Activity of dsRNA-dependent protein kinase in rat lymphoid cells under the effect of X-ray irradiation

The activity of dsRNA-dependent protein kinase, which is the key enzyme of the interferon signal system, was studied in the rat spleen and thymus lymphocytes under the influence of X-ray irradiation at 0.5 and 1 Gy doses and interferon inducers administration. An increase of the enzyme activity was established in the presence of FGA, concanavaline A, poly(I).poly(C) in vitro. The effect is intensified under the irradiation by 0.5 Gy dose. The protein kinase activity in lymphocytes is amplified in proportion to poly(I).poly(C) concentration, that was most pronounced in the irradiated animals. The comparative analysis of the action of interferon inducers on the dsRNA-dependent protein kinase activity was carried out. Two biological systems were used: in vivo (when the preparations were injected to the experimental animals) and in vivo (under the preincubation of isolated lymphocytes with the inducers). It was shown that the combined action of radiation and interferon inducers causes the stimulation of dsRNA-dependent protein kinase activity.     

Strain difference in mouse natural killer activity augmented by mouse interferon and poly I.C

The level of natural killer (NK) activity was found to vary considerably among several mouse strains. In vivo and in vitro, interferon (IFN) and IFN inducers have been shown to augment mouse NK activity. C3H/He mice showed high NK activity, DDD/1 and A/J mice low NK activity, and C57BL/6, BALB/c and DBA/2 mice intermediate NK activity after injection with polyinosinic polycytidylic acid (poly I. C.). The same NK activity correlation was observed in nontreated mice, but the NK activities were lower compared with the poly I. C.-injected mice. Moreover, the DDD/1 and A/J mice showed almost no augmentation of NK activity on injection with poly I.C. In vivo, C3H/He, BALB/c and C57BL/6 mice injected with IFN showed augmented NK activity, but DDD/1 mice showed no such reaction. In vitro, C3H/He, BALB/c and C57BL/6 mouse spleen cells treated with IFN also showed augmented NK activity, but DDD/1 mouse spleen cells showed almost none. F1 hybrids between high (C3H/He) and low (DDD/1) NK-activity strains showed high NK activity. Thus, activity is dominant over low activity. The segregation of (DDD/1 X C3H/He) Fl X DDD/1 back-cross mice suggested that the strain differences in NK activity are under polygenic control.     

Induction of xanthine oxidase and depression of cytochrome P-450 by interferon inducers: genetic difference in the responses of mice

Interferon and interferon inducing agents depress hepatic cytochrome P-450 systems. They also induce hepatic xanthine oxidase activity. It has been suggested that free radicals produced by xanthine oxidase may cause the loss of P-450. High titers of serum interferon are induced by poly IC (poly riboinosinic acid.polyribocytidylic acid) in both C57Bl/6J and C3H/HeJ mice; Newcastle disease virus (NDV) induces a high titer of interferon in C57Bl/6J mice but not in C3H/HeJ mice. The induction of xanthine oxidase activity by NDV in C3H/HeJ mice was less than half that seen in C57Bl/6J mice, thus demonstrating a relationship between the induction of xanthine oxidase, the depression of P-450 and a genetically determined difference in responsiveness of mice to interferon inducers.     

Delta 9-tetrahydrocannabinol decreases alpha/beta interferon response to herpes simplex virus type 2 in the B6C3F1 mouse

This study was undertaken to determine the effect of delta 9-tetrahydrocannabinol (delta 9-THC) on polyinosinic:polycytidylic acid [poly(I):poly(C)]-induced, and on herpes simplex virus type 2 (HSV-2)-induced, alpha/beta interferon in the B6C3F1 mouse. Animals were administered delta 9-THC, or the diluent, intraperitoneally for 4 consecutive days or at various time intervals prior to administration of the interferon inducer. Poly(I):poly(C) or HSV-2 was injected intravenously on Day 4. Animals receiving poly(I):poly(C) and treated with delta 9-THC at doses ranging from 5 to 100 mg/kg exhibited significantly lower titers of interferon than mice given poly(I):poly(C) and the diluent. Diminished interferon titers occurred in HSV-2-infected animals treated with delta 9-THC in doses exceeding 15 mg/kg when compared to virus-infected animals given the diluent. This suppression of early interferon persisted through 24 hr.     

Accumulation of 2'',5''-oligoadenylates in encephalomyocarditis virus-infected mice.

