Effect of interferon on the increased reduction of nitroblue tetrazolium (NBT)]

Human leukocyte interferon (HL-IF) enhanced the NBT reduction of human peripheral neutrophil in vitro. Dose relation between IF activity and the NBT reduction was recognized. Heat-inactivated HL-IF, HL-IF neutralized by anti-IF serum or heterologous IF could not increase the NBT reduction.     

Two inducers of cell differentiation enhance the 2'5' oligoadenylate synthetase activity in MSV transformed cells

The interferon induced 2′5′ oligoadenylate synthetase activity can be increased upon treatment of Moloney Sarcoma virus transformed cells with two inducers of cell differentiation: sodium n-butyrate and dimethyl sulfoxide. This effect does not seem to be the consequence of the inhibition of cell growth by butyrate since the basic level of the enzyme stayed the same in control cells whether growth was inhibited by the absence of serum in the medium or not. It did not seem either to be due to the induction of IFN by these compounds since we could not detect any antiviral activity in the supernatant of the treated cells. Treatment by interferon of the butyrate pretreated cells results in a higher enzyme activity and a higher antiviral state than in non-pretreated cells.     

Activated macrophages mediate interferon-independent inhibition of herpes simplex virus

Activated macrophages exhibit extrinsic antiviral activity (inhibition of virus replication in other cells) which may involve mechanisms similar to macrophage antitumor activity or macrophage-mediated immunosuppression. Peritoneal macrophages elicited in mice by Corynebacterium parvum vaccine suppressed the growth of herpes simplex virus (HSV) in infected cells by an interferon-independent mechanism. This was demonstrated by expression of activity against HSV-infected xenogeneic (Vero) cells. Culture supernatant fluids also did not mediate antiviral activity, and did not contain detectable levels of interferon (< 3 IU/ml). Moreover, antiviral activity was not affected by the presence of anti-mouse interferon IgG. Antiviral activity was expressed at 12–16 hr after infection, at the end of the first cycle of virus replication. Cell contact was required for optimal activity. No enhanced adsorption or phagocytosis of HSV by C. parvum macrophages could be detected nor was macrophage cytotoxicity responsible for the activity. Cytotoxicity (⁵¹Cr release) by macrophages for virus infected cells was low (< 6% specific cytotoxicity), and was not significantly higher with C. parvum macrophages than with resident macrophage controls. Although C. parvum macrophages were not cytotoxic at the macrophage-host cell ratio employed, they did significantly inhibit uptake of [³H]leucine by the host Vero cells. This suggests that inhibition of host cell metabolism by the macrophage, similar to macrophage immunosuppression, may be responsible for the antiviral activity in this system.     

Kaposi's sarcoma-associated herpesvirus viral interferon regulatory factor confers resistance to the antiproliferative effect of interferon-alpha

BACKGROUND: Kaposi's sarcoma-associated herpesvirus (KSHV) encodes a 442 amino acid polypeptide-designated viral interferon regulatory factor (vIRF) that displays homology to members of the interferon regulatory factor (IRF) family that bind to consensus interferon sequences and transactivate cellular genes that can modulate growth inhibition. Studies were conducted to determine whether vIRF affects the growth suppression mediated by interferon-alpha (IFN-alpha) in a human B lymphocyte cell line. MATERIALS AND METHODS: The human B lymphocyte cell line Daudi, which is sensitive to the antiproliferative effects of IFN-alpha, was stably transfected to express vIRF, and the proliferative response of vIRF expressing cells to IFN-alpha was compared with controls. The effect of vIRF on IRF- 1 transactivation was analyzed by co-transfection of an IFN-alpha-responsive chloramphenicol acetyltransferase reporter and expression plasmids encoding IRF-1 and vIRF. Electrophoretic mobility shift assays were conducted to determine whether vIRF interferes with the DNA binding activity of IRF-1. RESULTS: Daudi human B lymphocyte cells expressing vIRF were resistant to the antiproliferative effects of IFN-alpha, whereas wild-type Daudi or Daudi cells transformed with vector DNA were growth inhibited by IFN-alpha. The activation of an interferon-responsive reporter by IFN-alpha or IRF-1 was repressed by expression of vIRF. IRF-1 DNA binding activity was unaffected by vIRF, and vIRF alone did not bind to the interferon consensus sequence. CONCLUSIONS: These studies revealed that vIRF functions to inhibit interferon-mediated growth control of a human B lymphocyte cell line by targeting IRF-1 transactivation of interferon-inducible genes. Since KSHV is a B lymphotropic herpesvirus associated with two forms of B lymphocyte neoplasms, these effects of vIRF likely contribute to B cell oncogenesis associated with KSHV infection.     

