Characterization of human tumor necrosis factor produced by peripheral blood monocytes and its separation from lymphotoxin.

Cultures of human peripheral blood leukocytes (PBL) induced with phytohemagglutinin (PHA) and the phorbol ester 12-0-tetradecanoylphorbol 13-acetate (TPA) produced two types of cytotoxic proteins, indistinguishable in the in vitro assay employing murine L 929 cells as targets. One of these proteins had the antigenic and physicochemical properties of lymphotoxin (LT). We have identified the other cytotoxin as tumor necrosis factor (TNF), mainly on the basis of antigenic cross-reactivity demonstrated with antiserum to TNF, and also by its characteristic physicochemical properties and cell source. Unlike LT, PBL-derived TNF did not bind to Concanavalin A-Sepharose or to several other agglutinin-Sepharose columns specific for carbohydrate moieties common in glycoproteins. The molecular weight of native TNF determined by gel filtration was approximately 40,000 while SDS-PAGE revealed a single sharp peak of 16,500 +/- 500. When cultures of monocytes and lymphocytes separated by elutriation were stimulated with PHA and/or TPA, monocytes were the major source of TNF. In contrast, only lymphocytes produced LT. A mixture of antisera to TNF and LF neutralized all cytotoxicity of crude human lymphokine preparations for L 929 cells, suggesting that TNF and LT are either the only, or the major, cytotoxic proteins present in such crude lymphokine preparations demonstrable in this assay.

HGPRT- mutants of V79 cells that revert specifically by base pair substitution and frameshift mutations.

In order to determine the mutagenic specificity of mutagenic and carcinogenic agents in mammalian cells, a reversion system capable of distinguishing between frameshift mutations and various kinds of base pair substitutions would be useful. We report here a method for the isolation and characterization of HGPRT- Chinese hamster V79 cell mutants that might form the basis for such a system. Two mutants of different specificity have been partially characterized. DEW-1, isolated following N-methyl-N'-nitro-N-nitrosoguanidine (MNNG) treatment, is revertible by the base pair substitution mutagens MNNG and ethyl methanesulfonate (EMS), but not by frameshift mutagens. DSW-3, isolated following ICR-191 treatment, is specifically reverted by frameshift mutagens, but not by EMS or MNNG. With the further characterization of these and other mutants, it should be feasible to determine not only whether an agent is mutagenic in V79 cells, but also to determine the type(s) of mutation(s) it produces.

Interrelationships of human interferon-gamma with lymphotoxin and monocyte cytotoxin.

Crude preparations of interferon (IFN)-gamma derived from human peripheral blood leukocyte (PBL) cultures induced with 12-O-tetra-decanoylphorbol-13-acetate (TPA) and phytohemagglutinin (PHA) were more cytotoxic to HeLa cells than partially purified nautral or highly purified recombinant human IFN-gamma preparations. Conditioned media from PBL cultures contained, in addition to IFN-gamma, a mixture of cytotoxins, including classic lymphocyte-derived lymphotoxin (LT), and a TPA-induced cytotoxic activity produced by the adherent cell population (presumably monocytes). These two types of cytotoxins, indistinguishable in the mouse L929 cell LT assay, could be differentiated by an antiserum prepared against LT derived from the B lymphoblastoid cell line RPMI 1788. This antiserum neutralized lymphocyte-derived classic LT but failed to neutralize the activity of the monocyte-derived cytotoxin. Processing of conditioned media by sequential chromatography on silicic acid, Con A-Sepharose, and DEAE-Sephacel failed to separate IFN-gamma from the LT activity. However, this procedure did remove the monocyte-derived cytotoxic activity present in the original starting material, leaving predominantly classic LT. This LT showed a slightly basic isoelectric point (pI 7.6) which partially overlapped the more basic pI range of IFN-gamma. The two lymphokine activities also could not be completely separated by fast protein liquid chromatography or molecular sieve chromatography. LT in these partially purified preparations was associated with a protein having an apparent molecular weight of 58,000 on gel filtration. This form dissociated partially into a 20,000 mol wt species after denaturation with 0.1% NaDodSO4. IFN-gamma could be selectively removed from preparations containing both IFN-gamma and LT with the aid of monoclonal antibody to IFN-gamma. The addition of purified LT to purified E. coli-derived recombinant human IFN-gamma resulted in a marked synergistic enhancement of cytotoxicity for HeLa cells.

Stimulation of lymphokine production by teleocidin, aplysiatoxin, and debromoaplysiatoxin.

Previous studies showed that the tumor-promoting phorbol ester 12-O-tetradecanoylphorbol 13-acetate (TPA) and several structurally related tumor-promoting compounds stimulate lymphocytes to produce immune interferon (IFN-gamma) and interleukin 2 (IL-2). This study shows that three compounds structurally unrelated to TPA, previously shown to mimic TPA in some other biological activities, are similar to TPA in stimulating IFN-gamma and Il-2 production in cultures of human peripheral blood lymphocytes. The production of another lymphokine, termed lymphotoxin (LT), was also enhanced by TPA and the other three compounds examined. Maximal enhancement of lymphokine production was observed in cultures costimulated with TPA or one of the other tested compounds and phytohemagglutinin (PHA). TPA was separated from IFN-gamma during a multistep purification procedure.

