Interferon Production by Macrophages from Adult and Newborn Rabbits Bearing Fibroma Virus-Induced Tumors

Tumors were induced in adult and newborn rabbits by inoculation of fibroma virus. After 10 to 14 days, oil-induced peritoneal macrophages were harvested, purified, and tested in vitro for interferon synthesis after stimulation with specific and nonspecific viruses. Peritoneal macrophages from adult rabbits that had initiated tumor regression produced high levels of interferon (titers ranged from 160 to 640) after stimulation with fibroma virus, whereas macrophages from normal adult rabbits failed to produce significant levels of interferon under the same conditions (titers ranged from <10 to 10). Furthermore, fibroma-immune macrophages responded to vaccinia virus and Newcastle disease virus with higher levels of interferon than did normal macrophages. In contrast, macrophages from newborn tumor-bearing rabbits that showed no evidence of tumor regression failed to respond to fibroma virus stimulation with higher levels of interferon (titers ranged from <10 to 10). These macrophages did, however, yield significantly more interferon than newborn control macrophages when stimulated with a good interferon inducer, Newcastle disease virus (titers ranged from 10 to 80). These data suggest that interferon production may be an expression of macrophage activation to fibroma antigens and that macrophage activation is impaired in newborn rabbits with progressive growing tumors.     

Chronic production of interferon in carrier mice congenitally infected with lymphocytic choriomeningitis virus

Congenital infection of mice with lymphocytic choriomeningitis virus leads to a lifelong virus carrier state. Here we provide evidence for the presence and action of interferon in such mice. The level of circulating interferon in adult carrier mice is 8–16 NIH units/ml of plasma. This interferon is acid stable and is capable of inducing 2–5A synthetase in mouse L 929 cells but not in human HeLa cells. Control, noinfected mice show less than 1 NIH unit/ml of plasma. In accord with these results, adult carrier mice have higher levels of the interferon-mediated pppA(2′p5′A)_n synthetase (2–5A synthetase) in their liver and spleen than normal mice. Congenitally infected newborn mice also have higher levels of 2–5A synthetase in their liver in contrast to newborn control mice. These results in congenitally infected newborn and adult mice suggest that interferon may play a role, at least in part, in the pathogenesis of infection.     

Stimulating effect of mercuric chloride and nickel sulfate on DNA synthesis of thymocytes and peripheral lymphoid cells from newborn guinea pigs

The metal allergens mercuric chloride and nickel sulfate were found to stimulate DNA synthesis of different in vitro cultured lymphoid cells from newborn guinea pigs. In contrast to earlier findings in adult animals (where spleen cells were most consistently stimulated), in newborn animals thymocytes were the most clearly stimulated lymphoid cells. When separating thymocytes by peanut agglutinin agglutination, both agglutinated and nonagglutinated cells were stimulated, indicating that both functionally immature and mature thymocytes are the target cells for this effect of metal allergens.     

The story of DNase II: A stifled death‐wish leads to self‐harm

DNase II is an endonuclease which plays a fundamental role in the degradation of DNA from both apoptotic cells, and nuclei extruded from red blood cells during erythropoiesis: important tasks, considering that everyday 10⁸–10⁹ cells undergo apoptosis, and 10¹¹ red blood cells are produced in the adult human. The DNase II‐null mouse demonstrates embryonic lethality due to type I interferon‐mediated erythroid precursor cell death triggered by undegraded nucleic acids. However, the mechanisms leading to such cytotoxicity are poorly understood. A study in the current issue of the European Journal of Immunology investigates the role of the death ligand TRAIL in this process. Although TRAIL is shown to be dispensable for the interferon‐induced apoptosis of erythroid cells in DNAse II^−/− embryos, the authors have developed a useful strategy for further exploring this question in future studies. Interestingly, earlier studies by the same group showed that crossing the DNase II‐null mouse with a mouse deficient for the type I interferon receptor can rescue the lethal anaemia observed in the DNase II‐null embryos, but only at the cost of developing autoimmunity.     