Separation of human leukocyte interferon components by concanavalin A-agarose affinity chromatography and their characterization.

Human leukocyte interferon (HL-IF), produced by mixed leukocytes infected with Newcastle disease virus, was resolved into three distinct fractions when chromatographed on concanavalin A-agarose. The major portion (70--75%) of interferon appeared in the breakthrough (BT fraction). The bound interferon (25--30%) was displaced from the column as two peaks: the first was eluted with 0.01 M methyl alpha-D-mannoside, yielding 15-20% of the interferon activity (alpha-MM fraction), and the second by including ethylene glycol (70%) in the eluant, yielding the remaining 5--15% of the interferon (EG fraction). No interferon was retained when HL-IF produced in the presence of glycosylation inhibitors (tunicamycin or 2-deoxy-D-glucose) was chromatographed on concanavalin A-agarose, suggesting that the fraction of interferon retained by this lectin is glycosylated. The three fractions of interferon (BT, alpha-MM, and EG) were characterized by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, cross-species antiviral activity, and neutralization by specific antisera. The BT fraction contains exclusively the 16 000 molecular weight component of human leukocyte interferon. The majority of the alpha-MM fraction (90%) is the 21 000 molecular weight component. However, the EG fraction contains the 16 000 and 21 000--23 000 molecular weight components in essentially equal proportions. On the basis of cross-species antiviral activity and neutralization by specific antisera, the BT and alpha-MM fractions are leukocyte-type interferon and the EG fraction seems to be primarily of fibroblast type.     

Inhibition of human herpes simplex virus type 2 by interferon gamma and tumor necrosis factor alpha is mediated by indoleamine 2,3-dioxygenase

Genital herpes simplex virus type 2 (HSV-2) is a significant clinical problem. Infection in pregnancy may result in disseminated infection of the newborn with encephalitis. We analyzed the antiviral effects induced by interferon-gamma (IFN-gamma) and tumor necrosis factor alpha (TNF-alpha) in cervix carcinoma cells (HeLa) and astrocytoma cells (86HG39). We found that replication of HSV-2 in HeLa cells and in 86HG39 cells is inhibited after stimulation of the cells by IFN-gamma and TNF-alpha. The antiviral effect of IFN-gamma is enhanced in the presence of TNF-alpha, while stimulation by TNF-alpha alone did not induce antiviral activity. We found that IFN-gamma induces a strong activation of the tryptophan-degrading enzyme indoleamine 2,3-dioxygenase (IDO) and in addition, that the IFN-gamma-induced IDO activity was enhanced in the presence of TNF-alpha. Furthermore, we found that the induction of IDO activity is responsible for the inhibition of herpes simplex virus replication, since the presence of excess amounts of l-tryptophan abrogates the antiviral effect induced by IFN-gamma and the combination of IFN-gamma and TNF-alpha. We therefore conclude that the antiviral effect against HSV-2 mediated by type II interferon and TNF-alpha are dependent on IDO activation.     

Comparison of antiviral and antitumor activity of activated macrophages.

The antiviral and antitumor activity in vitro of normal, stimulated, vaccinia virus “immune”, and activated peritoneal macrophages were compared. Activated (pyran or corynebacteria induced) PEC exhibited both antitumor and antiviral activity. Stimulated (thioglyocollate) and vaccinia virus “immune” PEC inhibited virus growth but did not possess antitumor activity. Normal (unstimulated) PEC were relatively ineffective in either activity. The antiviral activity was nonspecific, being expressed against herpes simplex and EMC viruses in addition to vaccinia. Although a possible role for interferon was suggested by the lack of activity of mouse PEC on vaccinia virus growth in heterologous FLK cells, definitive proof was not obtained. The activity was most pronounced against multiple cycles of viral infection initiated at a low multiplicity of infection. Single cycle virus growth was not affected, suggesting that the major inhibition was on subsequent cycles of virus growth.     

