Mouse leukemia: depression of serum interferon production.

Production of circulating interferon is significantly impaired in AKR/J mice after development of lymphoblastic leukemia and in Balb/c mice with clinical signs of Friend erythroblastic leukemia. This alteration has been observed with three interferon inducers, each one known to elicit an interferon response in different cells.

Depression of circulating interferon response in Balb-c mice after urethan treatment.

Urethan, when given to female Balb/c mice, impaired the capacity of these animals to produce circulating interferon. The effect appeared rapidly after a single injection of either 1 or 1.5 milligrams of urethan per gram of body weight and was of short duration. The possibility that this inhibition of the production of interferon plays a role in the enhancement of viral leukemia by urethan should now be considered.

Delayed-type hypersensitivity to sheep red blood cells: inhibition of sensitization by interferon.

Interferon, when given or induced 24 hours before contact of mice with sheep red blood cells, prevented sensitization, and no delayed-type hypersensitivity reaction could be elicited 4 days later, after challenge with the antigen, as shown by the absence of footpad swelling in treated animals.

Strain dependence of the antiproliferative action of interferon on murine erythroid precursors.

Electrophoretically pure mouse interferon inhibits erythropoietin-dependent proliferation of committed erythroid precursors (CFU-E) obtained either from adult mouse bone marrow or from 14-day fetal mouse livers. The degree of inhibition is significantly influenced by the genotype of the cell donor; about ten times as much interferon is required to inhibit proliferation of CFU-E from C57BL/6 than is needed for comparable inhibition of CFU-E from BALB/c or Swiss mice. These strain-dependent results point to the existence of genes that influence the degree of the inhibitory effect of interferon on cell multiplication.

Interferon-gamma.

During the past year, the most striking progress in our understanding of interferon-gamma has been made in the elucidation of the three-dimensional structure of the molecule and in the analysis of the molecular structure and the functional domains of the interferon-gamma receptor. A greater insight into the molecular mechanisms of gene activation by interferon-gamma has been gained, and the immunoregulatory role of interferon-gamma has been better defined in studies dealing with its effects on B-cell function and with its production by the various T-cell subsets. Finally, the effects of interferon-gamma on intracellular parasites is an important aspect of interferon-gamma activity that is giving rise to several clinical trials.

Circulating Interferon Production in the Mouse : Origin and nature of cells involved and influence of animal genotype.

A radiobiological study of circulating interferon production in the mouse was undertaken in the hope of elucidating the site(s) of circulating interferon production. After total body X-irradiation of the animals, different radiosensitivities of circulating interferon production were observed with different viral inducers. Myxovirus-induced circulating interferon production was especially radiosensitive. Moreover, a study of interferon production in syngeneic and xenogeneic radiochimeras demonstrated that cells producing NDV (Newcastle disease virus)-induced circulating interferon were derived from hematopoietic stem cells. In addition, treatment of mice with antilymphocyte serum significantly reduced NDV- and Sendai virus-induced circulating interferon, as opposed to other inducers. Taken together, these results strongly suggest that the lymphocyte is the major source of myxovirus-induced circulating interferon. A survey of interferon production in 12 inbred mouse strains, using NDV as inducer, revealed the existence of low and high producers. A Mendelian analysis carried out with low producing Balb/c and high producing C57BL indicated that the difference between low and high interferon producers was caused by a single, autosomal, codominant factor.

Development of methods for somatic cell gene therapy directed against viral diseases, using retroviral vectors carrying the murine or human interferon-beta coding sequence: establishment of the antiviral state in human cells.

