Interferon: effects on the immune response and the mechanism of activation of the cellular response.

The discovery of interferon in 1957 by Drs. Isaacs and Lindenmann led to major revisions in the concepts of man's defenses against viral infections. There are at least two types of interferon. Along with their antiviral properties, they have recently been shown to exert a suppressive effect on the humoral and cellular immune response; they affect both B and T lymphocytes. A variety of substances, including virus, polyribonucleotides, and mitogens for T lymphocytes, are good interferon inducers. T lymphocytes seem to be necessary for these inducers to exert their immunosuppressive effects. The immunosuppressive effects of interferon inducers suggests that interferons may be mediators of suppressor T lymphocyte effects. In the virus system, interferon does not exert its antiviral effects by direct action on the virus, but rather derepresses a cell gene that results in the production of an antiviral protein. This antiviral protein is probably the mediator of inhibition of virus replication. This is a complex sequence of events that results in the interaction of interferon with the cell membrane and the resulting production of the antiviral state in the cell. This review will examine the various steps of this involved process.     

Antiviral activity of mutant interferons. A new approach to the study of structural-functional organization of interferons

The developed approach to investing the structure-functional organization of interferon has been developed consisting in: 1) fusing genes of interferon and alpha-peptide of beta-galactosidase, the resultant protein having the interferon properties and being determined by the beta-galactosidase alpha-complementation test; 2) constructing mutant genes of interferon by the localized mutagenesis; 3) determining the mutant interferon activity; 4) deducing the amino acid sequence of mutant interferon by sequencing mutant genes; 5) analyzing structure-functional organization of interferon. In accordance with this approach, ten mutant interferons with up to 15 changes of amino acid substitutions are obtained and their antiviral activity is determined. The role of some amino acid residues in antiviral activity of interferon alpha 2 is revealed.     

Interferon-induced proteins in human fibroblasts and development of the antiviral state.

Treatment of human fibroblasts with interferon induces the synthesis of several proteins, as detected by incorporation of [35S]methionine followed by analysis of cell extracts by polyacrylamide gel electrophoresis. The induction of these proteins had features in common with the development of the antiviral effect of interferon, such as (i) sensitivity to actinomycin D and cycloheximide when these compounds were added together with interferon, (ii) insensitivity to actinomycin D if the actinomycin D was added 2 h after the addition of interferon, (iii) similar dependence on interferon concentration, and (iv) species specificity for interferon. When interferon treatment was given in the presence of cycloheximide and actinomycin D was added before the removal of cycloheximide, all four proteins were induced, thus suggesting that their inductions are coordinated. Labeling for 2-h periods at varying time intervals after the addition of interferon revealed that the synthesis of these proteins was induced within a few hours, peaked at different time intervals, and was soon followed by a marked decline, suggesting that the mRNA's for these proteins have short half-lives. Moreover, this decline occurred despite the fact that the cells were continuously exposed to interferon, and there was no measurable loss of interferon activity in the medium. This suggests that the induction of these proteins is transient and is apparently subject to further control.     

Increase in the sensitivity of leukemia cells, incubated in interferon, to the effect of immune serum]

L-1210 AND P-388 leukemic cells were incubated in three types of interferon, i.e. L-cell interferon and two types of lymphocyte interferon (induced in the lymph node lymphocytes of intact mice and the lymphocytes obtained on the 10th day after intraperitoneal injections of 5-10(7) L-1210 cells). "False" interferon obtained by the method analogous to that of obtaining L-cell interferon, excluding the viral induction, was used for control. Cells incubated in interferon proved to be more sensitive to the action of the cytotoxic antibodies than those treated with "false" interferon. Treatment of the cells with lymphocytic interferon induced on the lymphocytes of immune mice increased their sensitivity even more in comparison with the cells treated with interferon obtained from intact mice.     