Effect of interferon on the inhibition of malignant cell growth by human peripheral leukocytes. III. Effect of systemic administration

Human leukocyte interferon preparation (HuIFN-alpha LE) was given to the patients with cancer or with chronic hepatitis. Spontaneous tumor cell growth inhibition by human peripheral lymphocytes (STGI) and NK activity were enhanced by the systemic administration of HuIFN-alpha LE, although there were differences in the kinetics between the two activities after one time administration or by the repeated administration. This suggests that IFN acts indirectly on the tumor cells by the medium of normal lymphocytes or NK cells, and that tumor cell growth inhibition is different from NK activity.     

Differentiation of the immunosuppressive and antiviral effects of interferon.

Virus-induced (virus-type) interferon suppression of the in vitro antibody response of mouse (C57B1/6) spleen cells to sheep red blood cells was blocked by 5 × 10^?5M 2-mercaptoethanol (2-ME). The blockade was not due to a direct effect on interferon since 2-ME was capable of blocking the suppression when added to cultures up to 48 hr after interferon. 2-ME blockade of virus-type interferon immunosuppression was not due to the immunoenhancing property of 2-ME. Similar protective effects of 2-ME were observed during immunosuppression by virus-type interferon inducers, but not T-cell mitogen inducers of interferon (immune interferon). The data suggest that the immunosuppressive properties of virus-type and immune interferon preparations involve different mechanisms. Virus-type interferon inhibited DNA synthesis in unstimulated spleen cell cultures and in 2-ME stimulated cultures, and the degree of inhibition of DNA synthesis appeared to be related to the immunosuppressive property of interferon in the absence or presence of 2-ME. 2-ME did not affect the antiviral properties of either virus-type or immune interferon in nonlymphoid cells. Further, the induction of virustype interferon in spleen cells was neither inhibited nor enhanced by 2-ME, while the induction of immune interferon was enhanced. This enhancement is consistent with 2-ME enhancement of the immunosuppressive effects of immune interferon inducers.There are two possibilities for 2-ME blockade of the immunosuppressive effect of virus-type interferon, while not affecting the antiviral property. Firstly, the immunosuppressive and antiviral properties of virus-type interferon may involve different mechanisms at the subcellular level. Secondly, the selectivity of the blockade by 2-ME could be due to the fact that spleen cells are the target cells in immunosuppression, while L cells are the target cells in inhibition of virus replication. Thus, virus-type interferon may suppress the immune response at the level of the macrophage and 2-ME may reverse this effect by replacing a blocked macrophage function.     

The role of interferon-beta 1 and the 26-kDa protein (interferon-beta 2) as mediators of the antiviral effect of interleukin 1 and tumor necrosis factor

This study confirms our earlier finding that human interleukin (IL)-1 beta exerts an antiviral effect on diploid fibroblasts and on MG-63 osteosarcoma cells. It also extends the observation in that a similar effect was noted on aged but not freshly trypsinized HEp-2 cells, and that not only IL-1 beta but also IL-1 alpha and tumor necrosis factor (TNF)-alpha exerted similar antiviral effects on cells. The antiviral effects of these cytokines were neutralized by addition to the assay system of an antibody that was specific for interferon (IFN)-beta 1, indicating that IFN-beta 1 or a structurally or functionally related substance is involved in the antiviral activity observed. Both IL-1 and TNF were able to induce production of the 26-kDa protein, also known as IFN-beta 2, hybridoma/plasmacytoma growth factor (HPGF) or B-cell stimulatory factor-2 (BSF-2) and previously proposed as an alternative to IFN-beta 1 for mediating the antiviral effect of TNF. However, no good correlation was found between the antiviral effects of TNF and its potential to induce production of the 26-kDa protein. Furthermore, the anti-IFN-beta 1 serum which neutralized the antiviral activity of IL-1 and TNF did not cross-react with the 26-kDa protein. Conversely, the antiviral effect of IL-1 and TNF was only weakly neutralized by an antibody that did react with the 26-kDa protein and showed low cross-reactivity with IFN-beta 1. These observations, together with the low specific activity of the 26-kDa protein as an antiviral agent (less than 10(5) U/mg protein) provide strong arguments against this protein and in favor of IFN-beta 1 (or still another IFN-beta 1-related molecule) as the ultimate mediator of the antiviral effect of IL-1 and TNF.     

