Induction of interferon-specific enzymes in cultures of cells sensitive and resistant to interferon

Induction of 2'-5'-oligoadenylatesynthetase (2-5A synthetase) by interferons and theophylline by means of activation of cAMP-system in interferon susceptible and resistant cell lines were studied. In interferon resistant cell lines the basal activity of 2-5A synthetase exceeded the level of the same enzyme in interferon susceptible cell lines. Activity of 2-5A synthetase is increased in interferon susceptible cell lines by interferon treatment, but the activity of the enzyme is not altered in interferon resistant cell lines. Among the studied cell lines the induction of 2-5A synthetase by theophylline was possible only in L929 cell line. The common mechanism for the absence of 2-5A synthetase induction by interferon and theophylline in interferon resistant cells is discussed.     

Interferon-induced 2-5A synthetase activity in human peripheral blood mononuclear cells after immunization with influenza virus and rubella virus vaccines.

The interferon-induced enzyme 2-5A synthetase can be a sensitive indicator of activation of the human interferon system during viral infection or interferon therapy. To determine the response of the human interferon system to viral antigens, the level of 2-5A synthetase activity was monitored in peripheral blood mononuclear cells of healthy adults before and after immunization with influenza or rubella virus vaccine. The influenza virus-vaccinated individuals demonstrated increases in enzyme activity on days 1 and 11 in vivo, whereas those vaccinated with rubella virus vaccine showed an increase only on day 11. The difference in the day 1 in vivo 2-5A synthetase response in the two vaccinated groups could be demonstrated by in vitro incubations of peripheral blood mononuclear cells isolated approximately 90 days postvaccination with the two vaccines. The day 11 increase of enzyme activity in the rubella virus group showed a positive correlation with an increase in serum antibody titer, suggesting activation of the interferon system during antibody production in vivo after human exposure to virus antigens. The demonstration of increased 2-5A synthetase activity at specific times postimmunization in this investigation indicates that the interferon system is involved in the human in vivo response to virus vaccination.     

2',5'-Oligoadenylate synthetase activity in lymphocytes from normal mouse.

The activity of 2',5'-oligoadenylate synthetase, an enzyme recently discovered in interferon-treated cells, was found in lymphocytes from normal mouse spleen that had received neither exogenous interferon nor its inducers. The oligoadenylate synthesized by lymphocyte cell extracts inhibited protein synthesis in rabbit reticulocyte lysates. The oligomers were composed mainly of trimer and were resistant to digestion by T2 ribonuclease. The level of the enzyme in lymphocytes was about 20 to 30% of that in L929 cells treated with interferon. The activity of the enzyme was further enhanced in lymphocytes in vitro by addition of interferon. The 2',5'-oligoadenylate synthetase was distributed among several lymphoid tissues, but was not detected in cell extracts from brain or liver. The enzyme may play an important role in the regulation of the immune system.     

Epidermal growth factor-urogastrone action: Induction of 2′, 5′-oligoadenylate synthetase activity and enhancement of the mitogenic effect by anti-interferon antibody

Epidermal growth factor-urogastrone (EGF-URO), early in the course of stimulating DNA synthesis in quiescent human fibroblasts, also causes a three to five-fold elevation of the activity of intracellular 2′,5′-oligoadenylate synthetase (2,5A synthetase), an enzyme that has been previously implicated in the antiproliferative effects of interferon. The increase in synthetase activity precedes DNA synthesis by approximately six hours, with maximal synthetase activity either preceding or coinciding with maximal DNA synthesis. EGF-URO stimulation does not result in the secretion of detectable amounts of interferon (IFN) into the growth medium and anti-human IFN-β antibodies do not block the EGF-URO-mediated rise in 2,5A synthetase activity. Thus, the elevation of 2,5A synthetase can be attributed to the action of EGF-URO itself, and not to IFN. Nonetheless, in the presence of anti-human interferon-antibodies, the time course of EGF-URO-stimulated DNA synthesis is prolonged both in human and in Syrian hamster embryo fibroblasts; the effects of the antibody were reversed in both cell strains by the addition of human IFN-β (HuIFN-β). The data suggest a role for 2′,5′-oligoadenylate synthetase in the process of EGF-URO-mediated mitogenesis and point to the possible production of interferon-related cell-associated regulators during the course of EGF-URO action.     

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.     

Induction and maintenance of 2'',5''-oligoadenylate synthetase in interferon-treated chicken embryo cells.

