Differences between ATP-mediated cytotoxicity and cell-mediated cytotoxicity.

There has been considerable recent interest in the possible role of extracellular ATP in cell-mediated cytotoxicity. To investigate the similarities and differences between ATP-mediated lysis and CTL-mediated lysis, we studied in detail the ATP-mediated lysis of EL-4 cells, which are highly susceptible to lysis by extracellular ATP. ATP-mediated lysis was time and concentration dependent; most lysis occurred within 4 to 6 h of incubation. The kinetics of ATP- and cell-mediated lysis were similar. However, in contrast to CTL-mediated lysis, ATP-mediated lysis of EL-4 cells did not appear to be accompanied by characteristic chromosomal degradation (apoptosis). In order to compare these cytotoxic processes in greater detail, ATP-resistant clones were derived by growing EL-4 cells continuously in the presence of high concentrations of ATP. These cloned EL-4 lines showed marked resistance to ATP-mediated cytotoxicity across a wide range of concentrations but were as susceptible as the parent EL-4 cells to cell-mediated cytotoxicity by an alloreactive effector population from a MLC. Thus, there appear to be a number of differences between ATP-mediated and cell-mediated cytotoxicity in this system; most notable is the identification of cell lines that are resistant to ATP but susceptible to CTL-mediated lysis.     

Clustered ergot alkaloids modulate cell-mediated cytotoxicity.

Dimers of agroclavine (1) and terguride (2), as well as a series of terguride oligomers, for example trimers (5, 6), tetramer (7), hexamer (8) and functionalized tergurides for further complex clustering were synthesized. Terguride oligomers were screened for their direct cellular toxicity on lymphoma cell lines in vitro and for their immunomodulating activities, represented by the natural killer (NK) cell-mediated cytotoxicity, as the most sensitive screening marker during immune responses. Dimers linked via aromatic spacer showed a high toxicity (1 microM) to lymphoma cells, which was not detected in other derivatives. In vitro and ex vivo experiments performed on mouse spleen lymphocytes in the presence of terguride oligomers demonstrated an immunosuppressive effect of dimers with aromatic spacer (4c-d) and NK cell stimulatory effect of terguride hexamer (8) and trimer with aliphatic spacer (5c). There is a considerable evidence that indolic part of molecule contributes to immunosuppressive action of terguride, which is potentiated in dimers carrying aromatic linker. This effect can be reversed by higher oligomerization of the respective alkaloids.     

Murine trophoblast resists cell-mediated lysis. II. Resistance to natural cell-mediated cytotoxicity

The susceptibility of murine trophoblast cells to natural cell-mediated cytotoxicity has been assessed. Primary short-term cultures of murine trophoblast cells isolated from 14-day placentas were found to be resistant to endogenous and interferon-activated natural killer (NK) cells and natural cytotoxic cells. That the relevant target structures are expressed on the surface of trophoblast cells and accessible to the effectors was demonstrated by their ability to inhibit the lysis of NK-sensitive target cells (YAC-1) in a dose-dependent manner. The lytic resistance of trophoblast cells was unaffected by neuraminidase treatment, inhibition of protein synthesis, or extending the assay time to 12 hr. Moreover, trophoblast cells were resistant to antibody-dependent cell-mediated cytotoxicity when coated with an alloantibody capable of mediating their lysis in the presence of heterologous complement. Neither the preincubation of effector cells in concentrated trophoblast culture supernatants nor the direct exposure of effectors to monolayers of trophoblast cells inhibited their NK lytic activity, indicating that the secretion of a suppressive factor or the direct inactivation of the NK cells was not responsible for the observed resistance to lysis. These observations, together with previous results showing the resistance of trophoblast to cytotoxic T cell-mediated lysis, reveal that murine trophoblast cells possess a resistance mechanism against several forms of cell-mediated lysis. This feature of trophoblast cells at the maternal-fetal interface is likely to play an important role in protecting the fetoplacental allograft from immune rejection.     