Expression of interferon dependent resistance to influenza virus in mouse embryo cells

Adult but not newborn mice bearing the allele M chi display a specific resistance to in vivo infection with orthomyxoviruses. In vitro, cells isolated from adult M chi-animals exhibit a several hundred-fold higher sensitivity to the action of interferon (IFN) against influenza viruses than do cells from M chi-negative mice. We here tested whether or not cells from immature M chi-bearing animals would likewise express the virus-specific higher sensitivity to IFN. Cultured cells from 16-day gestation mouse embryos with and without M chi were equally permissive for an influenza virus when single cycle virus growth was measured. However, influenza virus plaques were smaller in M chi-cells. Treatment of cells with mouse interferon reduced viral protein synthesis, single cycle virus yields and the number of virus plaques more efficiently in M chi-cells than in non-M chi-cells. The smaller size of influenza virus plaques in M chi-cells not treated with IFN seems to be due to the action of endogenous IFN:inclusion of anti-interferon antibodies in the agar overlay during plaque formation resulted in plaques of approximately the size seen in control cells. When treated with the same dose of IFN, cells with M chi developed protection against influenza virus more rapidly than cells without M chi. However, after removal of IFN, the antiviral protection decayed more rapidly in cells in cells without M chi. No differences in sensitivity to IFN, viral plaque formation and kinetics of induction and decay of the antiviral state were observed between the two cell types when the rhabdovirus VSV was used as challenge. Thus, the allele M chi is expressed in cultured embryo cells much as in cells from adult animals, and susceptibility of newborn M chi-animals to influenza virus infection cannot be due to inability of their cells to respond to IFN appropriately.     

Analysis of cellular DNA synthesis during polyoma virus infection of mice: acute infection fails to induce cellular DNA synthesis.

It is widely believed that infection with various DNA viruses stimulates quiescent host cells to divide in preparation for virus replication. To examine this issue, the effects of acute polyoma virus infection on cellular DNA synthesis are observed in newborn mice. Using [3H]thymidine incorporation and fluorography of whole mouse sagittal sections, we observed clear, high-resolution images of organ-specific patterns of cellular DNA synthesis in newborn animals. No alteration in these patterns was observed during acute polyoma virus infection. Other methods, including measurements of [3H]thymidine-labeled DNA-specific activities in various tissues and in situ autoradiography, also failed to detect virus-induced alterations in cellular DNA synthesis. These results indicate that newborn animals have high endogenous levels of DNA synthesis and imply that acute polyoma virus infection may not be associated with further induced levels of cellular DNA synthesis.     

Modulation of immune responses in newborn and adult mice by interferon.

Interferon was found to have both suppressive and enhancing effects on the antibody response in newborn and adult mice. Evidence was obtained that these effects are primarily evoked during the initial steps controlling cell proliferation. Stimulation of thymus and spleen cells with a T-cell mitogen was enhanced by low doses and suppressed by high doses of interferon. Treatment of parental spleen cells with interferon before injecting them into immunized F1 hybrid mice resulted in an enhanced allogeneic effect. These results are compatible with the hypothesis that interferon affects T cells and has an immunoregulatory role, either by inhibiting the action of suppressor cells or by promoting immunological maturation.     

Studies on interferon priming: cellular response to viral and nonviral inducers and requirement of protein synthesis

Preexposure of L cells to mouse interferon (priming) and subsequent induction by Newcastle disease virus (NDV) resulted in earlier emergence of interferon and its mRNA by 3–4 hr, and a reduction of the final yield of secreted interferon. The effect of priming was most prominent in L cells stimulated by the addition of poly(I)-poly(C). Only cells that had been primed produced interferon and its mRNA in detectable amounts. When protein synthesis was blocked by the addition of cycloheximide, interferon mRNA was synthesized at the usual time and tended to accumulate in the cytoplasm, providing an estimate of the level of the primed state. By using this system, we were able to show that interferon priming was dependent on cellular protein synthesis.     

Role of age-related serum factors in the mechanism of interferon production

The activity (free and total) of cathepsin D and acid phosphatase was studied in cells of peritoneal exudate of mice of different ages in the process of interferon production in the presence of sera from newborn and adult animals. Cathepsin D release in newborn mice upon interferon induction is actively stimulated by serum factors of newborn animals altering lysosome permeability selectively for this enzyme alone. Another lysosomal enzyme, acid phosphatase, was more strongly bound to the structures and showed no such features. With an increased age of the donor the amount of stimulating factors in the serum decreases and that of inhibiting factors increases. Inhibition of cathepsin release from lysosomes by the serum factor of adult animals protects the synthesized interferon molecules from degradation and facilitates intensive interferon production in adult mice. A higher susceptibility of children to virus infections and inefficiency of the earliest defence system, interferon, may be due to degradation of newly synthesized interferon molecules by lysosomal cathepsin D.     