Levels of 2',5'-oligoadenylates (2-5A) in various tissues of murine encephalomyocarditis virus (EMCV)-infected mice were determined and compared with those found in pathogen-free mice and in mice treated with the interferon inducer poly(I).poly(C). In control, pathogen-free mice, liver, spleen, brain, and kidney tissues possessed levels of 2-5A below 1 pmol/g of tissue, demonstrating that 2-5A was not a major component of uninfected mouse tissue. All control tissues had low basal levels (0.3 to 2.0 pmol/h per g) of 2-5A synthetase, the enzyme responsible for 2-5A production. After mice were injected intravenously with the interferon inducer poly(I).poly(C), circulating interferon, 2-5A synthetase, and 2-5A were elevated with increasing doses of double-stranded RNA. The greatest response to poly(I).poly(C) occurred in the kidney, in which enzyme levels increased 5-fold and 2-5A levels increased 24-fold to 15 pmol/g. Mice that were infected with EMCV also possessed elevated levels of 2-5A and 2-5A synthetase in the four tissues examined, although the relative distribution differed from that observed with poly(I).poly(C), indicating that the interferon inducer affects the concentration and location of intracellular 2-5A. Brain, spleen, and kidney tissues from EMCV-infected mice contained seven- to eightfold more 2-5A than control tissues did. The nanomolar levels of 2-5A in the tissues of EMCV-infected mice provide evidence that 2-5A may play a role in the antiviral response in an intact animal. In both poly(I).poly(C)- and EMCV-treated mice, the levels of 2-5A recovered from the tissues were not directly proportional to the amount of 2-5A synthetase present. These results indicate that factors other than the level of 2-5A synthetase controlled the accumulation of 2-5A in tissues.     

Antitumour activity of endotoxin, concanavalin A and poly I: C and their ability to elicit tumour necrosis factor, cytostatic factors, and interferon in vivo

Summary Concanavalin A, endotoxin, poly I : C, and tumour necrosis serum (TNS) were compared for antitumour activity against Meth A sarcoma transplanted in syngeneic BALB/c mice and their capacity to induce tumour necrosis factor (TNF), heat-stable cytostatic factors, and heat-labile interferon in the blood of normal and Corynebacterium parvum-pretreated mice. All the agents induced hyperemia and inhibition of mitosis at 4 h, and by 24 h many tumours had a dark necrotic centre. Subsequent tumour growth was inhibited and in some of the treated mice tumours regressed completely. Poly A : U and normal mouse serum did not induce regression and their effects were less marked in all other respects, suggesting that these events may be linked. The necrotizing effects of concanavalin A and poly I : C are unlikely to be mediated by TNF, because neither agent could mimic endotoxin in eliciting RNase-resistant necrotizing and regressing activity in the serum of mice pretreated with C. parvum. Poly I : C did not induce strong cytostatic activity in the sera of C. parvum-treated mice, and for this and other reasons these factors are unlikely to be responsible for the observed effects. Concanavalin A, endotoxin, and poly I : C induced high levels of serum interferon but purified interferon had only weak antitumour activity in the Meth A system, suggesting that interferon may not be the mediator.From these and other data it is concluded that there is no clear relationship between the capacity of the agents to induce tumour necrosis and their capacity to elicit TNF, cytostatic factors, and interferon.     

Synthetic double-stranded RNA poly(I:C) combined with mucosal vaccine protects against influenza virus infection

The mucosal adjuvant effect of synthetic double-stranded RNA polyriboinosinic polyribocytidylic acid [poly(I:C)] against influenza virus was examined under intranasal coadministration with inactivated hemagglutinin (HA) vaccine in BALB/c mice and was shown to have a protective effect against both nasal-restricted infection and lethal lung infection. Intranasal administration of vaccine from PR8 (H1N1) with poly(I:C) induced a high anti-HA immunoglobulin A (IgA) response in the nasal wash and IgG antibody response in the serum, while vaccination without poly(I:C) induced little response. Intracerebral injection confirmed the safety of poly(I:C). In addition, we demonstrated that administration of poly(I:C) with either A/Beijing (H1N1) or A/Yamagata (H1N1) vaccine conferred complete protection against PR8 challenge in this mouse nasal infection model, suggesting that poly(I:C) possessed cross-protection ability against variant viruses. To investigate the mechanism of the protective effect of poly(I:C), mRNA levels of Toll-like receptors and cytokines were examined in the nasal-associated lymphoid tissue after vaccination or virus challenge. Intranasal administration of HA vaccine with poly(I:C) up-regulated expression of Toll-like receptor 3 and alpha/beta interferons as well as Th1- and Th2-related cytokines. We propose that poly(I:C) is a new effective intranasal adjuvant for influenza virus vaccine.     

