RNase F and 2′,5′-oligoA synthetase activities in mice after poly(I). poly(C) administration

The present study demonstrates the presence of considerable levels of 2′, 5′-oligoA synthetase activity in tissue extracts from mice. The interferon inducer, poly(I) .poly(C) , induced the synthetase activity in all the tissue extractsin vivo. Similarly, a significant amount of endonuclease RNase F activity is found to be present in these tissue extracts. But interferon induction does not seem to have any significant effect on RNase F activity.     

Mitogenic effect of double-stranded RNA in human fibroblasts: role of autogenous interferon

The synthetic double-stranded RNA polyinosinate-polycytidylate [poly(I).poly(C)] was mitogenic in cultures of human foreskin fibroblasts, as demonstrated by a stimulation of 3H-thymidine incorporation and an increase in cell density. Poly(I).poly(C) is a potent inducer of interferon (IFN)-beta in human fibroblasts. Single-stranded poly(l) or poly(C) were not mitogenic in human fibroblasts and did not stimulate IFN production. Antiserum to interferon (IFN)-beta, added to poly(I).poly(C)-stimulated cultures in order to neutralize endogenously generated IFN, markedly amplified the mitogenic action. Under similar experimental conditions, antiserum to IFN-beta did not enhance the mitogenic action of epidermal growth factor (EGF). Dexamethasone enhanced the mitogenic action of poly(I).poly(C) in a manner similar to antiserum against IFN-beta. This effect of dexamethasone correlated with its marked inhibitory action on poly(I).poly(C)-stimulated IFN production. Together with the results of other related studies, these findings support the notion of an evolutionary link between the generation of a mitogenic signal and IFN induction. In addition, these results support the concept that autocrine secretion of IFN-beta can exert negative feedback control of cell proliferation.     

The role of cell membrane in the antiviral effect of interferon

The mechanism of interferon action in human fibroblasts has been studied by use of both antisera to human fibroblast interferon and the antisera to the surface of human fibroblast cell. The anti-interferon serum completely neutralized the antiviral effect of human fibroblast interferon. Interferon antiserum prevented the intracellular antiviral state from developing when added to the medium of the cells in which interferon synthesis had already been induced by poly (I · C). This suggests that development of the antiviral state involves interferon interaction with the external part of the producing cell. Treatment with the serum directed against the surface of human fibroblast cells failed to inhibit the antiviral activity of human interferon in these cells. In addition, the effect of gangliosides on the antiviral activity of human interferon was studied and it was found that human interferon binds to gangliosides and that this interaction leads to inactivation of the antiviral effect of interferon. Pretreatment of human fibroblasts with gangliosides had no effect on the sensitivity of these cells to exogenous interferon.     

Proteins induced by various types of double-stranded RNA and interferon type I in human fibroblasts. Correlation with antiviral activity of the substances]

Protein induction by new antiviral preparations of dsRNAs (larifan, ridostin, rifastin and poly(A).poly(U)) and recombinant beta-interferon in human fibroblasts (M19) was studied. The common gene products: 88, 80, 68, 58, 56, 52, 50 and 26 kD were detected in the spectra of the induced cytoplasmic polypeptides. At the same time the sets of the induced proteins had individual distinctions in various preparations. Induction of the 56-kD protein was more essential in the action of dsRNAs than that of interferon. The antiviral activity of dsRNAs and interferon preparations correlated with a relative increase in the synthesis of proteins with molecular weights of 88, 80 and 58 kD. The study results are in agreement with the fact that the dsRNAs have interferon-independent pathways of antiviral action with participation of 56- and 58-kD protein genes.     

Synthesis of human fibroblast interferon by E. coli

A cDNA library was constructed using mRNA from human fibroblasts induced with poly(I):poly(C). A bacterial clone containing fibroblast interferon cDNA sequences was identified by hybridization to a cDNA probe synthesized using deoxyoligonucleotide primers which hybridize to fibroblast interferon mRNA specifically. Expression plasmids were constructed which permitted the synthesis in E. coli of 8 x 10(7) units of human fibroblast interferon per liter of culture. The bacterially produced fibroblast interferon is indistinguishable from authentic human fibroblast interferon by several criteria.     