Regulation of protein synthesis during postnatal maturation of mouse brain.

On a DNA basis, there is higher concentration of polysomes in the brain of newborn than in the brain of adult mice, but there is no maturation-dependent decrease in tRNA content during postnatal development. The amino acid incorporating activity of cell-free systems with polysomes or mitochondria from newborn brain exceeds that of adult controls significantly in contrast to a smaller incorporating rate of labelled amino acids into synaptosomal protein. Addition of polysomes isolated from newborn brain increases the amino acid incorporation by cell-free systems with adult brain tRNA and enzymes, whereas the polysomes from adult brain decrease the incorporating activity of newborn brain systems. The loading capacity of newborn brain tRNA exceeds that of the adult controls and the velocities of its aminoacylation are four times faster. Uncharged as well as precharged newborn brain tRNA increases the amino acid incorporating activity of tRNA-dependent cell-free systems with adult brain polysomes and enzymes. In contrast to polysomes and tRNA, the newborn brain enzymes involved in protein synthesis seem to be less active in cell-free amino acid incorporation than the enzyme fractions from adult brain. These data indicate that the different protein synthesizing activity in developing and adult mouse brain is the result not only of higher amino acid incorporating activities of the newborn polysomes, but also of a stimulated acceptance and transfer function of the newborn brain tRNA.     

The synthesis and actions of mouse and human interferons in mouse-human hybrid cells.

Lines of mouse-human hybrid cells segregating either mouse or human chromosomes were used for the analysis of various aspects of the production and actions of mouse and human interferons. In one of the hybrid cell lines capable of producing both mouse and human interferons, the proportion of the two interferon activities produced varied greatly under different inducing conditions, suggesting that there are differences in the triggering mechanisms of the two interferons. Generally both mouse and human interferon production could be enhanced (“superinduced”) by sequential treatment with cycloheximide and actinomycin D; however, in one of the lines producing both mouse and human interferon, only the latter could be superinduced. There was no correlation between the capacity of the lines to produce mouse or human interferons and the sensitivity to their action. However, there was good correlation between the sensitivity to the antiviral action and the priming action (i.e., enhancement of subsequent interferon production) by the two interferons. Thus, line 55-91F2 produced both mouse and human interferons, but was sensitive to the antiviral and priming actions of human interferon only. Line GM52 × BalbVC15 produced only mouse interferon but was sensitive to the antiviral and priming actions of both interferons.     

Ontogenetic peculiarities of the effect of endothelin-1 on tissue homeostasis of various cell populations in albino rats

The effects of endothelin-1 on tissue homeostasis in newborn and adult albino rats were studied by ³H-thymidine autoradiography, staining with AgNO₃, computer-assisted morphometry, and biochemiluminescence. In adult rats the ability of endothelin-1 to activate DNA synthesis in epithelial cells decreased compared to that in newborn animals; inversion of the stimulatory effects of endothelin-1 on the nucleolar apparatus of cardiomyocytes and pronounced intensification of free radical oxidation in the heart were also seen in adult animals. These differences can be related to ontogenetic peculiarities of endothelin-1 reception.     

Comparison of cytotoxic properties of neonatal and adult neutrophils and monocytes and enhancement by cytokines.

We studied cytotoxic capabilities of newborn polymorphonuclear leukocytes (PMNs) and monocytes and their enhancement by cytokines and antibodies. Umbilical cord PMNs were assessed for their ability to kill various target cells spontaneously, after activation with phorbol myristate acetate, in the presence of antiserum (antibody-dependent cellular cytotoxicity), and in the presence of dually specific antibody (heteroantibody-mediated cytotoxicity). Target cells included the K562 cell line (natural killer cell target), chicken erythrocytes (CRBCs), and herpes simplex virus-infected CEM cell lines. Newborn PMNs were equivalent to adult PMNs in their cytotoxic capacity in several cytotoxicity assays. Neither adult nor newborn PMNs lyse tumor cell targets (i.e., K562 cells) spontaneously, but both lyse K562 cells following activation with phorbol myristate acetate. Both adult and newborn PMNs lyse CRBCs and herpes simplex virus-infected CEM cells in antibody-dependent cellular cytotoxicity assays, and this lysis could be enhanced by the cytokines granulocyte-macrophage colony-stimulating factor and gamma interferon. PMN heteroantibody-mediated cytotoxicity, resulting from the use of an antibody with dual specificity to CRBCs and immunoglobulin G FcRII, was greater in newborn PMNs than in adult PMNs; however, monocyte heteroantibody-mediated cytotoxicity, resulting from the use of an antibody to CRBCs and monocyte immunoglobulin G FcRI, was lower in newborn monocytes than in adult monocytes. The percentage, but not the density, of PMNs expressing FcRII was significantly reduced in newborn PMNs compared with that in adult PMNs, while the percentages and densities of FcRI expression were equivalent in newborn and adult monocytes. We conclude that the cytotoxic capability in term newborn PMNs is equivalent to that in adult PMNs, that the activity of newborn PMNs can be enhanced by antibody and/or cytokines, and that PMNs can contribute to the newborn's ability to kill virus-infected cells.     