Treatment of primary cultures of chicken embryo cells with homologous interferon results in a substantial increase in the level of 2',5'-oligoadenylate synthetase activity that can be detected in cell extracts. This increase can be prevented by inhibitors of RNA or protein synthesis and is thus thought to represent the induction of an interferon-inducible gene, perhaps the 2',5'-oligoadenylate synthetase gene itself. To examine this response in greater detail, we studied its kinetics under the following conditions: (i) cessation of interferon treatment after different lengths of time, (ii) delayed inhibition of RNA or protein synthesis, and (iii) combinations of these treatments. The results showed that in cells treated continuously with interferon, the enzyme level reached a peak after 9 h of treatment and then decreased with a half-life of about 30 h, despite the continued presence of interferon. Removal of interferon during induction reduced the peak level of activity that was attained and somewhat accelerated its decline but did not otherwise affect the time-course of the response. On the other hand, removal of interferon after maximum induction clearly accelerated the decay of enzyme activity. This process could be delayed by inhibitors of protein synthesis, which effectively stabilized the induced enzyme. This behavior is reminiscent of other inducible enzymes, such as the steroid-induced tyrosine aminotransferase, and suggests that the level of 2',5'-oligoadenylate synthetase, which is also inducible by steroid hormones in some cell types, is subject to similar control mechanisms.     

Induction of (2′–5′) oligoadenylate synthetase activity during granulocyte and monocyte differentiation

Summary Variations in the (2′–5′) oligoadenylate synthetase (2–5 A synthetase) level were examined prior to and during the differentiation in culture of the human monocyte cell line U937 and the promyelocytic cell line HL60 in an attempt to reveal whether the enzyme is actively involved in hematopoietic cell maturation. The basal level of this enzyme was much higher in U937 than in HL60 cells. The activity of 2–5 A synthetase was enhanced in both cell lines in response to α, β interferons. During cell differentiation, ten markers were measured. The level of the enzyme rose during the process of cellular maturation in both cell lines. The 2–5 A synthetase activity observed in differentiated HL60 and U937 cells was comparable to that observed in mature normal granulocytes and monocytes respectively. Induction of U937 differentiation by chemicals was associated with detectable production of IFN. The increase in enzyme activity observed was mostly dependent on endogenous production of interferon, since it was inhibited by interferon antibodies. Kinetic studies showed that in U937 cells 2–5 A synthetase was expressed prior to several of the differentiation markers. The rise in the enzyme's level observed during the differentiation of HL60 cells was independent of endogenous production of interferon, since it was not inhibited by the addition of anti-interferon antibodies. These results suggest that different biochemical and molecular mechanisms are responsible for the induction of 2–5 A synthetase observed during the differentiation of hematopoietic cells. In any case, 2–5 A synthetase can be considered as a biochemical marker of cell status and differentiation in hematopoietic cells.     

(2'-5') oligoadenylate synthetase in chicken embryo erythrocytes and immature red blood cells

The appearance and induction of (2'-5')oligoadenylate synthetase (2-5A synthetase) in chicken embryo erythrocytes during development, and the activity and molecular size of this enzyme in immature red blood cells from anemic chickens were studied. Enzyme activity first appeared in the embryos on the 15th day of incubation, a marked increase being seen 1 or 2 days after hatching. In erythrocytes from early embryos without 2-5A synthetase activity, chicken interferon (5 IU/ml at most) induced the production of a large amount of the enzyme. In immature red blood cells from anemic chickens, only a small amount of 2-5A synthetase was detected in the nuclear fraction. The cytoplasmic fraction contained the smaller enzyme (about 45 kilodaltons), but the larger enzyme (85-120 kilodaltons) was scarcely detected in either fraction. The larger enzyme may be synthesized during the maturation of red blood cells.     

