Cytotoxicity of human peripheral lymphocytes in cell-mediated lympholysis; antibody-dependent cell-mediated lympholysis and natural cytotoxicity assays after mixed lymphocyte culture.

Lymphocytes that have been purified by Ficoll-Hypaque centrifugation lose antibody-dependent and natural cytotoxic activities upon culture in tissue culture medium supplemented with human plasma. However, stimulation of peripheral lymphocytes in the mixed leukocyte culture (MLC) appears to enhance killer (K) and natural killer (NK) activities in addition to generating cytotoxic T ymphocytes. Enhancement of NK and antibody dependent activities appears to correlate with cell division as measured by 3H-thymidine uptake. However, elimination of dividing cells in the MLC by addition of 5-bromodeoxyuridine has no effect on NK and K cells activities. Since this treatment abolishes cell-mediated lympholysis mediated by cytotoxic T lymphocytes, it is a useful probe for determining the relative activities of NK, K, and cytotoxic T lymphocyte effector cells after lymphocyte stimulation.     

Selective inhibition of fibroblasts by spermine in primary cultures of normal human skin epithelial cells

Summary Overgrowth with fibroblasts has been a major problem in the cultivation of normal human skin epithelium. In the present study it is shown that the addition of spermine to the culture medium in micromolar concentrations has a differential cytotoxic effect on fibroblasts allowing the cultivation of human skin epithelial cells in primary culture without fibroblastic overgrowth. Putrescine, another polyamine, is shown to be equally cytotoxic to fibroblasts and epithelial cells when added in millimolar concentrations; below this concentration range no cytotoxic effect could be demonstrated. This difference in cytotoxicity between spermine and putrescine is suggested to depend on the conversion of spermine, but not putrescine, and to highly cytotoxic products by an amine oxidase present in fetal bovine serum. This project was supported by the Novo foundation.     

T cell growth factor: parameters of production and a quantitative microassay for activity.

Several soluble factors have recently been associated with the proliferation and differentiation of thymus-derived lymphocytes. One of these factors present in medium conditioned by T cell mitogen-stimulated lymphocytes has the ability to promote the long-term culture of normal and antigen-specific cytotoxic T cells. We report a method to test for this proliferative stimulus in the form of a sensitive microassay based upon the tritiated-thymidine incorporation of continuous murine tumor-specific cytotoxic T cell lines (CTLL). The microassay requires microliter quantitites of sample fluid and is amenable to quantitative analysis. This highly reproducible, quantitative assay for T cell growth factor (TCGF) has allowed investigation as to the kinetics of TCGF generation and has revealed that T lymphocytes are required for its production. Further investigation has supported the notion that this nonspecies-specific factor is actively removed from tissue culture medium by the proliferation of either T cell mitogen-activated lymphocytes or CTLL.     

Lymphocytes from hepatic inflammatory infiltrate kill rat hepatocytes in primary culture. Comparison with peripheral blood lymphocytes

In the last few years it has become possible in the liver to isolate lymphocytes from inflammatory infiltrates and to culture them in vitro. Most of the lymphocyte clones obtained are CD 8+ cytotoxic cells, but interactions between these lymphocytes and hepatocytes in primary culture have not been analysed previously. In this study, cloned human T lymphocytes from liver biopsies and from the peripheral blood of patients with chronic hepatitis B or primary biliary cirrhosis, after phenotypical and functional characterization into CD 8+ or CD 4+ cytotoxic lymphocytes, were activated in an antigen-independent fashion by adding either anti CD 3 or anti CD 2/R-3 monoclonal antibodies to the cell suspension. The activated cells were then coincubated with rat hepatocytes in primary culture. The killing capacity of the activated lymphocytes was monitored by light and electron microscopy and by measurement of lactic dehydrogenase (LDH)-release into the culture medium. It was found that cytotoxic CD 8+, but not CD 4+ helper lymphocytes very effectively killed hepatocytes. The killing effect was dependent on the time of cocultivation and on effector-target (E/T) ratio. Total breakdown of the hepatocyte monolayer was achieved after 10-20 h coculture and at an E/T ratio of 10 to 1. As LDH-release in the culture medium reached about 80% of the total LDH-content, most of the hepatocytes were lysed by activated lymphocytes. Cytotoxic activity of clones obtained from different biopsies was comparable with that of clones from peripheral blood. Hepatocytes in primary culture seem to be very sensitive to the killing capacity of activated cytotoxic lymphocytes.     