Immunochemical measurement of conformational heterogeneity of poly(inosinic acid).

Several pure poly(I) preparations differed in: (a) their complement fixation reactivity with anti-poly(I) antiserum; (b) their ability to bind to a solid-phase anti-poly(I) antibody-Sepharose column; (c) their ability to inactivate serum complement; and (d) their reactivity with purified antibodies to double-stranded RNA. In particular, poly(I) samples that could induce interferon production differed from non-inducer poly(I)s; the inducers reacted weakly with anti-poly(I) antiserum and were the only ones that reacted with antibodies to double-stranded RNA. One inducer poly(I) did not inactivate complement, and differed from non-inducer poly(I) in quantitative aspects of poly(I) . poly(C) formation with varying amounts of poly(C). An additional type of poly(I) preparation reacted poorly with anti-poly(I) antiserum, did not react with anti-double-stranded-RNA antibodies and failed to induce interferon production. The varying forms of poly(I) were not interconvertible by boiling and rapid chilling. These results indicate that several different stable structural forms of poly(I) may result from a standardized synthetic procedure.     

Induction by synthetic polyribonucleotide poly(I) of differentiation of cultured mouse myeloid leukemic cells.

The effects of some synthetic polyribonucleotides on induction of differentiation of mouse myeloid leukemic M1 cells were examined. Poly(I) was found to be a potent inducer; on treatment with 100--200 microgram/ml of poly(I) for 2--4 days, M1 cells differentiated into cells resembling macrophages and granulocytes and developed phagocytosis and locomotive activities, Fc receptors and lysozyme activity. Poly(C) was less effective than poly(I) for induction of phagocytic activity, while the other single-stranded RNAs, poly(U) and poly(A), had no effect. Double-stranded RNAs, such as poly(I) . poly(C) and poly(A) . poly(U), were cytotoxic to M1 cells, and differentiation of the cells could not be detected even at the highest tolerable concentrations of these double-stranded RNAs.     

Induction of 2',5' oligo(A) synthetase in tumor-bearing mice with encephalomyocarditis (EMC) virus or poly(I)poly(C)

Infection of 13 month-old C3H mice with EMC virus or inoculation with the interferon inducer poly(I)poly(C) results in elevated levels of the enzyme 2',5' oligo(A) synthetase only in animals with spontaneous tumors (breast cancer or hepatomas). High enzymatic activities are detected in homogenates from liver, spleen, plasma and neoplastic cells of the animals with breast carcinomas and only in the neoplastic liver cells of the animals with hepatomas.     

Interferon induction by platinum(II)-poly(I).poly(C) complexes

The effect of the interaction between poly(I).poly(C) and cis-dichloro-diammineplatinum(II) (cis-Pt), its trans analogue and chloro-diethylene-triamminoplatinum(II) (dien-Pt) on interferon induction activity was investigated. The covalent monodentate fixation of the three compounds on N7 of inosine has different effects on the structure and thermostability of poly(I). poly(C) which is well reflected by the interferon induction activity of the samples. Thus, the sandwich stabilization by dien-Pt at low binding ratios is manifested by an increased interferon induction and a high resistance towards RNAase degradation. The destabilization of the duplex by cis-Pt decreases interferon induction, accompanied by an increase in RNAase sensitivity of the complexes. In the case of trans-Pt the duplex structure is little perturbed and interferon induction is essentially maintained.     

Sepharose-bound poly (I)-poly (C): interaction with cells and interferon production.

Poly(I).poly(C) covalently coupled to a matrix by one point fixation through its 3′ terminal stimulated both antiviral activity and interferon production in primary rabbit kidney (PRK) cells. This effect could not be accounted for by free polynucleotide released from the matrix into the medium. Penetration of the polynucleotide into the cells does not appear to be necessary for interferon production. A limited amount of matrix-bound poly(I).poly(C) was associated with the cells. Since it was sensitive to extraneous ribonuclease treatment, this poly(I).poly(C) was believed to be localized at the cell surface. Preliminary findings suggest that the binding of the polynucleotide to the cell is not directly proportional to the amount of interferon induced.     