Studies on the Mechanism of Interferon Action

Interferon does not inactivate viruses or viral RNA. Virus growth is inhibited in interferon-treated cells, but apart from conferring resistance to virus growth, no other effect of interferon on cells has been definitely shown to take place. Interferon binds to cells even in the cold, but a period of incubation at 37°C is required for development of antiviral activity. Cytoplasmic uptake of interferon has not been unequivocally demonstrated. Studies with antimetabolites indicate that the antiviral action of interferon requires host RNA and protein synthesis. Experiments with 2-mercapto-1(β-4-pyridethyl) benzimidazole (MPB) suggest that an additional step is required between the binding and the synthesis of macromolecules. Interferon does not affect the adsorption, penetration, or uncoating of RNA or DNA viruses, but viral RNA synthesis is inhibited in cells infected with RNA viruses. The main action of interferon appears to be the inhibition of the translation of virus genetic information probably by inhibiting the initiation of virus protein synthesis.     

IL-28 elicits antitumor responses against murine fibrosarcoma

IL-28 is a recently described antiviral cytokine. In this study, we investigated the biological effects of IL-28 on tumor growth to evaluate its antitumor activity. IL-28 or retroviral transduction of the IL-28 gene into MCA205 cells did not affect in vitro growth, whereas in vivo growth of MCA205IL-28 was markedly suppressed along with survival advantages when compared with that of controls. When the metastatic ability of IL-28-secreting MCA205 cells was compared with that of controls, the expression of IL-28 resulted in a potent inhibition of metastases formation in the lungs. IL-28-mediated suppression of tumor growth was mostly abolished in irradiated mice, indicating that irradiation-sensitive cells, presumably immune cells, are primarily involved in the IL-28-induced suppression of tumor growth. In vivo cell depletion experiments displayed that polymorphonuclear neutrophils, NK cells, and CD8 T cells, but not CD4 T cells, play an equal role in the IL-28-mediated inhibition of in vivo tumor growth. Consistent with these findings, inoculation of MCA205IL-28 into mice evoked enhanced IFN-gamma production and cytotoxic T cell activity in spleen cells. Antitumor action of IL-28 is partially dependent on IFN-gamma and is independent of IL-12, IL-17, and IL-23. IL-28 increased the total number of splenic NK cells in SCID mice and enhanced IL-12-induced IFN-gamma production in vivo and expanded spleen cells in C57BL/6 mice. Moreover, IL-12 augmented IL-28-mediated antitumor activity in the presence or absence of IFN-gamma. These findings indicate that IL-28 has bioactivities that induce innate and adaptive immune responses against tumors.     

Effect of nucleosides on interferon production and development of antiviral state induced by poly I.poly C.

Effect of nucleosides both on induction of antiviral state in chick embryo cells (CEC) or rabbit kidney cells (RK13) and on interferon production in RK13 or mouse fibroblast cells (L cells) by polyriboinosinic-polyribocytidylic acid (poly I.poly C) was studied. Addition of inosine or a fifty-fifty mixture of inosine and uridine at a final concentration of 0.1 mM to 10 mM to a growth medium enhanced development of antiviral state in CEC. The nucleoside effect was also observed in RK13 at 0.1 mM but not at a concentration higher than 1 mM. Interferon production in RK13 by superinduction (sequential treatment with metabolic inhibitors after exposure to poly I.poly C) was enhanced 1.5- to 4.0-fold by addition of the nucleoside mixture to the growth medium. When RK13 was pretreated with 10 units per ml of interferon and then superinduced by inhibitors, the enhancing effect of nucleosides on interferon production was not observed. Interferon production in L cells was potentiated a little by addition of 1 mM of the nucleoside mixture to the growth medium. The effect of nucleoside was not observed when the nucleosides were added after exposure to poly I.poly C. The nucleoside effect may be applicable for production of high titered interferon.     