Interferon and cytotoxic factor (cytotoxin) released in the blood of mice infected with Mycobacterium bovis BCG. III. Interferon and cytotoxin induced by the specific antigen as compared with those induced by bacterial lipopolysaccharide

The time course of development and decline of the ability of BCG-infected mice to produce interferon in the serum in response to the intravenous infection of purified protein derivative of tuberculin (PPD) was very similar to that of their systemic hypersensitivity to PPD. A cytotoxic factor (cytotoxin) was produced in parallel with interferon in the serum of BCG-infected mice after stimulation with PPD. The duration of the period in which cytotoxin-production responsiveness to PPD was definitely detectable was much shorter than that for interferon-production responsiveness although the periods for the maximum production of interferon and cytotoxin coincided. The kinetics of release of interferon in the serum of BCG-infected mice after stimulation with PPD did not parallel that of release of cytotoxin. The four kinds of activities, interferons and cytotoxins induced by PPD and lipopolysaccharide (LPS) in the serum of BCG-infected mice, were compared for their stability to heating at 56 C and to treatment at pH 2. The kinetics of inactivation of these four activities differed significantly, when the serum was either heated at 56 C or treated at pH 2. Interferon produced in response to LPS could be neutralized by anti-L cell(NDV) interferon rabbit serum as easily as L cell (NDV) interferon, 16 times as much antiserum was required to neutralize the same amount of interferon in response to PPD, but cytotoxins induced by PPD and LPS were not neutralized at all by the antiserum. From these findings it is thought likely that interferons and cytotoxins induced by PPD and LPS in the serum of BCG-infected mice are different substances, although the antigenic relationship between cytotoxins induced by PPD and LPS remains unknown.     

Natural killing can be independent of interferon generated in vitro

PBL produce interferon in response to culture with the tumor cell line K562, but this production of interferon does not correlate with natural cytotoxicity. A basal level of natural killing is independent of interferon generated in vitro. We base this conclusion on the following findings: (i) natural killing and interferon level are not temporally correlated; (ii) preincubation of lymphocytes at 37 °C greatly reduces their ability to produce interferon but does not affect their lytic capacity against K562; and, (iii) addition of an anti-interferon antibody has no effect on NK. We conclude that NK against K562 is not dependent upon or correlated with the level of interferon generated during in vitro assay.     

Analysis of Additional Interference Occurring After the Removal of Interferon

Cultures of chick cells exposed to interferon continued to decrease in virus-producing ability during incubation after the interferon was removed. The rate of development of the additional interference and the degree of viral interference finally manifested were dependent on the concentration of interferon to which the cultures were exposed and the time of exposure. Additional interference occurred also in infected cells. Additional interference was inhibited by actinomycin D and puromycin. The best explanation of additional interference is that it results from interferon that is fixed to the cells during their initial period of contact.     

Ability of the matrix protein of vesicular stomatitis virus to suppress beta interferon gene expression is genetically correlated with the inhibition of host RNA and protein synthesis

The vesicular stomatitis virus (VSV) matrix (M) protein plays a major role in the virus-induced inhibition of host gene expression. It has been proposed that the inhibition of host gene expression by M protein is responsible for suppressing activation of host interferon gene expression. Most wild-type (wt) strains of VSV induce little if any interferon gene expression. Interferon-inducing mutants of VSV have been isolated previously, many of which contain mutations in their M proteins. However, it was not known whether these M protein mutations were responsible for the interferon-inducing phenotype of these viruses. Alternatively, mutations in other genes besides the M gene may enhance the ability of VSV to induce interferons. These hypotheses were tested by transfecting cells with mRNA expressing wt and mutant M proteins in the absence of other viral components and determining their ability to inhibit interferon gene expression. The M protein mutations were the M51R mutation originally found in the tsO82 and T1026R1 mutant viruses, the double substitution V221F and S226R found in the TP3 mutant virus, and the triple substitution E213A, V221F, and S226R found in the TP2 mutant virus. wt M proteins suppressed expression of luciferase from the simian virus 40 promoter and from the beta interferon (IFN-beta) promoter, while M proteins of interferon-inducing viruses were unable to inhibit luciferase expression from either promoter. The M genes of the interferon-inducing mutants of VSV were incorporated into the wt background of a recombinant VSV infectious cDNA clone. The resulting recombinant viruses were tested for their ability to activate interferon gene expression and for their ability to inhibit host RNA and protein synthesis. Each of the recombinant viruses containing M protein mutations induced expression of a luciferase reporter gene driven by the IFN-beta promoter and induced production of interferon bioactivity more effectively than viruses containing wt M proteins. Furthermore, the M protein mutant viruses were defective in their ability to inhibit both host RNA synthesis and host protein synthesis. These data support the idea that wt M protein suppresses interferon gene expression through the general inhibition of host RNA and protein synthesis.     

