Natural Cytotoxicity to Murine Cytomegalovirus-Infected Cells Mediated by Mouse Lymphoid Cells: Role of Interferon in the Endogenous Natural Cytotoxicity Reaction

Lymphoid cells from unstimulated normal C57BL/6J mice were shown to lyse murine cytomegalovirus (MCMV)-infected syngeneic mouse embryo fibroblasts but not uninfected mouse embryo fibroblasts. This cytotoxicity by mouse effector cells was not restricted to MCMV-infected syngeneic cells since MCMV-infected xenogeneic rat heart fibroblasts were also lysed. Characterization of the effector cells mediating this cytotoxicity against MCMV-infected cells indicated that the effector cells are similar to described natural killer (NK) cells mediating lysis of tumor cells and virus-infected cells. Because of the described augmentation of NK activity by interferon, we examined the role of interferon in the NK reaction. Although low levels of virus-induced interferon were detectable in supernatants of MCMV-infected mouse embryo fibroblasts, no interferon was detectable in supernatants of MCMV-infected rat heart fibroblasts, a target significantly more sensitive to NK cytolysis than infected mouse embryo fibroblasts. We were able to augment the NK reaction against MCMV-infected cells by in vitro treatments with interferon. However, the amounts of interferon required for augmentation were significantly greater than the amounts generated by infected target cells. In vitro interferon-stimulated NK cells retained selective cytotoxic activity since they continued to remain incapable of lysing uninfected target cells. MCMV-infected rat heart fibroblasts induced more interferon and were also more susceptible to NK activity than MCMV-infected mouse embryo fibroblasts. In spite of this difference in interferon-inducing capacity, there was no augmentation of cytotoxicity of MCMV-infected mouse embryo fibroblasts when mouse splenocytes were cocultivated with both target cells. Finally, when production of interferon in the NK reaction was inhibited by the addition of actinomycin D, no reduction of NK activity was seen. Our findings suggest that native mouse NK cells can discriminate between MCMV-infected cells and uninfected cells, this ability leading to the selective lysis of the virus-infected cells. Furthermore, although we could demonstrate augmentation of NK activity by interferon, interferon activation of NK cells may not be a necessary precondition for the development of endogenous NK activity.     

Dipyridamole is an interferon inducer

2,6-Bis(diethanolamino)-4,8-dipiperidinopyrimido-[5,4-d]-pyrimidine (dipyridamole) induced interferon production in vitro in explanted mouse peritoneal leukocytes and to a lower degree in non-lymphoidal cell cultures of mouse (L cells primary embryo fibroblasts) and human (diploid embryo lung fibroblasts) origin. Dipyridamole induced interferon also in mice after intravenous administration. Peak interferon levels in the blood (128 IU/ml) were attained at 49 hr after injection of 0.1 mg dipyridamole per kg body weight and at 24 and 12 hr after injection of 0.6-1.8 and 16.7 mg/kg respectively. By its pH stability, thermostability and antigenic properties the interferon induced in mice, mouse peritoneal leukocytes and L cells corresponded to IFN-alpha and IFN-beta. This interferon-inducing capacity of dipyridamole may account for its broad-spectrum antiviral effect.     

Different antiviral activity and cell specificity of interferon preparations produced by mouse peritoneal cells at 37 degrees C and at 26 degrees C

Three sublines of mouse L cells and mouse embryo fibroblasts were used for determination of the antiviral activity of mouse interferons produced by nonadherent peritoneal exudate cells incubated either at 37 degrees C or at 26 degrees C. IFN produced at 37 degrees C or at 26 degrees C had the same antiviral activity in L Borgen, L929 cells. However, in MEC IFN-37 degrees had relatively higher activity than IFN-26 degrees. Of the interferon investigated only IFN-37 degrees exhibited antiviral activity in the established line of rat kidney cells. The IFN preparations showed no activity in the human and chicken cells. The studies on the sensitivity of viruses to both forms of IFN revealed that EMC and VSV viruses were equally sensitive to IFN-26 degrees C. However, the replication of EMC virus was more strongly inhibited by IFN-37 degrees than the multiplication of VSV virus.     