Mechanism of Interferon Action: Inhibition of Viral Messenger Ribonucleic Acid Translation in L-Cell Extracts

Encephalomyocarditis (EMC) virus ribonucleic acid (RNA) stimulated the incorporation of (14)C-amino acids into polypeptides in cell-free systems using preincubated S10 extracts from L cells. Incorporation was linear for over 2 hr. Analysis of the tryptic peptides derived from the polypeptide products formed in response to EMC RNA showed them to be virus specific. The major product, a polypeptide of 140,000 in molecular weight, migrated on sodium dodecyl sulfate-polyacrylamide gels with one of the virus-specific polypeptides present in EMC-infected cells. A minor component of molecular weight about 230,000 may correspond to the product of complete translation of the EMC virus genome. Little or no effect of interferon or vaccinia virus infection was observed in the preincubated, cell-free system. The EMC RNA-stimulated incorporation of (14)C-amino acids into polypeptides was not inhibited in extracts derived from L cells early in virus infection, from interferon-treated cells, or from cells subjected to both treatments. Interferon treatment did appear to have a slight inhibitory effect on chain elongation in this system. However, treatment of cells with highly purified interferon before virus infection caused a decrease of about 80% in the capacity of non-preincubated cell extracts to translate added EMC RNA. This effect did not extend to the translation of polyuridylic acid and could be reversed by preincubation of the extracts at 37 C for 20 min. The inhibition of translation was manifest at interferon concentrations as low as 5IU/ml, and in this respect closely paralleled the inhibition of virus growth. Inactivation of the antiviral activity of the interferon by heating or digestion with trypsin also abolished the effect on cell-free protein synthesis. The EMC-specific polypeptides formed in reduced amounts in extracts of interferon-treated vaccinia-infected cells were smaller than those formed in extracts of untreated, vaccinia-infected cells. Thus, inhibition of initiation or elongation of polypeptides, or both, can be demonstrated in cell-free systems employing non-preincubated extracts from interferon-treated, virus-infected cells. These results indicate that antiviral activity of interferon is directed against the translation of viral messenger RNA.     

The anti-chlamydial and anti-proliferative activities of recombinant murine interferon-gamma are not dependent on tryptophan concentrations

The effect of recombinant murine interferon-gamma on the growth of Chlamydia trachomatis was analyzed in a mouse fibroblast cell line (McCoy cells). Murine interferon-gamma had a very potent anti-chlamydial activity, although minimally affecting cellular proliferation. Over 95% inhibition of chlamydial inclusions was obtained at a concentration of 1 U/ml of interferon. At a concentration of 1 U/ml of murine interferon-gamma, there was minimal inhibition of the proliferative capacity of McCoy cells. Approximately 50% inhibition of cell growth was obtained with a concentration of 10 U/ml of interferon. Varying concentrations of tryptophan in the medium did not alter either the anti-chlamydial or the anti-proliferative activity of the interferon.     

Effects of diethyldithiocarbamate, an inhibitor of interferon antiviral activity, upon human natural killer cells