We are developing methods for somatic cell gene therapy directed against chronic and fatal virus infections, such as acquired immunodeficiency (AIDS), by transforming cells with a constitutively expressed interferon (IFN) coding sequence. Previous work from our laboratory has shown that stable antiviral expression (SAVE) can be obtained in murine BALB/c 3T3 cells and human U937 cells transformed with plasmids carrying either the murine or the human IFN-beta coding sequence placed under the expression control of a 0.6-kb Xho II-Nru I promoter region of the murine H-2Kb major histocompatibility complex (MHC) gene (Macé et al., 1991; Seif et al., 1991). In the present paper, we report the construction of murine (Mu) and human (Hu) IFN-beta-expressing retroviral vectors (pMPZen-MuIFN beta, pHMB-KbMuIFN beta) and the problems encountered. Because of the murine origin of commonly used packaging cells and the species specificity of IFN, it was evident that placing the murine IFN-beta sequence under constitutive expression control could result in the production of Mu IFN in the murine packaging system, and thereby lead to decreased vector production and also to enhanced resistance of target cells. Using a packaging cell line that releases a beta-galactosidase-expressing vector, we show that, as expected, Mu IFN-alpha/beta decreases vector production of murine packaging cells and also inhibits the transformation of target NIH-3T3 cells with this vector, but the presence of anti-Mu IFN antibodies rescues the viral titer of the packaging cells and restores the sensitivity of target cells to virus transformation. However, the same antibody treatment is unable to rescue the viral titer of psi-2 packaging cells producing autocrine Mu IFN-beta encoded by the pMPZen-MuIFN beta and pHMB-KbMuIFN beta vectors. Because of the species specificity of IFN, this problem is circumvented with the pMFG-HuIFN beta vector carrying the human IFN-beta sequence. In spite of the production of Hu IFN, murine psi-CRIP packaging cells are able to release retroviral vectors expressing Hu IFN-beta, and these amphotropic vectors can transform human MRC-5 cells and confer to these cells an enhanced resistance to vesicular stomatitis virus (VSV) infection.

Structure and expression of a new murine interferon-alpha gene: MuIFN-alpha I9.

A new murine alpha interferon gene, MuIFN-alpha I9, isolated from a BALB/c genomic clone, was characterized. It encodes a mature polypeptide of 167 amino acids (aa), presenting from 77 to 86% homology with the seven other MuIFN-alpha I aa sequences previously described. When compared to the latter, pre-IFN-alpha I9 has 13 distinctive aa, and, remarkably, ten of these occur in pairs. The coding region, fused to the SV40 early promoter and introduced into COS monkey cells, directed the transient secretion of an acid-stable functional IFN of 18-21 kDa. The production in this system reached levels of 300 000 units per 0.15 ml. A comparison of the aa sequence of different murine, rat, bovine, and human alpha and beta IFNs revealed certain common features allowing us to propose a putative secondary structure of the IFN proteins. A detailed analysis of results previously published by us and by others showed that the MuIFN-alpha I9 gene is, together with a least twelve other MuIFN-alpha I genes, located on chromosome 4.

Type I interferons.

Type I interferons (IFNs) constitute a family of structurally related proteins that are all derived from the same ancestral gene and act on a common cell-surface receptor. Contrary to many other cytokines, the production of type I IFNs is not a specialized function, and all cells in the organism can produce them, usually as a result of induction by viruses, via the formation of double-stranded RNA. Type I IFNs are indeed responsible for the first line of defense during virus infection and act through the induction of a great number of proteins. Of these, at least thirty have been characterized, and there are probably many more. In addition to their direct antiviral effect, type I IFNs exert a wide variety of other activities, such as for example the induction of various cytokines and the stimulation of different effector cells of the immune system. Due to these pleiotropic effects, recombinant interferons are used in the clinic to treat a variety of diseases, among which cancer, viral hepatitis and multiple sclerosis.

Immunoregulatory action of type I interferon in the mouse.

The effect of type I interferon production on immunity to the interferon-inducing virus was examined using the Newcastle disease virus [NDV]-mouse model and comparing If-1h and If-1l animals. The degree of cell-mediated immunity, as measured by delayed hypersensitivity [DH] to NDV, was influenced by the levels of interferon produced. Anti-interferon globulin given immediately after immunization decreased sensitization to NDV, whereas additional, exogenous, interferon, given to low interferon producers, stimulated sensitization to NDV. The alleles at the If-1 locus influenced the extent of DH to NDV, in that If-1h mice developed much stronger DH than did If-1l mice. However, results from recombinant inbred strains, F2 and backcross generations showed that for interferon production to stimulate DH to NDV, other genes, present in the C57BL/6 background but as yet not characterized, are required. Thus DH to NDV is determined on the one hand by the alleles at If-1, influencing interferon production, and on the other hand by a combination of several genes affecting the interaction of interferon with cells of the immune system.