Culture conditions affect cytotoxin production by Serratia marcescens

Abstract Cytotoxins have been implicated in the pathogenesis of bacterial infections. In this study, the influence of different culture conditions was evaluated on cytotoxin production by Serratia marcescens . Parameters such as culture media, incubation temperature, starting pH of culture medium, aeration, anaerobiosis, carbon sources, iron concentration in the culture media, and release of cell-bound toxin by polymyxin B were investigated. The data suggest that this cytotoxin is predominantly extracellular and is not induced by iron limitation. Aerobic culture with shaking resulted in higher cytotoxicity than static aerobic or anaerobic culture. Bacteria grown in glucose, sucrose or galactose were more cytotoxic than those grown in inositol or maltose. The culture conditions that were identified as optimal for cytotoxin production by Serratia marcescens were incubation temperature ranging from 30 to 37°C, in medium adjusted to pH 8.5, with shaking. This work will contribute to further studies on the identification of this cytotoxic activity.     

Analysis of the cytotoxic effects of light-exposed hepes-containing culture medium

Summary The addition ofN-2-hydroxyethylpiperazine-N′-2-ethanesulfonic acid (HEPES) to RPMI 1640 medium markedly increases the production of cytotoxic products during exposure of the medium to visible light. The cytotoxicity has been analyzed by measuring uptake of [³H]thymidine by murine thymocytes cultured in preirradiated medium containing 25 mM HEPES. Complete inhibition of thymidine uptake was produced by exposing 50% of the culture medium to light for 3 h before addition of cells. The HEPES-mediated effect requires only that HEPES and riboflavin be exposed to light; other medium constituents are not necessary. Hydrogen peroxide is a principal cytotoxic agent produced in this system. It is demonstrated that most, but not all, of the inhibition of thymidine uptake can be attributed to hydrogen peroxide.     

Lymphocytes from hepatic inflammatory infiltrate kill rat hepatocytes in primary culture

In the last few years it has become possible in the liver to isolate lymphocytes from inflammatory infiltrates and to culture them in vitro. Most of the lymphocyte clones obtained are CD 8 + cytotoxic cells, but interactions between these lymphocytes and hepatocytes in primary culture have not been analysed previously. In this study, cloned human T lymphocytes from liver biopsies and from the peripheral blood of patients with chronic hepatitis B or primary biliary cirrhosis, after phenotypical and functional characterization into CD 8+ or CD 4+ cytotoxic lymphocytes, were activated in an antigen-independent fashion by adding either anti CD 3 or anti CD 2/R-3 monoclonal antibodies to the cell suspension. The activated cells were then coincubated with rat hepatocytes in primary culture. The killing capacity of the activated lymphocytes was monitored by light and electron microscopy and by measurement of lactic dehydrogenase (LDH)-release into the culture medium. It was found that cytotoxic CD 8 +, but not CD 4 + helper lymphocytes very effectively killed hepatocytes. The killing effect was dependent on the time of cocultivation and on effector-target (E/T) ratio. Total breakdown of the hepatocyte monolayer was achieved after 10–20 h coculture and at an E/T ratio of 10 to 1. As LDH-release in the culture medium reached about 80% of the total LDH-content, most of the hepatocytes were lysed by activated lymphocytes. Cytotoxic activity of clones obtained from different biopsies was comparable with that of clones from peripheral blood. Hepatocytes in primary culture seem to be very sensitive to the killing capacity of activated cytotoxic lymphocytes. Supported by DFG grants Ra 362/5-2 and SFB 311 A7 (G.R.) and A5 (H.P.D.)     

Tumor necrosis factor is cytotoxic to human fibroblasts in the presence of exogenous arachidonic acid

Recombinant human tumor necrosis factor (TNF) stimulated the growth of confluent human fibroblasts (FS-4) in serum-free culture medium. However, TNF had a cytotoxic effect upon the growth of FS-4 cells in combination with arachidonic acid. When arachidonic acid was added to culture medium in the absence of TNF, however, it had no effect on the cell growth. Arachidonic acid inhibited the TNF-induced cell growth in a dose-dependent manner: it reversed the TNF-stimulated growth to the control level at a concentration of 10 microM and was cytotoxic to TNF-treated FS-4 cells at higher concentrations. This cytotoxicity of TNF was not observed in FS-4 cells treated with palmitic acid. Indomethacin, a cyclooxygenase inhibitor, decreased the cytotoxic effect that TNF exerted in the presence of arachidonic acid. These results suggest that TNF becomes cytotoxic to FS-4 cells when arachidonic acid present in the culture medium is converted to prostaglandins.     