The Inhibitory Effect of Staphylococcus epidermidis Slime on the Phagocytosis of Murine Peritoneal Macrophages Is Interferon-Independent

The extracellular slime produced by Staphylococcus epidermidis has been shown to interfere with several human neutrophil functions in vitro, such as chemotaxis, degranulation and phagocytosis. Slime production has been suggested as a useful marker for clinically significant infections with coagulase-negative Staphylococcus. Since the main role of macrophages in defense mechanisms is phagocytosis, the effect of slime on the phagocytic activity of macrophages was investigated. The phagocytic activity of murine peritoneal macrophages treated with slime in vitro decreased in a dose-dependent fashion. A similar decrease was also observed in macrophages isolated from mice that had previously received intraperitoneal injection of slime. To investigate whether interferon also plays a role in this process, mice were treated with interferon or an interferon inducer, polyinosinic-polycytidylic acid (poly I:C), together with slime before macrophage isolation. The slime-suppressed phagocytic activity of macrophages was partially relieved by both agents, and the recovery effect of poly I:C in slime-suppressed phagocytosis of macrophages in vivo might be attributed to the increased interferon level in peritoneal fluid and sera. However, when slime was given to poly I:C-pretreated mice, the phagocytic activity remained suppressed. Thus, it appears that slime is able to suppress the phagocytic activity of macrophages regardless of the state of macrophage activation by poly I:C. The results suggest that the inhibition of phagocytosis by S. epidermidis slime may be independent from the activation of interferon.     

Interferon: ten stories in one. A short review of some of the highlights in the history of an almost quinquagenarian

This short review article on some pertinent observations in the unfolding story of interferon is dedicated to Professor Ilona Béládi on the occasion of her 80th birthday. This by no means covers the whole story on interferon. It just highlights some of the more striking findings made with interferon (or its inducers) over a time span of almost 50 years since its original discovery (in 1957) by Isaacs and Lindenmann. These observations concern (i) the induction of interferon by synthetic polyanions such as polyacrylic acid and polymethacrylic acid; (ii) the prolonged antiviral activity shown by polyacrylic acid in vivo; (iii) the interferon-inducing ability of double-stranded RNAs such as poly(I) x poly(C) and (iv) mismatched derivatives thereof (i.e. ampligen); (v) the cloning and expression of interferon-beta, and (vi) its usefulness in the treatment of multiple sclerosis; (vii) the potential of (pegylated) interferon-alpha in the treatment of hepatitis C and (viii) the therapy/prophylaxis of SARS; (ix) the efficacy of interferon (inducers) in the experimental treatment of flavivirus encephalitis and enterovirus myocarditis; and, finally, (x) the role of interferon in the activity shown by S-adenosylhomocysteine inhibitors such as 3-deazaneplanocin A against experimental Ebola virus infections in mice.     

Effect of double-stranded RNA and interferon on the antiviral activity of Atlantic salmon cells against infectious salmon anemia virus and infectious pancreatic necrosis virus

Type I interferons (IFN) establish an antiviral state in vertebrate cells by inducing expression of Mx and other antiviral proteins. We have studied the effect of Atlantic salmon interferon-like activity (AS-IFN) and poly I:C on the Mx protein expression and antiviral activity against infectious salmon anaemia virus (ISAV) and infectious pancreatic necrosis virus (IPNV) in the Atlantic salmon cell lines SHK-1 and TO. The double-stranded RNA poly I:C is an inducer of type I IFN in vertebrates. A cell cytotoxicity assay and measurements of virus yield were used to measure protection of cells against virus infection. Maximal induction of Mx protein in TO and SHK-1 cells occurred 48 h after poly I:C stimulation and 24 h after AS-IFN stimulation. TO cells pretreated with AS-IFN or poly I:C were protected from infection with IPNV 24 to 96 h after stimulation. Poly I:C or AS-IFN induced a minor protection against ISAV infection in SHK-1 cells, but no protection was induced against ISAV in TO cells. Western blot analysis showed that ISAV induced expression of Mx protein in TO and SHK-1 cells whereas IPNV did not induce Mx protein expression. These results suggest that ISAV and IPNV have very different sensitivities to IFN-induced antiviral activity and have developed different strategies to avoid the IFN-system of Atlantic salmon. Moreover, Atlantic salmon Mx protein appears not to inhibit replication of ISAV.     