Author index for volume 132

It has previously been shown that a strain of thymidine kinase (tk)-deficient mouse L-929 cells was unable to respond to murine β-interferon by induction of an anti-viral state and synthesis of double-stranded, RNA-dependent enzymes. Sensitivity to interferon can be restored by introducing into the cells a segment of Herpes simplex virus DNA containing the viral tk gene. It is shown here that not all Ltk(?) cell strains are resistant to interferon, suggesting that expression of a tk gene is not a prerequisite for response to interferon. Introduction of various genes into the resistant Ltk(?) strain, either alone or together with DNA containing the Herpes virus tk gene, leads to restoration of interferon sensitivity only when tk-containing DNA is inserted, showing that the activation of interferon responsiveness is not an artifact of the gene transfer, selection, and cloning procedures. The results imply that a component of the Herpes virus DNA introduced into the cells is able to activate interferon sensitivity.     

Electrophoretically pure mouse interferon has priming but no blocking activity in poly(I.C)-induced cells

The effect of interferon on its own synthesis was examined by exposing cells to electrophoretically pure interferon prior to induction with poly(I.C.) In C-243 cells, pretreatment with pure interferon, at any dose, up to 160,000 units, resulted in a very pronounced priming effect and no blocking was observed. Crude interferon did not display any blocking effect either, but its priming effect was much less pronounced than that of pure interferon. In mouse embryo fibroblast cultures, low doses of pure interferon had a slight priming effect, whereas higher doses, from 500 up to 50,000 units, had neither priming nor blocking activity. At a dose of 50,000 units the crude interferon preparation had blocking activity. When NDV instead of poly(I.C.) was used as inducer, pretreatment with pure interferon of both C-243 and mouse embryo fibroblast cells resulted in inhibition of interferon synthesis.     

Rescue of SV 40 from interferon-treated heterokaryons

Summary Simian virus 40 (SV 40)-transformed nonpermissive cells express only the early products of SV 40. Heterokaryons formed by fusion of these transformed cells with uninfected permissive cells support the activation of the resident viral genome leading to subsequent viral DNA replication, late protein synthesis and release of progeny virus. Pretreatment of heterokaryon cultures with either mouse or monkey interferon (IFN) before fusion with polyethylene glycol (PEG) produced a dose-dependent inhibition in the appearance of free viral DNA as well as production of infectious virus. The decreased yield of SV 40 in these cultures was similar to the inhibition which was observed in mouse or monkey cells incubated with homologous IFN prior to exogenous infection with SV 40. when IFN was added to the cultures at progressively later times after fusion with PEG, there was less inhibition of virus production. Although there was a comparable decrease in the production of virus by pretreatment with either mouse or monkey IFN, monkey IFN exerted the inhibition for a longer period of time when added after heterokaryon formation. These results demonstrate that IFN treatment applied even after initiation of SV 40 replication can still inhibit virus multiplication.With 1 Figure     

Suppression by Alpha-Fetoprotein of Murine Natural Killer Cell Activity Stimulated in Vitro and in Vivo by Interferon and Interleukin 2