CpG oligodeoxynucleotide induction of antiviral effector molecules in sheep

Immunostimulatory CpG oligodeoxynucleotide (ODN) can protect mice against infection by many pathogens but the mechanisms mediating disease protection are not well defined. Furthermore, the mechanisms of CpG ODN induced disease protection in vivo have not been investigated in other species. We investigated the induction of antiviral effector molecules in sheep treated with a class B CpG ODN (2007). Subcutaneous injection of ODN 2007 induced a dose-dependent increase in serum levels of the antiviral effector molecule, 2'5'-A synthetase. Peak levels of enzyme were observed 4 days following ODN injection and enzyme levels remained elevated for the following 3-5 days. Repeated ODN injections induced a more sustained elevation of serum 2'5'-A synthetase activity. Finally, formulation of ODN 2007 in emulsigen increased the level of serum 2'5'-A synthetase activity and this response was CpG-specific. Elevated serum 2'5'-A synthetase activity suggested that CpG ODN acted through the induction of either interferon (IFN)-alpha or IFN-gamma. ODN 2007 did not induce detectable levels of IFN-alpha or IFN-gamma when incubated with peripheral blood mononuclear cells, but both IFN-alpha and IFN-gamma were detected following stimulation of lymph node cells with ODN 2007. CpG ODN induction of 2'5'-A synthetase in vitro correlated with the secretion of both IFN-alpha and IFN-gamma. Furthermore, immunohistochemical staining of skin revealed a marked cellular infiltration at the site of ODN 2007 injection. This cellular infiltration was CpG-specific and consisted of primarily CD172(+) myeloid cells. Many of the cells recruited to the site of ODN 2007 injection expressed IFN-alpha and some IFN-gamma. These observations support the conclusion that localized cell recruitment and activation contribute to CpG ODN induction of antiviral effector molecules, such as interferon and 2'5'-A synthetase.     

Expression of interferon-induced genes in different tissues of mice.

In vivo responses to interferon (IFN) in mice were determined by measuring the steady-state levels of induced mRNAs following injection of IFN and poly(I)-poly(C). With cDNA probes for mouse 2'-5' oligoadenylate synthetase (2-5A synthetase) and 1-8, constitutive expression of the corresponding mRNA was detectable in different organs of normal C3H/He mice. These mRNA levels were increased by as much as 15-fold over control levels in various tissues, including the brain, after IFN and poly(I)-poly(C) treatment, coincident with increases in 2-5A synthetase enzyme activity. The basal activity level of this enzyme could be reduced in normal mice by treatment with anti-mouse IFN (alpha + beta) antibody. This treatment also reduced the levels of 2-5A synthetase and 1-8 mRNAs. Thus, physiological levels of circulating IFN maintain elevated levels of IFN-induced mRNAs in mice. Furthermore, changes in 2-5A synthetase enzyme activity reflect the changes in gene expression in vivo.     

Activation of 2'',5''-oligoadenylate synthetase activity on induction of HL-60 leukemia cell differentiation.

A 27-fold increase in 2',5'-oligoadenylate synthetase activity, an enzyme associated with the antiproliferative actions of interferon (IFN), was observed after treatment of HL-60 human leukemia cells with dimethyl sulfoxide (DMSO), an inducer of granulocytic differentiation of the cells. Enzyme activity was elevated after 24 h of exposure to DMSO, was maximal at 48 hours, and declined thereafter. A comparable increase was observed after treatment with 1 U of alpha interferon (IFN-alpha) per ml or 8 U of beta interferon (IFN-beta) per ml. Elevated levels of expression of other IFN-inducible genes, including type I histocompatibility antigen (HLA-B) mRNA and 2',5'-oligoadenylate phosphodiesterase activity, were also observed with DMSO treatment. DMSO-treated HL-60 cells had an increased amount of a 1.8-kilobase mRNA for oligoadenylate [oligo(A)] synthetase when compared with that of control cells; both DMSO- and IFN-treated HL-60 cells also expressed 1.6-, 3.4-, and 4.3-kilobase mRNA. The increase in both oligo(A) synthetase activity and mRNA levels was inhibited by polyclonal antiserum to human IFN-alpha; however, no IFN-alpha mRNA could be detected in the cells. Antiserum to IFN-beta or gamma interferon (IFN-gamma) had no effect on oligo(A) synthetase expression or activity nor was there any detectable IFN-beta 1 or IFN-beta 2 mRNA in the cells. The anti-IFN-alpha serum did not block the elevation of HLA-B mRNA in DMSO-treated cells. These observations suggest that the increased expression of oligo(A) synthetase in DMSO-treated cells may be mediated by the release of an IFN-alpha-like factor; however, the levels of any IFN-alpha mRNA produced in the cells were extremely low.     