Treatment of mice with polyinosinic-polycytidilic polyribonucleotide reduces T-cell involvement in a localized inflammatory response to vaccinia virus challenge.

Mice inoculated intracerebrally with 10(3) PFU of vaccinia virus developed a nonfatal meningitis which was maximal 7 days after challenge. Intravenous administration of an interferon (IFN) inducer, polyinosinic-polycytidilic polyribonucleotide [poly(I)-poly(C)], on days 4 and 6 postinjection was associated with a three- to fourfold decrease in the number of T lymphocytes present in cerebrospinal fluid, reflected primarily by a decreased number of vaccinia virus-specific cytotoxic T-lymphocyte precursors. The lack of a concomitant reduction in the overall cytotoxic activity of cerebrospinal fluid cells directed against virus-infected target cells seemed to be largely due to an increase in natural killer cell activity. IFN was implicated as mediating the effect of poly(I)-poly(C) because high systemic levels of IFN were evident after injection, and neither the magnitude of the inflammatory response nor the T-cell levels were affected when poly(I)-poly(C)-treated mice were also given anti-IFN antiserum. However, the poly(I)-poly(C)-induced IFN did not seem to reduce the localized inflammatory response by affecting viral replication in brain tissue because the vaccinia virus titers present on days 6 through 8 of infection were similar to the titers in phosphate-buffered saline controls. These findings are consistent with either an effect of IFN on T-cell recruitment to the central nervous system or an inhibition of proliferation of cells participating in the response. These findings suggest that there is a potential source of complications for clinical protocols that use IFN or inducers to enhance T-cell function in various disease situations, and this effect of IFN may be a contributing factor to the immunosuppression often associated with many viral infections.     

Interferon induction in human lymphocytes by complexes of vinyl copolymers with polynucleotides

Analogues of the dsRNA poly(I).poly(C), namely double-stranded like complexes of poly(I) or poly(C), respectively, with corresponding vinyl nucleobases copolymerized with various vinyl compounds are highly effective interferon inducers in human lymphocyte cultures. Like poly(I).poly(C) they stimulate the liberation of an inhibitor(s) of interferon activity and, moreover, display mitogenic effect. With respect to the extent of these effects the various inducers differ considerably.     

Induction of innate immunity against herpes simplex virus type 2 infection via local delivery of Toll-like receptor ligands correlates with beta interferon production

Toll-like receptors (TLRs) constitute a family of innate receptors that recognize and respond to a wide spectrum of microorganisms, including fungi, bacteria, viruses, and protozoa. Previous studies have demonstrated that ligands for TLR3 and TLR9 induce potent innate antiviral responses against herpes simplex virus type 2 (HSV-2). However, the factor(s) involved in this innate protection is not well-defined. Here we report that production of beta interferon (IFN-beta) but not production of IFN-alpha, IFN-gamma, or tumor necrosis factor alpha (TNF-alpha) strongly correlates with innate protection against HSV-2. Local delivery of poly(I:C) and CpG oligodeoxynucleotides induced significant production of IFN-beta in the genital tract and provided complete protection against intravaginal (IVAG) HSV-2 challenge. There was no detectable IFN-beta in mice treated with ligands for TLR4 or TLR2, and these mice were not protected against subsequent IVAG HSV-2 challenge. There was no correlation between levels of TNF-alpha or IFN-gamma in the genital tract and protection against IVAG HSV-2 challenge following TLR ligand delivery. Both TNF-alpha(-/-) and IFN-gamma(-/-) mice were protected against IVAG HSV-2 challenge following local delivery of poly(I:C). To confirm that type I interferon, particularly IFN-beta, mediates innate protection, mice unresponsive to type I interferons (IFN-alpha/betaR(-/-) mice) and mice lacking IFN regulatory factor-3 (IRF-3(-/-) mice) were treated with poly(I:C) and then challenged with IVAG HSV-2. There was no protection against HSV-2 infection following poly(I:C) treatment of IFN-alpha/betaR(-/-) or IRF-3(-/-) mice. Local delivery of murine recombinant IFN-beta protected C57BL/6 and IRF-3(-/-) mice against IVAG HSV-2 challenge. Results from these in vivo studies clearly suggest a strong correlation between IFN-beta production and innate antiviral immunity against HSV-2.     

