Comparison of the antiviral action of different human interferons against DNA and RNA viruses

Abstract Five different interferon preparations were compared for their antiviral activity against Herpes simplex virus type 1 (HSV-1) and several RNA viruses. The interferons used were: interferon α from human buffy coats, interferon β from human fibroblasts, interferon γ from human lymphocytes after stimulation with phytohemagglutinin (PHA), lymphoblastoid interferon from Namalva cells IFN-α (Ly) and cloned α ₂ interferon produced by Escherichia coli containing the human gene for interferon α ₂. All preparations were able to protect monolayers of HeLa cells against HSV-1 infection when low multiplicities were used. The five IFN preparations were also tested against encephalomyocarditis (EMC) virus, poliovirus and vesicular stomatitis virus (VSV).     

Induction of Nitric Oxide Synthase in Rat C6 Glioma Cells

Abstract: We have examined the induction of nitric oxide syhthase (NOS) activity in the rat astrocyte-derived C6 glioma cell line. In contrast to the previous results with primary astrocyte cultures, incubation of C6 cells with bacterial endotoxin lipopolysaccharide (LPS; 1 μg/ml for 24 h) did not stimulate NO₂ production. However, addition of either tumor necrosis factor-a (TNF-α) or interferon-γ (IFN-γ), cytokines that by themselves had no effect on NOS activity, imparted LPS responsiveness onto these cells in a dose-dependent manner (EC₅₀ values of 39 ng/ml of TNF-α and 9.4 U/ml of IFN-γ), and the effect of TNF-α could be further potentiated (twofold) by the presence of interleukin-1β. The simultaneous presence of TNF-α and IFN-γ yielded a greater response than either cytokine alone; however, the respective EC₅₀ values were not affected. A cytoplasmic extract from induced C6 cells catalyzed the Ca²⁺-independent conversion of l -arginine to l - citrulline, with an apparent K _m of 51.2 n M , and this activity could be blocked by l -arginine analogues in the potency order amino > methyl > nitroarginine. Immunoblot analysis revealed an apparent molecular mass of 125 kDa for the NOS protein induced in C6 cells. These results indicate that the combination of LPS plus cytokines can induce NOS activity in C6 glioma cells with properties similar to those of the enzyme expressed in primary astrocyte cultures.     

Modulation of tubulin mRNA levels by interferon in human lymphoblastoid cells.

Blot hybridization with labeled tubulin cDNA showed that treatment of Ramos cells, a human cell line of lymphoblastoid origin, with either alpha or beta interferon (IFN) induced a marked increase in the amount of tubulin mRNA sequences. The level of tubulin mRNA sequences increased rapidly after exposure of cells to IFN-alpha and reached a maximum after 1 h of treatment, which was four times the control level. Treatment with IFN-beta induced a maximal increase after 4 h; the amount of tubulin mRNA sequences was seven times higher than the control level. The mRNA extracted from IFN-treated and nontreated cells was translated in vitro in a reticulocyte lysate cell-free system containing [35S]methionine. Electrophoretic analysis of the labeled cell-free products showed an increase in the amount of translatable tubulin mRNA that parallels the time course of induction of tubulin mRNA sequences. Two-dimensional gel electrophoresis of the labeled protein products directed by mRNA indicates that IFN caused a more pronounced increase in the level of alpha-tubulin than beta-tubulin mRNA. Treatment with colchicine, which disrupts the cell microtubules, caused a marked decrease in the tubulin mRNA content. Concomitant treatment of the cells with colchicine and IFN abolished the interferon-dependent induction of tubulin mRNA.     

Down-Regulation of the Noradrenaline Transporter by Interferon-α in Cultured Bovine Adrenal Medullary Cells

Abstract: The aim of this study was to investigate the effect of long-term treatment with interferon (IFN)-α on the noradrenaline transporter of bovine adrenal medullary cells. Treatment of cultured adrenal medullary cells with IFN-α caused a decrease in uptake of [³H]noradrenaline by the cells in time (4–48 h)- and concentration (300–1,000 U/ml)-dependent manners. IFN-β also inhibited [³H]noradrenaline uptake to a lesser extent than did IFN-α, whereas IFN-γ had little effect. An anti-IFN-α antibody reduced the effect of IFN-α on [³H]noradrenaline uptake. Saturation analysis of [³H]noradrenaline uptake showed that the inhibitory effect of IFN-α was due to a reduction in the maximal uptake velocity ( V _max) values without altering apparent Michaelis constant ( K _m) values. Incubation of cells with IFN-α caused a translocation of protein kinase C from the soluble to the particulate fraction in the cells. The effect of IFN-α on [³H]noradrenaline uptake was diminished in protein kinase C-down-regulated cells. Incubation of cells with IFN-α for 48 h significantly reduced the specific binding of [³H]desipramine to crude plasma membranes isolated from cells. Scatchard analysis of [³H]desipramine binding revealed that IFN-α decreased the maximal binding ( B _max) values without any change in the dissociation constant ( K _D) values. These findings suggest that IFN-α suppresses the function of noradrenaline transporter by reducing the density of the transporter in cell membranes through, at least in part, a protein kinase C pathway.     