Natural killer cells generated from bone marrow culture

By using anti-Nk-1 antiserum, we detected a significant proportion of Nk-1+ cells in bone marrow (BM) with low lytic activity that can be slightly enhanced with interferon (IF). These BM Nk-1+ cells also bind to YAC-1 cells. Because Nk-1 antigen has been found to mark NK cells, BM appears to harbor immature precursors to natural killer (NK) cells. We therefore used concanavalin A (Con A)-conditioned medium to culture BM cells to induce differentiation of the putative NK precursors. After 3 to 4 days in culture, cytotoxic activity to YAC, that peaked at 6 to 7 days, was consistently generated, and the activity still could be detected after 10 to 14 days in culture. In contrast, experiments using spleen cultures, performed in a similar manner, showed peak activity after 4 to 5 days and the activity declined thereafter. The cytotoxic activity of cultured BM cells was also higher than that of cultured spleen cells. Cultures of BM cells from old mice have good cytotoxic activity. The cytotoxic cells generated were Nk-1+ and Qa-5+. Furthermore, these culture conditions did not maintain the proliferation of CFU-C cells.     

Effect of L-ornithine on proliferative and cytotoxic T-cell responses in allogeneic and syngeneic mixed leukocyte cultures

The effect of L-ornithine on cytotoxic and proliferative responses in mixed leukocyte cultures has been analyzed. The activation of cytotoxic T lymphocytes (CTL) was strongly inhibited when 9 X 10(-3) M L-ornithine was added at the initiation of the cultures. The CTL precursor cells were not completely and irreversibly inactivated, however, since the cells generated normal cytotoxic activity if resuspended after 6 days in fresh culture medium together with a fresh set of stimulator cells. Experiments in microcultures with nylon-wool-nonadherent T-cell-enriched spleen cells as responder cells and "plastic adherent cells" as stimulator cells revealed that the cytotoxic responses were almost completely suppressed if ornithine was added within the first 20 hr but were only partially suppressed if ornithine was added after 48 hr. Also, ornithine had only a mild suppressive effect on proliferative responses in allogeneic and syngeneic mixed leukocyte cultures. The strong suppressive effect of the cytotoxic response was therefore not explained by a general toxic effect of L-ornithine on the responding cells in the culture. The addition of interleukin 2 (IL-2)-containing EL-4 supernatants did not prevent but rather enhanced the suppressive effect of L-ornithine. This indicated that the inhibitory effect was not (exclusively) expressed at the level of the IL-2-producing helper T cells. Since activated macrophages have been reported to secrete arginase, it appears that L-ornithine may be part of a regulatory circuit that selectively regulates the development of cytotoxic effector T cells.     

SIN-1 cytotoxicity to PC12 cells is mediated by thiol-sensitive short-lived substances generated through SIN-1 decomposition in culture medium

As a generator of peroxynitrite (ONOO⁻), 3-morpholinosydnonimine (SIN-1) is widely used in the study of oxidative/nitrosative stress in cultured cells, although controversy exists regarding active species responsible for cytotoxicity. In this study, we report that unstable thiol-sensitive substances, generated from the reaction of SIN-1 with components in culture medium, play a crucial role in SIN-1 cytotoxicity in PC12 cells. Exposure of cells to culture medium obtained after almost complete SIN-1 decomposition at 37 °C for 2 h demonstrated almost the same degree of cytotoxicity as did fresh SIN-1. The cytotoxicity of SIN-1-decomposed medium largely depended on serum, decayed with time, and could be completely abolished by the addition of thiols. Degradation of synthetic ONOO⁻ in the culture medium did not reproduce the unstable cytotoxicity. The presence of superoxide dismutase (SOD) during SIN-1 decomposition prevented the formation of the cytotoxic substances, whereas SOD had no protection against the cytotoxicity itself, suggesting a crucial role of simultaneously generated superoxide and nitric oxide in the formation of the toxicants, but not in their cytotoxic action. The cytotoxicity of fresh SIN-1 is dramatically suppressed in a basal medium (Hanks balanced salt), suggesting that the cytotoxicity of fresh SIN-1 also requires components of culture medium. These results suggest that SIN-1 cytotoxicity in PC12 cells is mediated via the generation of cytotoxic substances in the medium during its decomposition.     