Hyper- and hypo-reactivity in mice with regard to the induction of virus inhibitory factor or interferon]

1. IF induction in mice with Newcastle disease virus produced hyperreactivity on subsequent induction with certain doses of poly I:C. 2. Laparotomy or intraperitoneal injection with air caused temporary hyporeactivity of spleen and lungs on IF induction with endotoxin. Laparotomy in splenectomised mice did not affect IF formation in lungs.     

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.     

Induction of natural killer cell activity of thoracic duct lymphocytes by polyinosinic-polycytidylic acid (poly(I:C)) or interferon

Natural killer (NK) cell activity of thoracic duct lymphocytes (TDL) was examined in normal mice and in mice treated with polyinosinic-polycytidylic acid (poly(I:C) and interferon (IFN). TDL from mice treated with phosphate-buffered saline (PBS) expressed little or no NK cell activity against YAC-1 target cells at effector-to-target ratios of up to 200:1, even after in vitro treatment with murine L-cell IFN. In contrast, TDL from poly(I:C)- or IFN-treated mice expressed significant NK activity, which correlated with the significantly higher NK activity of splenocytes from these mice compared to the NK activity of splenocytes from PBS-treated mice. These data indicate that although TDL from normal mice express no detectable NK cell activity, NK cell activity can be induced in TDL by in vivo treatment with poly(I:C) or IFN.     

An antiviral state induced in Chinook salmon embryo cells (CHSE-214) by transfection with the double-stranded RNA poly I:C

Type I interferons (IFN alpha and beta) convert vertebrate cells into an antiviral state by inducing expression of proteins that inhibit virus replication. In humans and mice, Mx proteins constitute one family of interferon-induced antiviral proteins. Mx genes have recently been cloned from Atlantic salmon and rainbow trout. Moreover, double-stranded RNA (dsRNA) and type I IFN-like activity have been shown to induce Mx protein in salmonid cells. Chinook salmon embryo cells (CHSE-214 cells) have been suggested to have a defect in the IFN-system because the dsRNA polyinosinic polycytidylic acid (poly I:C) failed to induce an antiviral state in the cells. We have studied this phenomenon more closely in the present work. CHSE-214 cells were either transfected with poly I:C or incubated with poly I:C without transfection reagent. The cells were then studied for Mx protein expression and protection against infectious pancreatic necrosis virus (IPNV) infection. The results showed that cells transfected with poly I:C were protected from IPNV infection, whilst cells incubated with poly I:C were not protected. Cells transfected with the double-stranded DNA poly dI:dC were also not protected against IPNV. Mx protein was expressed in CHSE-214 cells upon transfection with poly I:C, but not after incubation with poly I:C alone. Stimulation of CHSE-214 cells with supernatants from cells transfected with poly I:C, induced protection against IPNV, indicating production of type I IFN-like activity. These results suggest that CHSE-214 cells in fact are able to produce type I IFN, but may have defects in the mechanisms mediating uptake of poly I:C or may degrade unprotected poly I:C.     

Influence of Statolon on Resistance of Mice to Influenza

Various interferon inducers are known to elicit protection against lethal or infecting doses of certain viral agents. Because of the relatively high morbidity rate of influenza and its seasonal occurrence, we wished to determine whether statolon-induced interferon might be effective in controlling this disease. Mice were treated intraperitoneally with statolon and challenged with influenza A(2) virus by the intranasal route. Although interferon was present in the serum at the time of virus administration, no change in mortality rate was observed. There was, however, a significant increase in the mean survival time of treated animals. Similar results were obtained when Newcastle disease virus was used as the interferon inducer. To determine the effect of the route of challenge, other mice were treated with statolon or Newcastle disease virus and inoculated with mengovirus by the intranasal or intraperitoneal route. The results demonstrated that the treated mice were protected to similar degree against challenge by either route. It is suggested that the relative ineffectiveness of interferon in protecting mice against influenza is due to an intrinsic characteristic of the virus itself rather than the type of interferon induced or the route of virus challenge.     

Interferon-inducing and antiviral effects of inosiplex in combination with high-molecular interferon inducers

It has been shown that the immunostimulant inosiplex (IP) is capable of inducing interferon production in mice and of stimulating interferon induction by high-molecular inducers. The combined use of IP and poly (G)-poly (C) or dsRNA (RFf2) leads to a longer interferon circulation in the blood. All the combination schemes ensuring the effect of interferon production prolongation were tested for anti-viral activity. The prophylactic administration of the drugs permitted the attainment in mice of the increased resistance to experimental influenza.