Is there a plant interferon?

Plants appear to have a mechanism of acquired resistance to virus infection resembling the one which involves the interferon in mammals. In some virus-infected plants, anti-viral agents are produced which show similarities to animal interferon but it is not certain whether these are analogous to the anti-viral protein which is induced by interferon in animal cells. Acquired resistance to virus infection in plants also can be produced by a variety of non-viral agents—biotic and abiotic. In a few cases, interferon-like substances may mediate the resistance. Although many virus inhibitors of higher plant origin differ from the typical interferon because of their preformed (endogenous) character, some show other physico-chemical and biological similarities to the interferon. This is particularly true of a glycoprotein obtained fromPhytolacca spp. whose mechanism of virus inhibition closely resembles a mechanism postulated for the anti-viral protein induced by the interferon in animal cells. Many virus-plant combinations which produce local lesions and an acquired resistance to virus infection remain to be tested for their potential synthesis of induced anti-viral agents. These could be valuable in the therapy of plant and animal disease. The Caryophyllales (Centrospermae) are particularly promising in this regard.     

Regulation of the 2',5'-oligoadenylate level in mouse L cells

A study of pH dependence for ppp5'A2'p5'A2'p5'A hydrolysis in interferon treated and untreated mouse L-cells extracts led to the detection of two types of the 2'-phosphodiesterase activities: interferon dependent and interferon resistant. Several pH-optima were observed for hydrolysis of ppp5'A2'p5'A2'p5'A in cell extracts after their treatment with non-ionic detergent NP-40 or their differential centrifugation. The 2'-phosphodiesterase activity was found in the membrane fraction as well as in the cytoplasmic one. The presence of several pH-optima for 2'-phosphodiesterase activity in L-cells and changes of the level of this activity depending on the growth stage of cells and time of their interferon treatment indicate the complicated character of the regulation of 2'-5'-oligoadenylate's concentration and localization. The results obtained suggest that in mouse L-cells several 2'-phosphodiesterases or one enzyme in different forms may be present.     

Immunomodulating properties of interferon inducers

Data on the immunomodulating activity of interferon inductors are presented. It was revealed that the inductors increased the animal vaccinal response. Schemes for combined use of the interferon inductors and immunomodulators were developed. The immunomodulators were shown to increase the host interferon response evident from synergistic increasing of the interferon titers or prolongation of interferon circulation in blood of the animals. The efficiency of the schemes for combined use of the interferon inductors and immunomodulators was obvious from stimulation of the antibody production. As a result the time of the antibody circulation in blood increased. The effect of the combined use of the immunomodulators and interferon inductors was studied. The combined use of the preparations significantly increased the average life-span of the animals and the rate of their survival.     

Establishment and Maintenance of the Interferon-Induced Antiviral State: Studies in Enucleated Cells

Requirements for the physical presence of the cell's nucleus for the establishment and maintenance of the interferon-induced antiviral state were investigated. Enucleated chicken embryo fibroblasts were obtained by cytochalasin B treatment during centrifugation. The inhibition of vaccinia virus cytoplasmic DNA synthesis, monitored by autoradiography, was used to measure the antiviral activity resulting from interferon treatment. The antiviral state is not established in cells treated with interferon after removal of their nuclei. On the other hand, cells first treated with interferon for 6 or 12 h and then enucleated express the antiviral state. Furthermore, the antiviral state is maintained in enucleated cells for 16 h after enucleation. The antiviral state appears to be more stable in enucleates than in the residual nucleated cells found in the same cultures. Single cells of antiviral populations are found to be either fully permissive or fully restrictive to vaccinia DNA synthesis. The effect of an increasing intracellular multiplicity of infectious virus is to overcome the antiviral cell's block against viral DNA synthesis.     