Effects of DL-alpha-difluoromethylornithine on Leishmania donovani promastigotes

Alpha-difluoromethylornithine (DFMO), an irreversible inhibitor of ornithine decarboxylase, has been demonstrated to be an effective agent against a variety of parasitic protozoa but not against Leishmania spp. In this report, we show that Leishmania donovani promastigotes in continuous culture are sensitive to the growth inhibitory and cytotoxic effects of DFMO. Incubation of the promastigotes with DFMO obliterates intracellular putrescine pools and depletes spermidine concentrations, which correlates with the onset of growth inhibition. The effects of DFMO on the growth and the intracellular polyamine pools can be reversed completely by the addition of 10 microM putrescine to the culture medium. These results suggest that the treatment of leishmaniasis may be amenable to chemotherapeutic manipulation by DFMO.     

A method for the continuous cultivation of mammary epithelium

Summary Established cell lines of mammary epithelium have been obtained from mice, rats, and hamsters. Maintenance and replication of the epithelium in serial subcultures were dependent on their periodic treatment with collagenase. Because collagenase is not cytotoxic and has maximum efficiency at neutral pH in isotonic saline solutions containing calcium and magnesium, this enzyme can be introduced directly into the culture medium; cells have been maintained for 3 days in such a medium with serous enrichment at no detriment to them. Up to 10% concentrations of serum have not interfered with enzymatic activity. Supported by United States Public Health Service Grant CA-08515 and CA-08740 from the National Cancer Institute. General Research Support Grant FR-5582 from the National Institutes of Health, and Grant-in-Aid Contract M-43 from the State of New Jersey.     

Effects of DL-Alpha-Difluoromethylornithine on Leishmania donovani Promastigotes1

ABSTRACT. Alpha-difluoromethylornithine (DFMO), an irreversible inhibitor of ornithine decarboxylase, has been demonstrated to be an effective agent against a variety of parasitic protozoa but not against Leishmania spp. In this report, we show that Leishmania donovani promastigotes in continuous culture are sensitive to the growth inhibitory and cytotoxic effects of DFMO. Incubation of the promastigotes with DFMO obliterates intracellular putrescine pools and depletes spermidine concentrations, which correlates with the onset of growth inhibition. The effects of DFMO on the growth and the intracellular polyamine pools can be reversed completely by the addition of 10 μM putrescine to the culture medium. These results suggest that the treatment of leishmaniasis may be amenable to chemotherapeutic manipulation by DFMO.     

The use of a CHO cell culture in studying Vibrio cholerae grown under different conditions]

Studies of biological activity of cholera vibrios in cultures of chinese hamster ovary cells (CHO) have revealed their strong dependence on culture conditions. Elongation of CHO cells is caused only by choleragenic strains. Under stationary conditions of culture the vibrios were found to release haemolisin into the medium and had a cytotoxic effect. Most of cytotoxic supernatants exhibited a neuraminidase activity. Proteolytic activity was less dependent on the vibrio culture conditions. Strains with a high proteolytic activity caused rounding of the CHO cells.     

Characterization of DNA lesions produced by HgCl2 in cell culture systems

HgCl2 is extremely cytotoxic to Chinese hamster ovary (CHO) cells in culture since a 1-h exposure to a 75- microM concentration of this compound reduced cell plating efficiency to 0 and cell growth was completely inhibited at 7.5 microM . The level of HgCl2 toxicity depended upon the culture incubation medium and has previously been shown to be inversely proportional to the extracellular concentration of metal chelating amino acids such as cysteine. Thus, HgCl2 toxicity in a minimal salts/glucose maintenance medium was about 10-fold greater than the toxicity in McCoy's culture medium. The HgCl2 toxicity in the latter medium was 3-fold greater than that in alpha-MEM which contains more of the metal chelating amino acids. When cells were exposed to HgCl2 there was a rapid and pronounced induction of single strand breaks in the DNA at time intervals and concentrations that paralleled the cellular toxicity. The DNA damage was shown to be true single strand breaks and not alkaline sensitive sites or double strand breaks by a variety of techniques. Consistent with the toxicity of HgCl2, the DNA damage under an equivalent exposure situation was more pronounced in the salts/glucose than in the McCoy's medium and more striking in the latter medium than in alpha-MEM. Most of the single strand breaks occurred within 1 h of exposure to the metal. We believe that the DNA damage caused by HgCl2 leads to cell death because the DNA single strand breaks are not readily repaired. DNA repair activity measured by CsCl density gradient techniques was elevated above the untreated levels at HgCl2 concentrations that produced little measurable binding of the metal to DNA or few single strand breaks assessed by the alkaline elution procedure. DNA repair activity decreased at HgCl2 concentrations that produced measurable DNA binding and single strand breaks. These irreversible interactions of HgCl2 with DNA may be responsible for its cytotoxic action in cells.     