Coproduction of interleukin-2 and interferon-gamma in human mononuclear cells.

Interleukin-2 (IL-2) and immune interferon (IFN-gamma) production was studied in Ficoll-hypaque purified human mononuclear cells derived from plateletpheresis residues. As previously reported, costimulation by 5 ng/ml of 12-O-tetradecanoylphorbol-13-acetate (TPA) and 5 microgram/ml of phytohemagglutinin (PHA) caused a marked enhancement of IFN-gamma levels compared to the yields obtained with PHA alone. IL-2 levels were also increased 50-300 times by this induction protocol. Mezerein (MZN), a compound structurally related to TPA, was found to be similar to TPA in enhancing IFN-gamma and IL-2 levels. In addition to TPA and MZN, four other related phorbol esters caused a stimulation of IFN-gamma and IL-2, with the production of IL-2 paralleling the production of IFN-gamma. Kinetic experiments indicate that low levels of IL-2 first became detectable 6 h after induction, whereas IFN-gamma could be demonstrated only later. Furthermore, IL-2 production reached a plateau earlier than IFN-gamma production in the TPA/PHA treated cultures. Dialysis at pH 2 abrogated the IFN-gamma activity but not the IL-2 activity. A three-step purification procedure developed for IFN-gamma isolation effectively separated IFN-gamma from IL-2. Our results show that there is a close correlation between the magnitude of IL-2 and IFN-gamma production under the experimental conditions employed. Physical separation of the two lymphokines is important for future studies on the interactions between IFN-gamma and IL-2 in various cellular immune reactions.

Demonstration of recovery from the potentially mutagenic effects of ultraviolet light by replication-inhibited Chinese hamster V79 cells.

The effects of replication-inhibiting conditions on the ability of Chinese hamster (V79) cells to recover from the potentially mutagenic effects of ultraviolet (UV) light were investigated. V79 cells were synchronized by a new technique using a low concentration of hydroxyurea (0.2 mM), which provided a mildly-toxic, nonmutagenic method for producing large quantities of synchronized cells needed for these studies. The protocol developed for this study involved UV-irradiation of synchronized V79 cells which were blocked at the Gl/S boundary. Following UV-irradiation, the cells were either allowed to enter S phase immediately or were blocked for increasing periods of time by the addition of more hydroxyurea. The former cells contained the highest frequencies of ouabain-resistant mutants, while cells whose replication was blocked following UV-irradiation showed decreasing mutation frequencies with respect to time. Therefore, V79 cells are able to demonstrate liquid holding recovery from potentially mutagenic UV-lesions. Since the UV-induced mutation frequency was reduced by almost 50% following 6 h of 'liquid holding', the mutagenic lesions seem to be removed at a faster rate than has previously been reported for the removal of pyrimidine dimers from these cells.

Inhibition of DNA synthesis is not sufficient to cause mutagenesis in Chinese hamster cells.

Experiments were designed to test whether the inhibition of DNA synthesis in Chinese hamster V79 cells would result in increased mutagenesis by a mechanism similar to "SOS repair" in E. coli. Treatment of cells for 16 hours with excess of the deoxynucleosides TdR, UdR, AdR and GdR was mutagenic, whereas treatment with hydroxyurea demonstrated no mutagenic effect. The mutagenicity of TdR could be reversed by the addition of CdR. In E. coli, inhibition of DNA synthesis by a short exposure to hydroxyurea resulted in the induction of lambda prophage and increased mutagenesis. These results show that whereas the presence of a stalled replication fork in E. coli can result in mutagenesis via induction of the "SOS system", the same phenomenon does not seem to occur in Chinese hamster V79 cells. The mutagenic mechanism of high concentrations of deoxynucleosides in Chinese hamster V79 cells is likely to be due to replication errors which result from alterations in deoxynucleotide pools.

Effects of inhibitors of de novo protein synthesis on UV-mutagenesis in Chinese hamster cells. Evidence against mutagenesis via inducible, error-prone DNA repair.

Experiments were designed to test whether UV-mutagenesis in animal cells requires an inducible error-phone DNA-repair system similar to the "SOS system" in E. coli. Chinese hamster (V79) cells were exposed to either 5,6-dichloro-1-beta-D-ribofuranosylbenzimidazole (DRB, a transcription inhibitor), cycloheximide, or puromycin for various times (3-6 h) following UV-irradiation (2-8 J/m2). Post-irradiation treatment with DRB resulted in a reproducible enhancement of UV-induced mutagenesis, whereas post-irradiation treatment with either cycloheximide or puromycin resulted in decreased UV-mutagenesis. Thus, the frequency of UV-mutagenesis does not appear to be dependent on an inducible error-prone DNA-repair pathway, since all 3 agents share the ability to inhibit de novo protein synthesis. In order to understand the effects of these inhibitors on mutation frequency, DNA synthesis in the presence of these agents was examined. DRB stimulated DNA synthesis in both irradiated and unirradiated cells. On the other hand, cycloheximide and puromycin caused an immediate inhibition of DNA synthesis in both irradiated and unirradiated cells. Therefore, it appears that UV-mutagenesis reflects changes in post-irradiation DNA synthesis rather than post-irradiation de novo protein synthesis.