Effect of Antilymphocytic Serum on Circulating Interferon in Mice as a Function of the Inducer

MOST investigators concerned with interferon synthesis in vivo have used the experimental procedure described by Baron and Buckler¹, in which circulating interferon is induced by intravenous administration of viruses. When interpreting results, however, it is difficult to know which cells are responsible for circulating interferon synthesis in the animal. Using a radiobiological approach, we have shown that after an intravenous injection of virus, interferon released into the blood stream of mice originates in cell populations of varying radiosensitivities, depending on the virus inoculated². Myxo-virus-induced circulating interferon production is characterized by high radiosensitivity, for serum interferon titres are decreased by more than 90% in C3H/He mice after one total body X-irradiation of 250 r. Moreover, the species specificity of interferon has enabled us to show that circulating interferon induced by Newcastle disease virus (NDV) is of donor type in xenogeneic radiochimaeras, from which we concluded that cells responsible for interferon synthesis with this virus originate from haemopoietic stem cells^3,4. Both granulocytes and lymphocytes fulfil the criteria of very radiosensitive elements derived from haemopoietic stem cells^5,6. We wish to report that myxovirus-induced circulating interferon production is selectively depressed after administration of antilymphocyte serum (ALS).     

Enhancement of delayed-type hypersensitivity and induction of interferon by the lipophilic agents DDA and CP-20,961

The lipophilic amines dimethyl dioctadecyl ammonium bromide (DDA) and N,N-dioctadecyl-N′, N′-bis(2-hydroxyethyl)propanediamine (CP-20,961) are compared on their capacities to induce interferon, nonspecific protection to viral infection, and enhancement of delayed-type hypersensitivity (DH). DDA, a well-known adjuvant for the induction of DH is a moderate interferon inducer like CP-20,961. On the other hand, CP-20,961, a known interferon inducer and resembling in structure DDA, is shown to enhance DH to inactivated Semliki Forest virus (SFV). Nonspecific protection to challenge with a lethal dose of either SFV or encephalomyocarditis (EMC) virus was induced on injection of both compounds.     

Correlation Between the Antiviral Effect of Interferon Treatment and the Inhibition of In Vitro mRNA Translation in Noninfected L cells

When noninfected L-cell suspension cultures are treated with interferon (specific activities superior to 10(6) reference units per mg of protein), the cell-free cytoplasmic extracts obtained are inactive for the translation of exogenous natural mRNAs. The dose-response curve shows that comparable amounts of interferon are required to produce a 50% reduction of Mengo virus multiplication in vivo and Mengo RNA translation in vitro. With higher doses of interferon, Mengo RNA translation is completely abolished, while poly U translation and endogenous protein synthesis are only slightly affected. The inactivation of Mengo RNA translation is reversible; after removal of interferon, normal translation activity is regained together with the ability to support Mengo virus multiplication. Fractionation of the cell-free extracts shows that the effect is localized in the fraction which can be washed off the ribosomes by high salt. These results establish that interferon induces a block in genetic translation in noninfected L cells.     

Cytokine receptor activation at the cell surface

Cytokines are well recognized for the pleiotropic nature of their signaling and biological activities on many cell types and their role in health and disease. Recent years have seen a steady stream of new cytokine receptor crystal structures including those that are activated by GM-CSF, type I interferon, and a variety of interleukins. Highlights include the observation of a dodecameric signaling complex for the GM-CSF receptor, electron microscopy imaging of an intact gp130/IL-6/IL-6Rα ternary receptor complex bound to its signal transducing Janus kinase and visualization of novel cytokine recognition mechanisms in the interleukin-17 and type I interferon families. This increasing knowledge in cytokine structural biology is driving new opportunities for developing novel therapies to modulate cytokine function in a diverse range of diseases including malignancies and chronic inflammation.     