Characterization of antiviral activity of spotted souslik (Spermophilus suslicus) interferons

The antiviral activity of three types of spotted souslik interferons So IFN alpha, So IFN beta and So IFN gamma was studied. Fibroblasts of lungs (SL), kidneys (SK) of adult sousliks and skin fibroblasts of foetuses (SE) were equally sensitive to three types of souslik interferons. In contrast to So IFN beta and So IFN gamma, So IFN alpha, exhibited a high (100% of activity in homologous cells) cross species antiviral activity in mouse embryo fibroblasts (MEF) and a low (3% to 10% of the activity) in bovine embryo fibroblasts (BEF) and human embryo fibroblasts (HEF). The development kinetics of antiviral activity not only depended on the interferon type but also on the temperature of incubation. In comparison with So IFN gamma, So IFN alpha and So IFN beta activated earlier the maximal antiviral state. Low incubation temperature (26 degrees C) did not decrease but only delayed the antiviral activity of spotted souslik interferons. Mixed preparations of So IFN gamma with So IFN alpha or So IFN beta exhibited synergistic antiviral activity at physiological (37 degrees C) and low (26 degrees C) temperatures. The development of antiviral activity of So IFN beta as well that of Mu IFN alpha/beta was inhibited by plant lectins which reacted with the cell membrane compounds. All three types of souslik interferons were completely destroyed by trypsin and boiling at 100 degrees C for 1 min. and partially by SDS. Their sensitivity to shaking, beta-mercaptoethanol and mouse antisera against So IFN beta was different in relation to the interferon type.     

Heterospecific activity of rat interferon.

In the experiments performed in vitro and in vivo it has been found that the rat and rat embryo fibroblasts cultured in vitro after the induction with virus produce interferon which displays the antiviral activity not only in the homologous cells but also in the heterologous ones. When analysed by chromatography on Sephadex G-100 it was shown that the rat serum contains two interferon populations differing in the molecular weight and both active in the homologous and heterologous cells. The interferon with the heterospecific activity has been used in the experimental therapy of mice infected with Encephalomyocarditis Virus (EMC), Vesicular Stomatitis Virus (VSV) and Influenza Virus, and was found to be effective.     

Antiviral action of swine leukocyte interferon in mouse experiments

Swine leukocytes had previously been found to produce interferon which has an antiviral effect not only in swine cells but also in human cells. Preliminary experiments in tissue cultures showed the culture of primarily trypsinized mouse embryo fibroblasts to be as sensitive to swine interferon as human diploid cells. The experiment studying the antiviral effect of swine leukocyte interferon in the animals demonstrated it to protect mice against the pathogenic A/Aishi/68 (H3N2) strain; with a reduction of virus doses to 10 the protective effect of swine interferon increased 2-fold as compared with the experiments using 100-1000 virus doses. Inhibition of virus reproduction in lung tissues of experimental mice inoculated with A/Moscow/23/78 (H1N1), A/Wisconsin/19/67 (Hsw1N1) and A/Aishi/68 (H3N2) strains as compared with the controls. The experimental results suggest that the swine interferon produces the antiviral effect both in tissue culture and experimental animals.     

Comparison of the activity of small molecular interferon inducers: Tilorone and acridine drugs

The antiviral activity and induction of interferon-like substance by mepacrine (quinacrine, Atabrine) and Acranil in mice, described previuosly, was studied in more detail and compared with tilorone. The serum substance induced by Acranil was characterized as mouse interferon. Acranil, given parenterally, proved to be as strong as tilorone regarding interferon stimulation, in spite of the presence of only one side chain in the Acrainl molecule. Mepacrine was found to be a wealer interferon inducer than either tilorone or Acranil. The mode of interferon induction by Acranil and tilorone (effect of metabolic inhibitors, hyporesponsiveness to repeated doses, correlation between acute toxicity and antiviral activity, failure of effectiveness in chicks and chicken embryo tissue culture) was found comparable. However, the body temperature reactions of mice to the drugs were different: a striking hypothermic effect of tilorone, a lower one of mepacrine and the absence of body temperature depression by Acranil was observed.     

Rat leukocyte interferons: production of an acid-labile interferon after induction with Newcastle disease virus

Rat leukocytes produce three interferon species after infection with Newcastle disease virus. Two of the three interferon species (22-24,000 and 30,000 daltons) were acid and heat stable, protected rat and mouse cells from virus-mediated cytopathic effect (CPE), and were neutralized by antisera to mouse L-cell (alpha/beta) interferon. The third, 30,000-dalton interferon species was heat and acid labile; protected rat, mouse, human, and bovine cells from virus-mediated CPE; and was neutralized by antisera to mouse L-cell interferon and human leukocyte interferon. Thus, the rat leukocyte interferon system is similar to the human system in the kinetics of interferon production, the production of multiple interferons, heterologous antiviral activity, molecular weight, and in the production of an acid-labile species. The rat leukocyte interferon system therefore represents an important model for examining the normal production and action of acid-labile interferons and their possible role in the regulation of leukocyte function and cellular differentiation.     