Metabolic and physiologic studies of nonimmune lymphoid cells cytotoxic for fibroblastic cells in vitro

An in vitro reaction between mouse lymphoid cells and target fibroblastic cells in wells of microtest plates, which appears to simulate the in vivo rejection of hemopoietic allografts, has been analyzed for metabolic and physiologic requirements. Protein synthesis was required for only the first few hours of culture. Inhibition of RNA synthesis and alteration of cell surface charge with various agents were without obvious effects. Metabolic slowing at 4 °C or deviation of the pH of the culture medium suppressed the reaction. Thymus cells, which are not cytotoxic in this system, significantly but not completely inhibited the cytotoxicity of lymph node cells. Antiserum directed against target cells specifically protected them from the cytotoxic lymphoid cells in the absence of complement.Precursors of cytotoxic lymphoid cells were radiosensitive, unlike the cytotoxic cells themselves. BALB/c anti-C57BL/6 spleen cell serum and ⁸⁹Sr both are able to prevent rejection of marrow allografts in vitro. Lymphoid cells incubated with this antiserum plus complement lost much of their cytotoxicity but were still effective at high ratios of aggressor to target cells. Lymphoid cells of mice treated with ⁸⁹Sr were effectively cytotoxic but lost practically all of their cytotoxicity afer incubation with the antiserum plus complement. Thus, it appears that this reaction detects two different cytotoxic lymphoid cells, either of which can function in vitro. Both cell types may need to cooperate in vivo during marrow allograft rejections.     

Proliferative response of human diploid fibroblasts to intermittent light exposure

Three- to four-hour exposure to fluorescnt light, one to three times weekly, reproducibly enhanced the proliferation rate of human diploid fibroblasts. This enhancement was observed in WI-38 and a line from whole embryo mince at late population doubling level (PDL) as well as in a line from adult skin at early PDL. Single or multiple exposures of short duration stimulated proliferation, whereas exposures of long duration were cytotoxic. This proliferative response is reversible, and is mediated through the culture medium, Dulbecco Vogt's supplemented with 10% fetal bovine serum. Apparently light produces some mitogenic substance(s) in the culture medium that accumulates in the cells and is toxic or growth-stimulatory depending on its concentration per cell. Another possibility is that light produces in the medium both cytotoxic and growth-stimulatory substances.     

Cytotoxic immune cells with specificity for defined soluble antigens. I. Assay with antigen-coated target cells

An in vitro cytotoxic system is described, in which immune cells specific for a given soluble antigen exert a specific cytotoxic effect on target cells to which this antigen has been covalently linked. The nature of the target cell is important in this system. When antigen-coated P 815-X2 mastocytoma cells and antigen-coated chicken red blood cells were incubated for several hours in culture medium at 37 °C, the presence of membrane-bound antigen could still be demonstrated on the latter, but not on the former target cells. This might be the reason why antigen-specific target cell destruction by specific immune cells was observed only with antigen-coated chicken red blood cells as target cells. The specificity of the cytotoxic effect was controlled in each experiment in a criss-cross way by using two non cross-reacting antigens both as immunogens and for coating the target cells. Specific cytotoxicity was demonstrable with both guinea pig and mouse immune cells and with different kinds of antigens: foreign proteins, hapten-heterologous protein conjugates and hapten-autologous protein conjugates.     

In vitro growth of cytotoxic human lymphocytes. I. Growth of cells sensitized in vitro to alloantigens.

Human lymphocytes sensitized to allogeneic determinants in vitro were continuously cultured on medium prepared from phytohemagglutinin-(PHA-P) stimulated human mixed lymphocyte cultures. With such human-conditioned medium (HCM), human peripheral lymphoid cells could be grown in culture for over 90 days with a doulbing time of about 54 hr. Cytotoxic lymphocytes could be grown in culture for this time with no loss of cytotoxicity or change in cytotoxic specificity.