Biochemical mechanisms of realization of antiviral and interferon-inducing activity of amixine and its analogs

Antiviral and interferon inducing activity of the amixine and its some derivatives, as well as their influence on the proteolytic enzymes activity, monooxygenase activity of the microsomal fraction, level of the lipids peroxidation were studied. Lack of correlation between antiviral and interferon inducing activity in the investigated series of compounds was found. Vice versa, the good correlation between interferon inducing activity and the elastase-like activities inhibition ability of the compounds was observed. It allows to state the assumption, that only one ability of compounds to induce of an interferon doesn't suffice for obtaining of high titres of interferon, and while their rather high antiproteolitic activity is necessary. It's shown, that except for one compound the influence of amixine and its derivatives on the red-ox enzymes activity well correlates with their ability to the interferon-inducing. All presented above allows to attribute amixine and its derivatives to polymodal antiviral agents.     

Differential production of IFN-gamma, analyzed at the single-cell level, by specific subsets of human NK and T cells from healthy and HIV(+) subjects

BACKGROUND: Interferon gamma is a cytokine that plays a central role in immunity, and is physiologically secreted by T and NK cells under appropriate stimuli during the immune response. By means of flow cytometry, we performed a single cell analysis of interferon gamma producing NK cells and their surface phenotype in normal and HIV(+) individuals that show several defects of cytokine production and cellular immunity. METHODS: PBMC or purified NK cells were stimulated for 1-12 h with PMA/ionomycin in the presence of monensin, subsequently stained for surface CD56 and CD3 or CD8, and for intracytoplasmic IFN-gamma, and analysed by flow cytometry. RESULTS: Our results show that CD56(+) NK cells are more efficient interferon gamma producers than T cells. Moreover, within the CD56(+) NK cell population, those that co-express low density CD8 are the best producers. Finally, we show that NK cells during HIV infection are more massively recruited to interferon gamma production than those from normal subjects. CONCLUSIONS: Both in the normal and HIV(+) subjects, a higher percentage of NK cells than T cells can produce IFN-gamma although differences can be identified within the NK cells subset in terms of IFN-gamma production. The production of IFN-gamma is fully achievable in the HIV(+) subjects, which is consistent with their elevated plasmatic levels of the cytokine. The possibility that NK cells that produce interferon gamma could represent a functionally distinct population committed to the production of this cytokine, is discussed.     

Changes in the conformation of the interferon beta gene during differentiation and induction

DNase I sensitivity was used to investigate the chromatin conformation of the interferon beta gene during differentiation of the mouse teratocarcinoma cell line PC13 . These cells do not produce interferon upon viral induction in their undifferentiated state, but do so on differentiation from stem cells to endoderm. Only in induced differentiated cells were the interferon beta genes digested by DNase I. A similar effect was seen in a line of human cells ( MG63 ) upon induction. We conclude that it is induction of interferon production that brings about the change in the DNase I sensitivity of these genes, rather than differentiation.     

Comparison of the antiviral action of different human interferons against DNA and RNA viruses

Abstract Five different interferon preparations were compared for their antiviral activity against Herpes simplex virus type 1 (HSV-1) and several RNA viruses. The interferons used were: interferon α from human buffy coats, interferon β from human fibroblasts, interferon γ from human lymphocytes after stimulation with phytohemagglutinin (PHA), lymphoblastoid interferon from Namalva cells IFN-α (Ly) and cloned α ₂ interferon produced by Escherichia coli containing the human gene for interferon α ₂. All preparations were able to protect monolayers of HeLa cells against HSV-1 infection when low multiplicities were used. The five IFN preparations were also tested against encephalomyocarditis (EMC) virus, poliovirus and vesicular stomatitis virus (VSV).