Type I and type II interferons: differential antiviral actions in transformed cells

In transformed mouse embryo cells, type II interferon had much less antiviral activity than type I interferon. In non-transformed cells, the two interferons had similar high activity, Reversal of the phenotype of Moloney sarcoma virus (MSV) transformed cells by sodium butyrate restored their sensitivity to the antiviral action of type II interferon. Additional evidence for a role of the cytoskeletal network in the action of type II interferon is that its antiviral effect is reduced by cytochalasin B, colchicine or vinblastine. MSV-transformed cells, selected for their resistance to the antiviral action of type I interferon, were sensitive to type II interferon. These differences in the effects of type I and II interferon on transformed cells are at present unexplained, but suggest that they have at least partially separate mechanisms of action.     

Interferon induces an antiviral state in ganglioside-deficient transformed mouse fibroblasts

The antiviral activity of mouse fibroblast interferon against vesicular stomatitis virus was investigated in L-929 mouse fibroblasts and the ganglioside-deficient L-929 mutant cells (ATCC clone NCTC 2071). Although it has been widely reported that gangliosides serve as primary receptors for interferon at the cellular membrane, only a small difference in interferon sensitivity was observed between the wild-type L-929 and the ganglioside-deficient NCTC 2071 cells. It was not possible, however, to overcome this difference by administration of exogenous gangliosides.     

Studies on the mode of action of IME--an inhibitor of interferon activity from mouse embryo tissues.

IME--inhibitor of interferon activity from mouse embryo tissues does not inactive interferon by direct contact; it does not inhibit the production of interferon and it does not affect the replication of challenge virus. IME shows no species-specific activity since it exhibits anti-interferon action not only in homologous interferon system but also in the heterologous rat, chick and human interferon systems. The interferon-induced antiviral state as well as the efficient cell's metabolism appear to be necessary for the expression of activity of IME.     

Comparative study on the anticellular and antiviral effects of interferon and the haemopoietic stem cell inhibition factor.

Pretreatment with crude interferon preparations obtained from suspension cultures of bone marrow, spleen and thymus cells or from mouse L-cell cultures or with mouse serum interferon preparations did not change the colony-forming activity of bone marrow cells on syngeneic transplantation to lethally irradiated mice. Preparations of L-cell culture interferon, dialysed and purified by carboxymethyl-Sephadex (G-25) column chromatography, showed an inhibitory effect on exogenous colony formation by bone marrow cells. The results suggested the presence in crude interferon preparations of a substance either inhibiting the anticellular effect of interferon or stimulating colony formation. The factor produced by thymus cells following their treatment with antilymphocyte serum inhibited colony formation by bone marrow cells and, unlike interferon, possessed no antiviral activity when tested in cell cultures.     

Induction of interferon by group C arboviruses

Summary Several group C arboviruses are able to induce interferon in chick embryo fibroblasts, primary human amnion and mouse L cells.With 1 Figure     

Reversible inhibition of interferon-induced antiviral state by deoxyadenosine.

Deoxyadenosine reversibly inhibited the development of antiviral state (AVS) in chick embryo fibroblasts stimulated by interferon. Studies on cellular macromolecular syntheses in deoxyadenosine-treated cells suggested that the inhibition was related to nucleolar RNA synthesis. Reference experiments with actinomycin D and cordycepin (3'deoxyadenosine) also implied causal- or interrelation between AVS development and nucleolar RNA synthesis.     

Differences in mouse interferons according to cell source and mode of induction.

