-deoxynojirimycin (MdNM; 2 mM) and castanospermine (CS; 100 micrograms ml-1). With the latter drugs, incorporation of fucose in complex-type glycosylpeptides was dramatically reduced. The effect of SW on fucose-labeled glycosylpeptides of embryonic chick heart was similar to that observed on MO4 cells.(ABSTRACT TRUNCATED AT 250 WORDS)

Reciprocal lymphoid cell homing studies using wild and LPR (lymphoproliferation) mutant C57BL mice.

The injection of lymphoid cells from adult lpr mice into normal and athymic congenic mice does not transfer the lpr (lymphoproliferation) syndrome. We studied whether this phenomenon could be due to abnormal homing. The lymphoid cells from lpr donors do not show a marked deficiency of migration to lymphoid organs in comparison with cells from Wild donors and a T-cell lymphoma BL/VL3. The lymphoid organs of lpr recipients do not show an intrinsic abnormality as homing sites for lymphoid cells. The data reveal some minor migration preferences: the lpr cells migrate better than Wild cells into lpr lymph nodes (including athymic lpr hosts), whereas Wild cells migrate slightly better than lpr cells into Wild lymph nodes. In spite of such minor preferences, Wild cells can efficiently migrate into lpr lymphoid organs, as well as lpr cells into Wild lymphoid organs. Thus, the lack of transfer of lymphadenopathy in Wild recipients cannot be attributed to an alteration of lpr cell homing.

The effect of prenatal or early postnatal irradiation on the production of anti-arsonate antibodies and cross-reactive idiotypes.

Preliminary studies on the long-term effects of prenatal and early postnatal irradiation on the immune response to arsonate were performed using A/J mice. Pregnant mice were irradiated (0.5 Gy, X-rays) or sham-irradiated on a single occasion during gestation (between day 5 and 18 post-conception). Alternatively, newborn mice received the same treatment between day 2 and 7 after birth. Mice were immunized with keyhole limpet haemocyanin-arsonate (KLH-Ars) in adjuvant from 2 months after birth. The levels of specific antibodies to arsonate (anti-Ars) were measured by radioimmunoassay. In addition, the Ars-related cross-reactive idiotype (CRIA) was measured by the haemagglutination technique. In the primary response the titre of anti-Ars was reduced in animals that had been irradiated between day 12 and 15 of gestation. In the second response, in contrast, they had increased levels of anti-Ars. After immunization with KLH-Ars, high levels of CRIA were observed in all groups. However, in mice irradiated 18-20 days after conception the level of CRIA was often much higher than the level of anti-Ars, indicating that a large proportion of the CRIA-positive molecules were not specific for Ars. Thus, in this particular case, some specificity of the immune response was lost after irradiation. The expression of recurrent idiotypes may be a sensitive indicator of immunological perturbations after irradiation.

Effects of radioprotective glycans on the arrest of blood-borne lymphoma cells.

The mode of action of radioprotective glycans is not understood. In view of the known importance of cell migration in haematology, on the one hand, and of carbohydrates in homing processes, on the other, we have investigated the effect of several glycans on blood-borne arrest of lymphoma cells. Radioprotective glycans (but not heparin) modified the arrest of injected cells in the spleen. Altered recirculation and homing processes may play a role in the radioprotective properties of glycans.

Membrane carbohydrates of lymphoid cells: the receptor for interleukin 2.

The contribution of sialic acids and of N-linked sugars to the biological activity of the receptor for IL 2 has been evaluated by treating activated cells with Neuraminidase or by growing them in the presence of inhibitors of N-linked glycosylation or processing. After treatment with Neuraminidase, Con A-activated spleen cells had not lost their ability to bind IL 2. The IL 2-absorbing capability was, however, strongly reduced after trypsinisation. 6 hours after Trypsin treatment, this property was again expressed. Proliferation of the IL 2-dependent CTLL cells was normal in the presence of Swainsonine but strongly impaired in the presence of Tunicamycin. Glycosylation of the IL 2 receptor may thus be required, but integrity of the sugars is not critical.

Effect of inhibitors of glycosylation and carbohydrate processing on invasion of malignant mouse MO4 cells in organ culture.

Inhibitors of glycosylation and carbohydrate processing were used to investigate the role of carbohydrates exposed at the cell surface in invasion. Malignant mouse MO4 cells were confronted with embryonic chick heart in organ culture, an assay shown to be relevant for a number of aspects of invasion in vivo. Tunicamycin (1.0 microgram/ml), 2-deoxy-D-glucose (100 mM), beta-OH-norvaline (1.0 mM), and Monensin (0.1 microgram/ml) reversibly inhibited the invasion of MO4 cells. At these concentrations the drugs also inhibited the growth of MO4 cells. 1-Deoxynojirimycin (10mM), swainsonine (0.4 microgram/ml), and Marcellomycin (0.1 microgram/ml) permitted invasion. Marcellomycin also reversibly inhibited the growth of MO4 cells. These results show that drugs known to interfere with the glycosylation or processing of carbohydrate chains of glycoproteins in different ways have different effects on the invasion of MO4 cells in vitro.

Modification of blood-borne arrest properties of lymphoma cells by inhibitors of protein glycosylation suggests the existence of endogenous lectins.