Natural killer (NK) cells are 'spontaneously' cytotoxic cells thought to be involved in surveillance against tumour cells, rejection of virally infected cells, and regulation of haematopoietic stem cell differentiation and antibody synthesis. Fetus-derived alpha-fetoprotein (AFP) has been shown to regulate certain T cell-mediated immune reactions in vitro and in vivo. The lack of NK activity in newborn mice with high endogenous levels of AFP, together with the presence of cells expressing NK surface markers, also suggests that AFP may regulate NK activity. In this study we compared the effects of AFP on spontaneous versus activated murine NK activity. The lytic ability of both freshly prepared splenic NK cells and those arising after incubation for 24 h with interferon, Poly I:C, or T-cell growth factor (TCGF) was not affected by AFP if the latter was present only during the killing phase. However, if AFP was added at the beginning and retained for the duration of the 24-h in vitro lymphokine stimulation, the subsequent NK activity induced by interferon, Poly I:C, and TCGF was found to be significantly suppressed. This inhibition is both dose- and time-dependent. Delayed addition experiments showed that when AFP is present during the first 6 h of in vitro stimulation it will suppress interferon and TCGF-boosted NK activity by 50-80%. The AFP-mediated inhibitory effect on lymphokine-stimulated NK activity is not the result of increased death of effector cells nor, in the case of interferon and polyribonucleotides, of non-specific binding of AFP to the enhancing agents. In vivo injections of Poly I:C or TCGF failed to increase neonatal NK function, while administration of interferon did cause slightly higher levels of NK activity. However, spleen cells from newborn animals cultured for 24 h in the presence of lymphokines resulted in markedly elevated NK function and this in vitro activation could be suppressed by purified fetus-derived AFP. Thus, the in vivo pattern of NK activation in newborns with high endogenous levels of AFP was very similar to that of adult NK stimulation in vitro when exogenous AFP was added.     

The mechanism of interferon induction in mouse spleen cells stimulated with HVJ.

This study showed that functional viral RNA and the penetration of virus into the cell are needed for interferon induction in L cells, while simple contact of the viral glycoprotein with the cell surface appears to be sufficient for interferon induction by the same HVJ in mouse spleen cells. Thirty minutes of uv irradiation resulted in complete loss of the interferon-inducing ability of HVJ in mouse L cells. In contrast to this result, HVJ irradiated for 2 hr could induce interferon in mouse spleen cells as efficiently as untreated HVJ. These findings showed that the actual inducer of interferon in mouse spleen cells was not viral nucleic acid, but some other viral component. When HVJ was treated with potassium periodate at 37° for 1 hr, infectivity for eggs and the hemolytic and neuraminidase activities of the virus were not detectable, but a considerable portion of its hemagglutinating activity was retained. The binding to erythrocytes of this inactivated HVJ, which showed no interferon-inducing ability in both L and mouse spleen cells, was restored in mouse spleen cells but not in L cells. The results indicated that hemolytic and neuraminidase activities are not essential for interferon induction in mouse spleen cells and that hemagglutinating activity might be closely related to interferon induction in the cells, although the presence of hemagglutinating activity alone is not sufficient for interferon induction in the cells. It seems that structural integrity of hemagglutinin on the erythrocyte surface may be important for interferon induction. HeLa cell-grown HVJ, which is characterized by its inability to penetrate into tissue culture cells, was found to stimulate interferon production in mouse spleen cells but not in L cells. This suggests that the process of virus penetration may be essential for induction of interferon in L cells. Interferon was produced in mouse spleen cells incubated with membranous particles with HVJ glycoproteins, but interferon activity could not be detected in the culture fluids of L cells. Aggregation of the glycoproteins by an antibody enhanced its interferon-inducing ability in mouse spleen cells. These results showed that the actual inducer of interferon in mouse spleen cells is not viral nucleic acid, but viral glycoproteins of HVJ, and that the size of its membranous structure is related to interferon inducibility. The mechanism of interferon induction by influenza virus in mouse spleen cells is similar to that of interferon induction by HVJ.     

Interferon Sensitivity of Venezuelan Equine Encephalomyelitis Virus

Two strains of Venezuelan equine encephalomyelitis virus, which differ in virulence for mice, have been studied for their production of and sensitivity to chick and mouse interferon. Little interferon was produced by chick cells in response to the virulent Trinidad strain or the attenuated TC-83 strain without either aging or priming the cultures. Consistent differences in the production of chick interferon were not found between the two strains. Plaque variants of the Trinidad strain produced higher titers of mouse interferon than the TC-83 strain in both primed and control L-cell cultures. The TC-83 strain was found to be more sensitive than the Trinidad strain to the inhibitory effects of interferon. The greater sensitivity of the TC-83 strain was observed at both high and low multiplicities and for both chick and mouse interferons. These results are consistent with the hypothesis that interferon sensitivity may have a role as a determinant of virulence in some virus-host systems.