Do cells treated with human interferon survive virus infection?

Abstract HeLa cells pretreated with human lympho-blastoid interferon (Hu IFN-α (Ly)), at concentrations up to 100 IU / ml and infected with moderate multiplicities of encephalomyocarditis (EMC) virus (10 PFU / cell) died 1 or 2 days after infection. However, if cells were repeatedly treated with high doses of IFN (800 IU / ml) they survived infection by EMC virus for at least a month. Cells survived Semliki Forest virus (SFV) infection when even lower IFN concentrations were used. By contrast infection of IFN-treated HeLa cells with other RNA-containing viruses, such as poliovirus, vesicular stomatitis virus (VSV), Newcastle disease virus (NDV) and reovirus type 3 resulted in cell death. Similarly, infection with a number of DNA-containing viruses such as adenovirus type 5, Herpes simplex virus types 1 and 2 (HSV-1 and HSV-2) and vaccinia virus killed cells. The results are discussed in the light of different models for the molecular mechanism of action of interferon.     

Tumor Necrosis Factor-α (TNF-α), Interferon-γ, and Interleukin-6 but Not TNF-β Induce Differentiation of Neuroblastoma Cells: The Role of Nitric Oxide

Abstract: Tumor necrosis factor-a (TNF-α), interferon-γ (IFN-7), and interleukin-6 (IL-6), but not TNF-β, can induce the in vitro differentiation of the neuroblastoma cell line N103 in a dose-dependent manner. Differentiation of N103 was accompanied by the arrest of cell growth and neurite formation. The induction of neuroblastoma cell differentiation by TNF-α and IFN-γ can be specifically inhibited by a nitric oxide (NO) synthase inhibitor, l -N^G-monomethylarginine. In contrast, the differentiation of N103 cells by IL-6 was not affected by l -N^G-monomethylarginine. These results indicate that TNF-α and IFN-γ, but not IL-6, induce the differentiation of neuroblastoma cells via NO. This is confirmed by the finding that the culture super- natants of N103 cells induced by TNF-α and IFN-γ, but not that by IL-6, contained high levels of NO₂⁻, the production of which was inhibited by l - N ^G-monomethylarginine. Furthermore, the differentiation of N103 cells can be induced directly in a dose-dependent manner by the addition of nitroprusside, a generator of NO, into the culture medium. These data therefore indicate that NO may be an important mediator in the induction of neuronal cell differentiation by certain cytokines such as TNF-α and IFN-γ and that neuronal cells, in addition to the macrophagelike brain cells, can be induced by immunological stimuli to produce large quantities of NO.     

Interferon-γ, interleukin-1 and tumour necrosis factor-α synthesis during experimental murine staphylococcal infection

Abstract Several exotoxins of Staphylococcus aureus were shown to modulate the host immune system by stimulation of monokine release. BALB/c mice infected intravenously (i.v.) with live cells if S. aureus , strain Cowan 1, had a detectable serum level of TNF-α at 3, 4 and 5 h after injection. When S. epidermidis (strain F3380, clinical isolate) was used to infect mice, the level of TNF-α was lower (the detection limit of the cytotoxicity assay with WEHI cells was 40 pg ml⁻). Kinetics of TNF synthesis was different from that observed in experimental infections caused by Gram-negative bacteria. Similarly to TNF-α, IL-1α appears in a measureable level at 3 h after i.v. injection of bacteria. The highest serum level of IFN-γ was observed 12 h after infection with both S. aureus and S. epidermidis . A quantity ten times more of S. epedermidis than of S. aureus cells was required to induce similar levels of TNF-α and IFN-γ administered in vivo in four daily doses followed by infection of S. aureus resulted in increased elimination of bacteria from the spleen, liver and peritoneal cavity of mice.     

Elevated levels of (2''-5'')oligoadenylic acid polymerase activity in growth-arrested human lymphoblastoid Namalva cells.