Electrophoretically pure mouse interferon exerts multiple biological effects.

Electrophoretically pure (EP) mouse interferon (IF), was examined for a number of biological effects of previously ascribed to crude or partially purified interferon preparations. The following effects were observed: inhibition of antibody formation in vitro; inhibition or enhancement of sensitization to sheep erythrocytes in the mouse, depending on time of administration of IF, and dosage of antigen; inhibition of the expression of delayed type hypersensitivity in vivo; enhancement of natural killer cell activity in vitro and in vivo; inhibition of mouse tumor cells multiplication in vitro; inhibition of the growth of a transplantable tumor in mice. EP mouse IF had a very pronounced priming effect but had no blocking activity.

Stable antiviral expression (SAVE) as an approach to somatic cell gene therapy directed against HIV infection.

We are developing methods for somatic cell gene therapy directed against infection with human immunodeficiency virus by enhancing the antiviral resistance of target cells through the constitutive production of interferon-beta. Cells that have been transformed by plasmids or retroviral vectors carrying the human interferon-beta gene placed under the expression control of a murine H2Kb promoter fragment become resistant to HIV infection. Part of this enhanced resistance is due to inhibition of virus entry into the transformed cells, a hitherto unreported mechanism of interferon action.

Alpha/beta interferons fail to induce antiviral activity from within the nucleus.

Electrophoretically pure murine alpha/beta interferons (IFN-alpha/beta) were microinjected directly into the nuclei of mouse L cells, each nucleus receiving 10 fl containing about 20,000 (IFN) molecules, an amount sufficient to induce the antiviral state when added to the culture medium of control cells. Three, six or 24 h after intranuclear delivery, the cells were challenged with vesicular stomatitis virus or Semliki Forest virus and the appearance of cytopathic effects was scored for each individual cell. The scoring of more than 1,000 intranuclearly injected cells in nine different experiments showed unambiguously that the intranuclear delivery of IFN-alpha/beta did not induce the antiviral state. The results argue strongly against the physiological importance of high-affinity nuclear binding sites for native IFN that have been recently described (V. M. Kushnaryov, H. S. MacDonald, G. P. Lemense, J. Debruin, J. J. Sedmak, and S. E. Grossberg, Cytobios 53:185-197, 1988). Together with earlier results of other groups describing the lack of IFN activity after intracytoplasmic injection (Y. Higashi and Y. Sokawa, J. Biochem. 91:2021-2028, 1982; G. Huez, M. Silhol, and B. Lebleu, Biochem. Biophys. Res. Commun. 110:155-160, 1983), these results lend weight to the hypothesis that the binding of IFN-alpha/beta to the plasma membrane receptor is sufficient to set into motion the complex mechanism of transmembrane signalling without requiring internalization of the bound IFN molecules.

Interferon synthesis in x-irradiated animals v. Origin of mouse serum interferon induced by polyinosinic-polycytidylic Acid and encephalomyocarditis virus.

The radioresistant cell systems producing serum interferon after intravenous administration of polyinosinic-polycytidylic acid [poly(I.C)] or encephalomyocarditis virus in mice were studied in rat-to-mouse radiation chimeras. Interferon induced by poly(I.C) became of donor type within 3 months after grafting of irradiated C3H/He mice with Wistar rat bone marrow cells; this indicated that it was made in cells derived from the hemopoietic system. In contrast, encephalomyocarditis virus-induced interferon remained of recipient type in xenogeneic chimeras up to 3 months after grafting, which indicated that the bulk of this interferon originated from a cell population not derived from the hemopoietic system. To ascertain that the respective radiosensitivities of the systems producing rat interferon in chimeras corresponded to that of normal mice, some rat-to-mouse chimeras were subjected to a second X irradiation 1 month after the first irradiation and restoration. Circulating interferon production was studied 4 days later. As expected, the re-irradiation strongly depressed rat serum interferon production induced by Newcastle disease virus but had no effect on rat interferon synthesis induced by poly (I.C). These results point to a macrophage origin for the bulk of poly(I.C)-induced circulating interferon.