Human natural cell-mediated cytotoxicity

Summary The contribution of natural cell-mediated cytotoxicity (NCMC) to tumor-directed cytotoxicity is unknown. This study was undertaken to correlate changes in NCMC to a single target cell (K-562) with responses to therapy in patients with small cell carcinoma of the lung, resectable stage I non-small cell carcinoma of the lung, and stage I and II melanomas. Data from these studies suggested that these patients have depressed levels of NCMC to K-562 compared with a normal age-matched control population. However, NCMC levels appear to fluctuate with tumor burden, being highest in patients with large tumor masses and lowest in patients with no clinical evidence of tumor following a successful response to therapy.     

Lysis of dengue virus-infected cells by natural cell-mediated cytotoxicity and antibody-dependent cell-mediated cytotoxicity

Peripheral blood mononuclear cells (PBMC) from humans without antibodies to dengue 2 virus lysed dengue 2 virus-infected Raji cells to a significantly greater degree than uninfected Raji cells. The addition of mouse anti-dengue antibody increased the lysis of dengue-infected Raji cells by PBMC. Dengue 2 immune human sera also increased lysis of dengue-infected Raji cells by PBMC. These results indicate that both PBMC-mediated cytotoxicity and antibody-dependent cell-mediated cytotoxicity (ADCC) can cause significant lysis of dengue-infected Raji cells. The lysis of infected Raji cells in the ADCC assay correlated with the dilution of dengue-specific antibody which was added, indicating the dengue virus specificity of the lysis of dengue virus-infected Raji cells. Alpha interferon (IFN alpha) was detected in the culture supernatant of PBMC and dengue-infected Raji cells. However, enhanced lysis of dengue-infected Raji cells by PBMC may not be due to the IFN produced, because neutralization of all IFN activity with anti-IFN alpha antibody did not decrease the lysis of dengue-infected cells, and effector cells pretreated with exogenous IFN alpha also lysed dengue-infected cells to a greater degree than uninfected cells. The effector cells responsible for lysis of dengue virus-infected Raji cells in the natural killer and ADCC assays were analyzed. Nonadherent PBMC caused more lysis than did adherent cells. Characterization of nonadherent cells with monoclonal antibodies showed that the predominant responsible effector cells were contained in OKM1+ and OKT3- fraction in the natural killer and ADCC assays.     

Mistletoe viscotoxins increase natural killer cell-mediated cytotoxicity.

Mistletoe extracts have immunomodulatory activity. We show that nontoxic concentrations of Viscum album extracts increase natural killer (NK) cell-mediated killing of tumor cells but spare nontarget cells from NK lysis. The compounds responsible for this bioactivity were isolated from mistletoe and characterized. They have low molecular mass and are thermostable and protease-resistant. After complete purification by HPLC, they were identified by tandem MS as viscotoxins A1, A2 and A3 (VTA1, VTA2 and VTA3, respectively). Whereas micromolar concentrations of these viscotoxins are cytotoxic to the targets, the bioactivity with respect to NK lysis is within the nanomolar range and differs between viscotoxin isoforms: VTA1 (85 nm), VTA2 (18 nm) and VTA3 (8 nm). Microphysiometry and assays of cell killing indicate that, within such nontoxic concentrations, viscotoxins do not activate NK cells, but act on cell conjugates to increase the resulting lysis.     

Antibody dependent cell-mediated cytotoxicity against Trypanosoma cruzi.

This paper describes in vitro antibody dependent cytotoxicity against Trypanosoma cruzi epimastigotes by normal mouse splenic lymphocytes. Cytotoxicity was expressed as the percentage reduction in the number of motile parasites upon incubation with lymphocytes at 37 degrees C in a defined medium. Failure of the non-motile parasites to regain motility and their ensuing degeneration of 28 degrees C in liver infusion tryptose (LIT) medium confirmed loss of motility as a criterion of cytotoxicity. Incubation of T. cruzi cruzi at 37 degrees C for 18 h in a defined medium per se did not interfere with motility but was followed by a lag phase of the growth curve in LIT medium at 28 degrees C. The lag phase was prolonged for T. cruzi which had previously been incubated at 37 degrees C in the absence of cells.     