The dynamics of 2',5'-oligoadenylate synthetase activity in the nuclear and cytoplasmic fractions of human fibroblasts treated by double-stranded RNAs, by their complexes with DEAE-dextran and by type-I recombinant interferons. The ratio of double-stranded RNA-activated and -nonactivated froms of the enzyme

Novel original preparations of double-stranded RNAs (dsRNAs), i.e. larifan, ridostin and rifastin, and recombinant alpha 2- and beta-interferons promising for the clinical use were studied. The size and morphology of the dsRNAs in the preparation composition, the dynamics of their induction of interferon and the antiviral state in human fibroblasts and the effect of the DEAE dextran polycation on the activity of the dsRNAs were specified. For the first time the dynamics of 2',5'-oligoadenylate synthetase activity in the nuclei and cytoplasm of the human fibroblasts treated with the dsRNAs of different origin and their complexes with DEAE dextran was defined. To elucidate the specific features of the mechanism of antiviral action of dsRNAs and interferon, the relation of the 2',5'-oligoadenylate synthetase activity to dsRNAs was investigated. In the cells treated with dsRNAs and DEAE dextran there were an early activation of the enzyme and predominance of the enzyme activated forms requiring no addition of poly I.poly C to the reaction mixture. The results were indicative of possible intracellular activation of its isoforms, similar to that in the cells treated with interferon and contaminated with viruses. All the tested preparations of dsRNAs and interferons induced an increase in the activity of 2',5'-oligoadenylate synthetase both in the cytoplasm and the nuclei of human fibroblasts. The same ability was observed in DEAE dextran which is likely to be one of the causes of the increase in dsRNAs antiviral activity under its effect.     

2'-Phosphodiesterase activity in human cell lines treated or untreated with human interferon

2'-Phosphodiesterase activity was investigated, by measuring either the disappearance of (2',5')oligo(adenylate) or the release of 5'AMP, in several human cell lines (RSa, IFr, HEC-1, WGAr and HeLa) possessing different sensitivities to interferon, and treated or untreated with human interferon. In various cell lines whose (2'-5')oligo(adenylate) synthetase was normally induced by interferon treatment, both kinetic studies and measurements at different enzyme concentrations indicated that 2'-phosphodiesterase activity remained unchanged after interferon treatment. This observation was confirmed over a broad range of substrate concentrations (1-25000 nM). The activity of 2'-phosphodiesterase was dependent on Mg(OAc)2. Our results indicate that in various human cell lines the modulation of (2'-5')oligo(adenylate) metabolism by interferon does not involve an increase of 2'-phosphodiesterase activity.     

Age-related differences in the induction of 2-5A synthetase and 2-5A dependent binding protein activities by interferon in guinea pig peritoneal macrophages

2-5A synthetase and binding protein activities in peritoneal macrophages have been compared between young (6 month) and old (22-24 month) guinea pigs. Enzyme activities are lower in aged animals with a 17% and a 31% reduction in synthetase and binding protein activities, respectively. In addition, the response to the addition of mouse fibroblast interferon by macrophages from these two age groups is also substantially different. Whereas addition of interferon to young guinea pig macrophages elicits a 3.8- and a 1.7-fold increase in the synthetase and binding protein activities, only a marginal elevation in these two enzyme activities is found with interferon-treated old guinea pig macrophages. Analysis by thin layer chromatography demonstrates a marked difference in the relative distribution of the various oligomeric forms of 2-5A synthesized by young or old guinea pig macrophages. The binding protein in old animals appears to be significantly more thermolabile than the corresponding activity from young animals. The altered response to interferon and the difference in enzymatic properties in aged animals may represent part of the mechanisms involved in the progressive loss of the adaptative ability of an organism to environmental changes during senescence.     

Effect of mouse interferon alpha/beta on the expression of H-2 (class I) antigens and on the levels of 2'-5' oligoadenylate synthetase activity in interferon-sensitive and interferon-resistant Friend leukemia cell tumors in mice

DBA/2 mice were injected intraperitoneally (i.p.) with interferon-sensitive 745 or interferon-resistant 3C1-8 Friend erythroleukemia cells (FLC) and then injected i.p. with mouse interferon alpha/beta. Interferon enhanced the expression of histocompatibility (H-2) antigens on individual 745 FLC within the peritoneum, but did not alter the expression of H-2 antigens on individual 3C1-8 FLC. Likewise, interferon treatment resulted in an increase in the level of 2'-5' oligo-adenylate (2-5A) synthetase activity in 745 FLC, but did not affect the level of activity in 3C1-8 FLC. These results provide evidence that the phenotype of interferon sensitivity or resistance of FLC does not change within the peritoneum. An incidental finding was that the basal level of 2-5A synthetase activity of in vivo passaged 745 cells was greater than that of 3C1-8 FLC. The finding that injection of mice bearing 745 FLC with antibody to mouse interferon alpha/beta reduced the level of 2-5A synthetase activity in these cells, but did not alter the level of 2-5A activity in 3C1-8 FLC, suggests that endogenous interferon in the peritoneum may have been the responsible factor.     