Characterization of interferons produced by peripheral blood mononuclear cells from healthy subjects in response to Corynebacterium parvum or poly I: poly C

The biological significance of acid labile interferon alpha is presently unknown. We examined the putative production of acid labile interferon in vitro from human peripheral blood mononuclear cells induced with Corynebacterium parvum or poly I: poly C. Both agents induced up to 1200 IU/ml interferon, and the interferon was 80 to 90% acid labile. The interferons were typed by antibody neutralization of their antiviral activity. Contrary to previous reports, C. parvum induced predominantly interferon gamma, which is normally acid labile, whereas poly I: poly C induced an acid labile interferon alpha activity with characteristics similar to those of acid labile interferon alpha reported in serum in certain human diseases.     

Control of Interferon Synthesis: Effect of Diethyl-aminoethyl-Dextran on Induction by Polyinosinic-Polycytidylic Acid

Interferon production in cultures of rabbit kidney cells (RKC) stimulated with 10 to 250 mug of polyinosinic-polycytidylic acid (poly I.poly C) per ml peaked at 3 to 4 hr after the exposure of cells to inducer and rapidly declined thereafter. On the other hand, RKC stimulated with poly I.poly C (10 or 2 mug/ml) in the presence of diethylaminoethyl (DEAE)-dextran (100 or 20 mug/ml, respectively) produced a protracted interferon response, with the release of interferon continuing for over 24 hr. The kinetics of interferon production in RKC stimulated with lower concentrations of the mixture of poly I.poly C and DEAE-dextran were similar to the response produced by poly I.poly C alone (10 to 250 mug/ml). Only the responses that terminated early were paradoxically enhanced by treatment with low doses of actinomycin D or with cycloheximide. Cells stimulated with 50 mug of poly I.poly C/ml showed hyporesponsiveness to a second interferon induction with poly I.poly C when restimulated 7 hr after primary induction. This hyporesponsiveness could be overcome by restimulating with higher concentrations of the poly I.poly C-DEAE-dextran complex. The results are compatible with the hypothesis that the early termination of interferon production and hyporesponsiveness to repeated induction with poly I.poly C are due to a cellular repressor exerting negative control on interferon synthesis, and that the increased cellular uptake of poly I.poly C in the presence of DEAE-dextran may effectively neutralize the repressor. These results also suggested that the often observed different kinetics and the varied effects of inhibitors of ribonucleic acid or protein synthesis on interferon responses in various cells and in cells stimulated with different inducers (such as with viruses as compared with polynucleotides) need not imply the existence of fundamentally different mechanisms of interferon production.     

Induction of poly(I):poly(C)-binding 50 K protein and 2',5'-oligoadenylate synthetase in interferon-treated mouse L929 cells

A new protein retained by poly(I):poly(C)-Sepharose was induced together with dsRNA-dependent enzymatic activities, a protein kinase and 2',5'-oligoadenylate synthetase (2,5A synthetase), in interferon-treated mouse L929 cells; it had an apparent molecular weight of 50,000 (50 K) and was not phosphorylated by the protein kinase. The kinetics of the induction of the poly(I):poly(C)-binding 50 K protein were similar to those of dsRNA-dependent protein kinase and 2',5'-oligoadenylate synthetase, and their inductions were all dependent on the interferon dose added, though a relatively higher dose was required for the 50 K protein. When the interferon preparation was heated to 100 degrees C in the presence of sodium dodecyl sulfate, its effect on cells of inducing the activity of 2',5'-oligoadenylate synthetase was preserved completely, indicating that the interferon molecule itself is responsible for the induction of the synthetase. Since the induction of the enzymatic activity was inhibited by addition of either actinomycin D or cycloheximide, it may not be an activation of a latent enzyme but a de novo synthesis of the enzyme.     

The binding of poly(rI) - poly(rC) to human fibroblasts and the induction of interferon.