The requirement for intact carbohydrates of glycoproteins at the cell surface was investigated after treatment of lymphoma cells with compounds which interfere at different steps in N-linked glycosylation: swainsonine and 1-deoxynojirimycin act at different levels during the processing, so that complex oligosaccharides cannot be formed; 2-deoxyglucose, beta-hydroxynorvaline, and tunicamycin completely prevent the formation of N-linked (high-mannose as well as complex) oligosaccharides. The role of sialic acid was investigated by treating the cells with neuraminidase. These treatments resulted in altered patterns of surface-labelled glycoproteins after SDS-polyacrylamide gel electrophoresis. Blood-borne arrest of lymphoma cells in the spleen was sensitive to neuraminidase and to treatments interfering with the processing of complex N-linked oligosaccharides. It is suggested that carbohydrates are signals for cellular interactions involved in the recirculation and homing behaviour of lymphoid cells and probably interact with endogenous lectins at their site of homing.

Integrity of glycoprotein complex sugars is required for homing but not for several other membrane-mediated functions.

In order to correlate the biochemistry of cell surface carbohydrates with cell function, we have treated cells with swainsonine and followed the biochemical and functional modifications induced by this compound. After treatment with swainsonine, surface glycoproteins had a lower apparent molecular weight and a higher isoelectric point. This is compatible with the replacement of complex carbohydrates by hybrid or high-mannose carbohydrates. Several functional tests were unaffected. However, swainsonine-treated cells displayed an altered pattern of in vivo homing, suggesting that carbohydrates play a role in this process.

Surface proteins of radiation-induced and radiation leukemia virus-induced thymic lymphosarcomas in mice.

Thymic lymphosarcomas (TLS) were induced in C57BL mice by X-rays or by Radiation Leukemia Virus (RadLV) and their surface glycoproteins (gps) compared after cell-surface radioiodination and polyacrylamide gel electrophoresis (SDS-PAGE). All lymphocytic antigens tested (T200, 170/100, Thy-1) and proteins with apparent molecular weight (Mr) around 120,000 and 100,000 were present on all tumours, as well as retrovirus--encoded proteins but considerable variation in the Mr of several serologically-related proteins was observed. Therefore, the TLS in C57BL mice form a heterogeneous group, suggesting that T cells can be transformed at different stages of maturation. The possibility that transformation allows or even triggers differentiation is also entertained.

The Thy-1 glycoprotein on nerve cells in culture: electron-microscopic analysis and biochemical characterization.

The distribution and some biochemical properties of the Thy-1 glycoprotein, an antigen common to brain and thymus-derived lymphocytes, were analyzed in primary nerve cell cultures from mouse embryos. The Thy-1 antigen was detected on the plasma membrane of most neurons and fibroblast-like cells and on some astrocytes. The labelling pattern was homogeneous, except on some neuronal cell bodies where the distribution varied from a few patches to a homogeneous labelling. The Thy-l molecules immunoprecipitated from either nerve cell cultures or from fibroblast-like cells from the meninges had similar molecular weights and isoelectric points. In contrast, Thy-1 from thymocytes was more heterogeneous in molecular weight and in isoelectric point range.

A new neuronal marker identified by phosphorylcholine-binding myeloma proteins.

The difficulty of working with the intact brain in vivo has led to the increasing use of nerve cell cultures in neurobiology. However, dissociated cells cannot be unambiguously identified by morphological criteria before the third week in culture, for it is not until then that the basic morphology and size of neurones become stable so that these and other cell types can be easily distinguished. However, cultured neurones can be identified by various cytochemical techniques based on (1) the detection of neurotransmitters or receptors for transmitters, (2) the presence of the Thy 1 antigen and the receptor for tetanus toxin, which are present on the membrane of most neurones, and (3) the presence in neurones of neurone-specific enolase (NSE), a cytoplasmic enzyme, which can only be identified on fixed specimens. Furthermore, other cell types in culture can also be specifically labelled. For instance, antisera to galactocerebroside bind selectively to oligodendrocytes, and antibodies to a neural tumour bind selectively to Schwann cells. We report here the selective interaction of phosphorylcholine-binding myeloma proteins (PC-BMP) with mouse neurones in culture and in suspension. Phosphorylcholine (PC) is found as part of lecithin and sphingomyelin molecules in variable amounts in eukaryotic and prokaryotic membranes, including plasma membranes.

Iodination of cell membranes results in cytologic fixation.

Cells grown as adhering monolayers have been iodinated using the lactoperoxidase technique with glucose and glucose oxidase instead of H2O2. A high degree of cytologic fixation, as evidenced by electron microscopy, resulted from this treatment even in the absence of lactoperoxidase.

Role of antigenic structure in cell to cell cooperation.

Two synthetic polypeptides which differ only in the order of amino acids in their NH2-terminal side chains, namely, (Tyr-Tyr-Glu-Glu)-poly(DLAla)- -poly(LLys) and (Tyr-Glu-Try-Glu)-poly(DLAla)- -poly(LLys), were found to be under different genetic control. By three different in vivo systems for thymus-derived cell depletion, it was demonstrated that (Tyr-Tyr-Glu-Glu)-poly(DLAla)- -poly(LLys), which represents the random poly(Tyr,Glu)-poly(DLAla)- -poly(Lys) in the pattern of immune responses and in the quality of antibodies they elicit, is thymus-dependent whereas (Tyr-Glu-Tyr-Glu)-poly(DLAla)-poly(LLys) does not require thymus-derived cell help for efficient antibody production. Therefore, the two ordered polypeptides which are similar chemically differ in parameters, not yet determined, which affect their capability to trigger bone marrow-derived cells.