Oligo(2'-5')adenylate synthetase in human lymphoblastoid cells

The enzyme oligo(2′–5′)adenylate synthetase, when activated by double-stranded RNA, polymerizes ATP into the novel oligonucleotide (2′–5′)ppp(Ap)_nA. We describe conditions for assay of this enzyme in crude extracts of a human lymphoblastoid cell line, Namalwa. The production of (2′–5′)ppp(Ap)_nA by Namalwa extracts was 3–5 times greater than the production by extracts of interferon pretreated mouse L cells, and 700 fold higher than the production by extracts of untreated mouse L cells. The relatively high level of oligo(2′–5′)adenylate synthetase in Namalwa cells was not attributable solely to their constitutive secretion of low levels of interferon. Analysis of the size distribution of the oligomers formed at different times suggested that the enzyme can add ATP to a free pppApA. Infection by Newcastle disease virus or treatment with interferon raised the apparent synthetase levels only marginally. Experiments that employed antibody to interferon suggested that the interferon must be externalized from the NDV-infected cell to induce maximal synthetase levels.     

Subcellular distribution of 2'',5''-oligoadenylate synthetase in differentiating Friend leukemia cells

The occurrence of distinct (2'-5')(A)n-synthetase activities has recently been documented in cytoplasmic and nuclear extracts of several interferon (IFN)-treated cell lines. Since a role has been proposed for (2'-5')(A)n synthetase in the control of cell growth and differentiation, we examined the subcellular distribution of (2'-5')(A)n-synthetase activity both in IFN-treated undifferentiated Friend leukemia cells (FLCs) and during dimethyl-sulfoxide (DMSO)-induced erythroid differentiation of FLCs. Both the nuclear and cytoplasmic (2'-5')(A)n activities were modulated to the same extent by IFNs and DMSO. No evidence for a causal relationship between enzyme activation and FLC differentiation was found.     

Induction and function of 2',5'-oligoadenylate synthetase in differentiation of mouse myeloid leukemia cells

The activity of 2′,5′-oligoadenylate synthetase (2-5A synthetase), known to be induced by interferon, was detected in mouse myeloid leukemic M1 cells only when they differentiated to phagocytic cells after incubation with conditioned medium (CM) from rat embryo cells. However, no interferon activity occurred in culture fluids of CM-treated M1 cells, although some activity was detected in the cell extracts. When anti-interferon serum was added to M1 cell cultures, the induction of 2-5A synthetase by CM was suppressed. These results suggest that CM stimulated the M1 cells to produce a minute amount of interferon, which was reponsible for induction of the 2-5A synthetase activity. On the other hand, development of the phagocytic activity of M1 cells could not be influenced by addition of antiserum. Interferon added exogenously per se neither induced phagocytic activity of M1 cells, nor did it enhance the CM-induced differentiation of the cells. Moreover, dexamethasone, which induced differentiation of M1 cells, was not capable of inducing 2-5A synthetase. These results indicate that interferon and/or 2-5A synthetase plays no essential part in the differentiation of M1 cells.     

Pharmacokinetic study of a human recombinant interferon (Re-IFN-alpha A) in cynomolgus monkeys by 2'-5' oligoadenylate synthetase assay

We evaluated 2'-5'Oligoadenylate (2-5 A) synthetase assay for pharmacokinetic study of human interferon (IFN) in cynomolgus monkeys. The enzyme was induced in primary cultures of cynomolgus monkey kidney (PMK) cells as well as in FL cells in response to human recombinant IFN-alpha A treatment. The enzyme activity increased with IFN dose and, in parallel with the enzyme elevation, developed the antiviral state of the cells. The enzyme activity induced in the peripheral blood lymphocytes peaked at 6 to 12 hr after iv or im administration. The peak level of the enzyme activity depended on the IFN concentration of the blood and the activity rapidly decreased as serum IFN was cleared from the blood. These results indicate that human recombinant IFN-alpha A induces 2-5 A synthetase in monkey cells both in vitro and in vivo, and that the enzyme assay can be used to quantitatively monitor the host response after IFN administration.