Human embryonic fibroblasts produce interferon when incubated at 37 degrees C after being treated at 4 degrees C with poly(rI) - poly(rC), either by addition of the double-stranded duplex or by sequential addition of the constitutent single-stranded polynucleotides. Cells which have been incubated with double-stranded poly(rI) - poly(rC) can be prevented from forming interferon by washing the cells with high concentrations of salt, immediately after adsorption of polynucleotides, or by incubation of the cells with single-stranded polynucleotides. The inhibition is probably due to displacement of the inducing molecule from the cell surface. Interferon production by cells treated sequentially with poly(rI) and poly(rC) is not inhibited by either of these treatments and the polynucleotides are not easily displaced from the cell surface.     

Treatment with poly I.C. enhances lipid peroxidation and the activity of xanthine oxidase, and decreases hepatic P-450 content and activities in mice and rats

Treatment of mice and rats with polyriboinosinic acid-polyribocytidylic acid (poly I.C., 5 mg/kg i.p.), a potent interferon inducer, decreased hepatic cytochrome P-450 system content and activities without influencing P-450-independent xenobiotic metabolizing enzymes. Treatment with poly I.C. decreased the content of P-450 by 28% in mice (P less than 0.05) and 30% in rats (P less than 0.05) but did not alter the activity of cytochrome c reductase. With treatment of poly I.C., the activity of XO increased 87% in mice (P less than 0.01) and 30% in rats (P less than 0.01). Lipid peroxidation was enhanced by 82% in mice (P less than 0.01) and 95% in rats (P less than 0.05). These results raise the possibility that a part of the depression of P-450 system content and activities by poly I.C. might be caused by enhanced lipid peroxidation associated with increased activity of XO.     

Interferon Induction in Rabbit Cells Irradiated with UV Light

UV irradiation of a continuous line of rabbit kidney cells (RK13) was used as a tool for the study of the mechanism of interferon induction. Irradiation of cells prior to their exposure to Newcastle disease virus (NDV) resulted in a dose-dependent decrease in interferon production. The inhibition of total cellular RNA synthesis by UV irradiation in uninduced cultures was similar to the inactivation curve of interferon production in NDV-induced cultures. In contrast, the production of interferon with polyinosinate-polycytidylate (poly[I].poly [C]) paradoxically was enhanced in cells irradiated with a wide range of doses of UV. However, in cells stimulated with poly(I).poly(C) and "superinduced" by the sequential addition of cycloheximide and actinomycin D, the rate of inactivation of interferon production by UV light was similar to that observed with NDV. These results are not inconsistent with the idea that both poly(I).poly(C) and NDV stimulate the same interferon gene(s), but indicate that the mechanism controlling its expression may be different for each inducer.     

Interferon: a mediator of indoleamine 2,3-dioxygenase induction by lipopolysaccharide, poly(I) X poly(C), and pokeweed mitogen in mouse lung

When C3H/He mice were treated with lipopolysaccharide, poly(I) X poly(C), or pokeweed mitogen, the serum interferon titer increased almost instantaneously (100-2000 units/ml), and then the pulmonary indoleamine 2,3-dioxygenase was induced 50- to 140-fold. The peaks corresponding to interferon induction always preceded (approximately 24 h) those corresponding to dioxygenase induction. In C3H/HeJ (lipopolysaccharide-nonresponder) mice, however, lipopolysaccharide was totally inert in induction of both interferon and dioxygenase, although treatment with poly(I) X poly(C) and pokeweed mitogen led to a remarkable increase in the serum interferon titer and the enzyme activity. When lymphocytes of C3H/HeJ mice were inactivated by X irradiation and then reconstituted by the transfer of spleen cells from C3H/He mice, both enzyme and interferon from C3H/HeJ mice thus treated were induced almost normally after the lipopolysaccharide treatment. In addition, murine interferon alpha/beta, which was injected intravenously in C3H/He or C3H/HeJ mice, almost instantaneously and dose-dependently induced the pulmonary enzyme, and at a dose of 10(5) units per mouse the enzyme activity was enhanced 20- to 26-fold in these two strains of mice. These results suggest that interferon, which is generated by the interaction of lymphocytes with lipopolysaccharide, poly(I) X poly(C), or pokeweed mitogen, is a mediator of indoleamine 2,3-dioxygenase induction in the mouse lung by these agents.