Mouse interferon induced by ultraviolet-irradiated Newcastle disease virus or polyriboinosinic-polyribocytidylic acid in T lymphocytes, B lymphocytes, macrophages, and primary mouse embryonic cell culture was studied. Irrespective of the inducer, interferons produced by T or B lymphocytes were relatively heat stable and of low antigenicity when reacted with antiserum against L-cell interferon (ALI), whereas interferons produced by macrophages and mouse embryo cells were heat labile and of high antigenicity against ALI. Mouse interferons induced by ultraviolet-irradiated Newcastle disease virus were separated into three components by chromatography on CH-Sepharose 4B. Interferons produced by T and B lymphocytes consisted primarily of component 1 (unbound fraction), whereas interferons produced by macrophages or mouse embryo cells consisted primarily of component 3 (eluted by 0.5 M NaCl). Component 1 was heat stable and of low antigenicity against ALI, properties characteristic of T- and B-cell interferon. Components 2 and 3 were heat labile and of high antigenicity against ALI, properties characteristic of macrophage and mouse embryo cell interferon. In contrast, interferon induced in mice sensitized with BCG differed from these interferons induced in B cells, T cells, macrophages, and fibroblasts in being extremely acid labile and nonreactive against ALI.     

Attempt to transfer priming by cocultivation of L cells and chick embryo fibroblasts.

Interferon-treated L cells did not transfer priming to chick embryo fibroblasts (CEF) during cocultivation. Nor could transfer be observed when CEF were treated with homologous interferon in the mixed cultures. The results indicate that the lack of transfer by cocultivation is another characteristic of priming different from antiviral resistance.     

Murine cytomegalovirus: induction of and sensitivity to interferon in vitro.

Moderate amounts of viral inhibitor were produced by mouse embryo (ME) cultures infected with two strains of plaque-purified murine cytomegalovirus (MCV). This inhibitor was shown to be interferon, based on the possession of similar properties. The growth studies of MCV in ME cells showed that interferon was produced as early as 4 h after infection, infectious virus was produced between 12 to 16 h, and cytopathic effect was produced between 16 to 18 h. Since MCV-induced interferon production and the subsequent development of antiviral state occurred early, the long eclipse period may be due to an interferon-mediated delay of virus replication. Pretreatment of ME cells with varying concentrations of interferon before infection with MCV did not result in increased interferon production, but at high pretreatment doses a slight inhibitory effect on interferon production was observed. In vitro sensitivity studies showed that small doses of MCV were highly sensitive to the antiviral action of interferon, but higher viral doses proved to be markedly resistant. Although the available evidence does not permit a definitive interpretation of the mechanism by which MCV may show differing sensitivities to interferon action, the presence of a small interferon-resistant fraction of virus-infected cells may account for the observations.     

Interferon production by individual L cells

Using a protected centre technique in which agarose prevents the diffusion of interferon from individual producing cells, we have shown that essentially every cell in a monolayer of mouse L cells can be induced to produce interferon by infection with Newcastle disease virus (NDV). The amount of interferon produced by individual cells appeared to be highly variable, even when cloned cells and viruses were used. U.v.-irradiated virus lost its capacity to induce interferon in L cells and to infect chick embryo fibroblasts at the same rate. A small proportion of cells (1 X 10-6 to 10 X 10-6) appeared to produce interferon constitutively. This fraction was increased threefold by u.v. irradiation of the cells, and up to 10-fold by exposing cells to the mutagen ethyl methane sulphonate.     

Production of alpha and gamma interferons by spleen cells from cytomegalovirus-infected mice.

Antigen-induced lymphocyte proliferation and the production of a murine immune or type II interferon (MuIFN-gamma) by spleen cells in vitro were used to examine the cellular immune response to a cytomegalovirus infection of mice. Lymphocyte blastogenesis was induced by interaction with cytomegalovirus-infected mouse embryo fibroblasts in spleen cells from mice infected at least 6 days previously with cytomegalovirus. The peak of the blastogenic response occurred after 72 hr in culture with antigen. MuIFN was detected in cultures of cytomegalovirus-infected mouse embryo fibroblasts and spleen cells from both normal and infected mice. The MuIFN produced by spleen cells from normal or infected mice early during the course of the infection (days 1 to 2) was predominantly viral or type I interferon (MuIFN-alpha). Peak titers of MuIFN-alpha were present 24 to 48 h after exposure to antigen in vitro and before the peak of the blastogenic response. In contrast, spleen cells from mice infected at least 6 days previously produced both MuIFN-alpha and MuIFN-gamma in culture with the infected mouse fibroblasts. MuIFN-alpha was present early in the culture, before peak blastogenic activity. Peak levels of MuIFN-gamma were detected as lymphocyte blastogenic activity subsided. These results indicate that the cellular immune system of the murine host is capable of responding to cytomegalovirus infection, the afferent limb by antigen recognition and the efferent limb by the production of the lymphokine MuIFN-gamma.