Inhibition of NK cell-mediated cytotoxicity by oxysterols.

Some of the oxidation products of cholesterol (oxysterols) have profound effects on plasma membrane structure and function. The present studies were undertaken to determine the effects of oxysterols on NK cell-mediated cytotoxicity. When mouse spleen cells were preincubated with certain oxysterols, NK cell cytotoxicity was inhibited without loss of effector cell viability. The strongest inhibition was observed with oxysterols that are oxidized at the C-5, C-6, or C-7 positions of the sterol nucleus. Among these, 7 beta-hydroxycholesterol caused more inhibition than 7 alpha-hydroxycholesterol suggesting that the spatial orientation of the hydroxyl group in the beta-position results in a greater perturbation in plasma membrane structure than that oriented in the alpha-position. In contrast, oxysterols that are oxidized at the C-20 and C-25 positions that are located on the C-17 acyl chain had little or no inhibitory effect, suggesting that oxidation in the cholesterol nucleus which is situated closer to the phospholipid headgroups at the lipid bilayer-aqueous interface results in a more profound effect on the plasma membrane physical structure. These results suggest that the lytic function of NK cell is sensitive to alterations in the physical state of its plasma membrane induced by oxysterols.     

Antibody-dependent cell-mediated cytotoxicity in humans. II. Energy requirement.

Antibody-dependent cytotoxicity mediated by human peripheral lymphocytes is an active energy-requiring phenomenon. In this study the relative importance of glycolysis and respiration was analyzed, by measuring the effect of various metabolic inhibitors (sodium azide, antimycin A, 2-deoxy-D-glucose, iodoacetate), alone or in combination, on effector cell efficiency and intracellular ATP level. The inhibition of both aerobic and anaerobic energy production in the effector cells completely abolished cytotoxicity. An inhibition of 50% was observed when the intracellular ATP concentration was decreased to levels corresponding to 30 to 50% of those in the untreated controls.     

Natural cell-mediated cytotoxicity in mice treated with total lymphoid irradiation (TLI)

Fractionated total lymphoid irradiation (TLI) of adult (BALB/c × C57BL/6)F₁ mice resulted in transiently augmented natural killer (NK) and natural cytotoxic (NC) cell activities. Thus, 1 day after completion of TLI, NK and NC activities in the spleens of treated mice were lower than controls but values increased and reached a maximum level of 23- to 190-fold above control at 6 days after irradiation, returning to normal levels 9 days later. Cytotoxicity was enhanced after removal of the plastic adherent population. No cytotoxicity was observed against P 815 target cells, which are sensitive to activated macrophages but not to NK. The significance of this modulation of natural cell-mediated cytotoxicity following TLI is discussed.     

Splenic suppressor cells and cell-mediated cytotoxicity in murine schistosomiasis.

An impairment of the capacity to generate alloantigen-specific cytotoxic T lymphocytes (CTL) was observed in mixed lymphocyte cultures (MLC) established with spleen cells from mice infected with Schistosoma mansoni. This impairment, which was observed as early as the eighth week of infection, could be abrogated by the fractionation of spleen cell suspensions by the carbonyl iron/magnet method prior to the establishment of MLC. Cocultivation of normal spleen cells with increasing numbers of splenocytes from S. mansoni-infected syngeneic mice resulted in a dosage-dependent suppression of CTL generation. This "infectious suppression" was not sensitive to antiserum against mouse thymic lymphocyte antigen (MTLA). The present studies suggest the role of a macrophage rather than a T cell as the suppressor cell in this model of cell-mediated immunity in schistosome-infected mice.