(2'-5')Oligoadenylic acid [(2'-5')An] polymerase activity was measured in extracts of human lymphoblastoid cells of the Namalva line cultured under different conditions. Exponentially growing cells had a relatively low polymerase activity level, whereas cells grown to limit density showed elevated levels. When fresh medium was added to growth-arrested cells, (2'-5')An polymerase activity decreased concomitantly with the initiation of active deoxyribonucleic acid synthesis. An increase in polymerase activity level was also observed after exponentially growing cells were transferred from medium containing 20% serum to fresh medium containing 0.2% serum. These cells diminished deoxyribonucleic acid synthesis and remained quiescent until 20% serum was again added. Polymerase activity level decreased as the cells entered into S phase. The addition of the inhibitor of deoxyribonucleic acid synthesis, hydroxyurea, to exponentially growing cells did not increase polymerase level, indicating that cells blocked in S phase and at the G1-S boundary maintained the basal level of this enzyme. Degradation of labeled (2'-5')An was measured in extracts of Namalva cells cultured under different conditions, but no significant differences among degradative activities were observed. Since (2'-5')An polymerase activity is one of the enzymatic activities induced by interferon, we measured interferon titers in Namalva cell medium. Less than 1 reference unit per ml was detected in cells grown under different conditions. Moreover, the increase in (2'-5')An polymerase activity level in cells transferred from 20 to 0.2% serum was not prevented by including anti-lymphoblastoid interferon antibody in the medium. These results suggest that the activity level of (2'-5')An polymerase is regulated in Namalva cells on the basis of the growth status of the cells and that this regulatory mechanism is apparently not activated by interferon.     

Cytokine- and Endotoxin-Induced Nitric Oxide Synthase in Rat Astroglial Cultures: Differential Modulation by Angiotensin II

Abstract: Recent studies have shown that the stimulatory effects of bacterial endotoxin [lipopolysaccharide (LPS)] on inducible nitric oxide (NO) synthase (iNOS) in astroglia are significantly reduced by the peptide angiotensin II (Ang II). In the present study we have compared the modulatory actions of Ang II on cytokine- and LPS-stimulated iNOS in astroglia cultured from adult rat brain. Incubation of astroglia with LPS (100 ng/ml; 24 h) and/or combinations of interleukin-1β (IL-1β; 10 ng/ml, 24 h), interferon-γ (IFN-γ; 100 U/ml, 24 h), or tumor necrosis factor-α (TNF-α; 100 ng/ml, 24 h) resulted in significant increases of iNOS mRNA, iNOS protein, and NO production, with the latter indicated by increased nitrite accumulation. The effects of LPS, IL-1β, and TNF-α were significantly decreased by coincubation with Ang II (100 ng/ml, 24 h). In contrast, Ang II did not alter the stimulation of iNOS mRNA levels and NO production elicited by IFN-γ. Therefore, Ang II differentially modulates the stimulatory actions of LPS and cytokines on iNOS, and subsequently NO production, in astroglia. These data suggest that Ang II may have an important modulatory role in intracerebral immune responses that involve production of NO by astroglia.     

Involvement of various combinations of endogenous inflammatory cytokines in Listeria monocytogenes-induced expression of inducible nitric oxide synthase in mice

Abstract Using an in vitro infection of spleen cells with Listeria monocytogenes , the relationship between endogenous cytokines and the expression of inducible nitric oxide synthase (iNOS) was examined. When all interferon (IFN)-γ, tumor necrosis factor (TNF)-α and interleukin (IL)-1 α, or the combination of IFN-γ with either TNF-α or IL-1 α were neutralized by antibodies, there was a significant reduction of iNOS expression and nitrite production in culture. However, there was no reduction of iNOS expression and nitrite production when these cytokines were individually neutralized. After the depletion of natural killer cells, there was no change in the expression of Listeria -induced iNOS and nitrite production although the IFN-γ production was abrogated. Neutralization of TNF-α and IL-1 α in natural killer cell-depleted culture resulted in the reduction of iNOS expression. Thus, various combinations of cytokines appeared to play an important role in iNOS induction by L. monocytogenes .     

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.     

IL-17 is involved in bone resorption in mouse periapical lesions

Periapical lesions are induced by bacterial infection of the dental pulp and result in destruction of the surrounding alveolar bone. Although various immunological studies concerning periapical bone resorption have been reported, the role of cytokines in the formation of periapical lesions remains unclear. In this study, the role of IL-17A in periapical lesions in mice was investigated. Normal C57BL/6, IFN-γ^−/−, TNF-α^−/−, and IL-17A^−/− mice were subjected to pulp exposure and infected with Prevotella intermedia (ATCC25611) and Porphyromonas gingivalis (ATCC33277) in the mandibular first molar. Periapical lesions were determined by μCT on day 21 after infection, and 3D visual construction was performed using 3D picture quantification software. The expression of IL-17A mRNA in periapical lesions was determined by the RT-PCR and real-time RT-PCR method. Periapical lesions developed in wild-type, IFN-γ^−/−, and TNF-α^−/− mice after infection with P. intermedia and P. gingivalis . However, periapical lesions were not observed in IL-17A^−/− mice. The expression of IL-17A mRNA was significantly induced in periapical lesions of wild-type mice after infection. These results suggest that IL-17A, but not IFN-γ or TNF-α, plays an important role in the formation of periapical lesions.     

Interferon gamma mRNA "superinduction" in human lymphocytes

Human peripheral blood mononuclear lymphocytes produce interferon gamma (IFN-gamma) in response to stimulation by mitogens. Previous studies on the kinetics of IFN-gamma mRNA production upon mitogen induction, showed that steady-state levels of mRNA increased to a maximum at 12-24 h post-induction after which they declined to levels not detectable by the assay used. We show here that in mitogen induced peripheral blood lymphocytes, inhibition of protein synthesis using three different inhibitors (cycloheximide, puromycin, pactamycin) resulted in an increase in the steady-state levels of IFN-gamma mRNA. The levels of mRNA in cells treated with inhibitor 16 h post-induction were up to 3-fold higher than in untreated cells. Superinduction was possible up to 40 h post-induction after which the steady-state levels of mRNA had declined to limits below detection; IFN-gamma mRNA was not superinduced by cycloheximide in the presence of actinomycin D.     

Field production and functional evaluation of chloroplast-derived interferon-alpha2b

Type I interferons (IFNs) inhibit viral replication and cell growth and enhance the immune response, and therefore have many clinical applications. IFN-α2b ranks third in world market use for a biopharmaceutical, behind only insulin and erythropoietin. The average annual cost of IFN-α2b for the treatment of hepatitis C infection is $26 000, and is therefore unavailable to the majority of patients in developing countries. Therefore, we expressed IFN-α2b in tobacco chloroplasts, and transgenic lines were grown in the field after obtaining United States Department of Agriculture Animal and Plant Health Inspection Service (USDA-APHIS) approval. Stable, site-specific integration of transgenes into chloroplast genomes and homoplasmy through several generations were confirmed. IFN-α2b levels reached up to 20% of total soluble protein, or 3 mg per gram of leaf (fresh weight). Transgenic IFN-α2b had similar in vitro biological activity to commercially produced PEG-Intron™ when tested for its ability to protect cells against cytopathic viral replication in the vesicular stomatitis virus cytopathic effect (VSV CPE) assay and to inhibit early-stage human immunodeficiency virus (HIV) infection. The antitumour and immunomodulating properties of IFN-α2b were also seen in vivo . Chloroplast-derived IFN-α2b increased the expression of major histocompatibility complex class I (MHC I) on splenocytes and the total number of natural killer (NK) cells. Finally, IFN-α2b purified from chloroplast transgenic lines (cpIFN-α2b) protected mice from a highly metastatic tumour line. This demonstration of high levels of expression of IFN-α2b, transgene containment and biological activity akin to that of commercial preparations of IFN-α2b facilitated the first field production of a plant-derived human blood protein, a critical step towards human clinical trials and commercialization.     

Expression of mRNA encoding IFNα in macrophages stimulated with Lactobacillus gasseri

Abstract The ability of Lactobacillus gasseri , a dairy lactic acid bacterium, to induce interferon (IFN) was investigated in murine macrophage cultures. IFN α was substantially induced by some strais of L. gasseri and the titers were the highest at a concentration of 100 μg ml⁻¹ of L. gasseri DSM20243^T. The expression of mRNA encoding IFN α was detected in spleen-macrophages (SP-M θ ) and Peyer's patch-adherent cells stimulated with L. gasseri DSM20243^T. Actinomycin D and cycloheximide added to SP-M θ cultures showed that the mRNA was synthesized by 0.5 h, and that IFN α was produced within 3 to 6 h after the stimulation with L. gasseri DSM20243^T. The results support the notion that dairy products containing L. gasseri can be 'physiologically functional foods'.     

Interferon α enhances expression of TAG-72 and carcinoembryonic antigen in patients with primary colorectal cancer

Interferons up-regulate the expression of human tumor-associated antigens in animal models and in vitro. The use of interferons may enhance the immunodetection and immunotherapy of tumors by monoclonal antibodies that detect tumor antigens. For this strategy to be effective, however, the interferon must have an effect at the site of the tumor. In this study, the induction by interferon (IFN) of two tumor surface antigens was evaluated in six patients with primary colorectal cancer. Patients were treated with IFN and 48 h later underwent resection of the tumor. The interferon treatment induced expression of a tumor-associated glycoprotein (TAG-72) in two patients without antigen expression prior to interferon but had no effect on one TAG-72-negative tumor. IFN did not induce expression of carcinoembryonic antigen (CEA) in the two patients whose tumors were CEA-negative prior to interferon. In all patients with heterogeneous expression of CEA and TAG-72 prior to IFN treatment, preoperative interferon increased the percentage of cells positive for CEA in two patients and TAG-72 in one patient. This study supports the addition of interferon induction to immunotherapy regimens directed at the CEA and TAG-72 cell-surface antigens.