In support of a postulated role of the Cu++-dependent enzyme, superoxide dismutase (SOD), in antiviral effects of interferon (IFN), a close correspondence was previously shown to exist between inactivation of cellular SOD and concomitant blockade of IFN antiviral activity in fibroblasts by the Cu++-chelating agent, diethyldithiocarbamate (DDC). To further define the extent of "anti-IFN" activity, we initiated studies of DDC effects on IFN stimulation in the NK cell system. Unexpectedly, DDC directly inhibited cytotoxicity mediated by unstimulated NK cells. Pronounced inactivation occurred rapidly (less than 30 min), but was spontaneously reversible in the absence of DDC. Neither cell viability nor lymphocyte binding to target cells was detectably affected. Preincubation of DDC with Cu++ or Zn++ failed to neutralize its inhibitory effects nor could function be restored in DDC-pretreated NK cells by subsequent addition of Cu++, Zn++, Mg++, or Ca++. DDC treatment that inactivated NK cells did not detectably alter lymphocyte SOD activity. Thus, inhibition was probably not attributable to chelating properties of DDC. N-ethyl maleimide (NEM) and para-( hydroxymercuri ) benzoic acid ( PMBA ), enzyme inhibitors that preferentially react with sulfhydryl groups, both inactivated NK cells in a time- and dose-dependent manner similar to that of DDC. Preincubation with the sulfhydryl compound, cysteine, neutralized in parallel fashion the capacity of NEM, PMBA , and DDC to inhibit NK cell activity. Thus, a previously unreported reactivity of DDC with sulfhydryl groups appeared to be the basis of inhibition. NK cells incubated 1 hr with IFN and subsequently cultured 17 to 23 hr without IFN were activated to an extent comparable to cells continuously incubated 18 to 24 hr with IFN. Exposure to IFN for 1 hr was therefore sufficient to commit NK cells to acquisition of a fully activated state. Whether preactivated by a 1-hr or 18- to 24-hr IFN treatment, activated NK cells retained the DDC-sensitive phenotype characteristic of fresh unstimulated NK cells. Thus, prolonged IFN treatment did not render NK cells resistant to DDC or preferentially activate a DDC-sensitive NK cell subset. An 18- to 24-hr incubation of DDC-pretreated cells in the continual presence of IFN resulted in the boosting of NK cell activity. However, the 1-hr IFN pulse treatment protocol was consistently ineffective in boosting when IFN was added just after DDC-pretreatment. These results strongly suggested that DDC temporarily rendered NK cells unresponsive to what, under normal circumstances, approximated an optimally potentiating IFN stimulus.(ABSTRACT TRUNCATED AT 400 WORDS)     

Affinity of human leukocyte interferon for polyribonucleotides.

Human leukocyte interferon (HL-IF)binds to AGPOLY(A)TM, AGPOLY(U)TM and AGPOLY(I)TM. The bound interferon could be displaced from all three polyribonucleotides by including sodium chloride in the eluant. The nature of interaction of HL-IF with polyribonucleotides is electrostatic and not hydrophobic since its binding was not prevented in the presence of 50% ethylene glycol. The binding of HL-IF on AGPOLY(I)TM is stronger at lower pH since an increase in ionic strength is required to displace it.     

Species specificity of interferon action: maintenance and establishment of the antiviral state in the presence of a heterospecific nucleus.

The expression of the interferon-induced antiviral state was studied in heterokaryons and cytoplasmic hybrids (cybrids). An autoradiographic assay for the antiviral state, in which the percentage of cells containing vaccinia viral DNA factories was determined, was used. The expression of the antiviral state was dominant in homokaryons and heterokaryons formed by fusion of interferon-treated cells with untreated cells. Cytoplasts derived from treated cells conferred resistance to virus growth on cybrids formed by fusing such cytoplasts with untreated cells. Treatment of L cell x HeLa cell heterokaryons with human interferon or mouse interferon was much less effective in inducing a detectable antiviral state than was similar treatment of parental cells with homospecific interferon. The antiviral state was fully induced when heterokaryons were treated simultaneously with both types of interferon. Cybrids formed by fusing L cell cytoplasts with HeLa cells or HeLa cytoplasts with L cells did not enter a detectable antiviral state after treatment with interferon specific for the cell type of the enucleated parent. However, treatment of cybrids with interferon specific for the cell type of the nucleated parent was effective in inducing a detectable antiviral state.     

Characteristics of the factors that suppress and stimulate interferon production

A repressor of interferon production is contained in the hyaloplasm of the chick embryo fibroblast cells in the state of hyporeactivity. A factor stimulating interferon production (FSIP) was found in the hyaloplasm of the cells subjected to single or double induction with poly (pI) x poly (pC) followed by exposure to actinomycin D. Both preparations were of the protein nature and possessed high biological activity. They repressed or stimulatd interferon production at a dilution of 1:512-1:1024. The preparations had tissue and inductor specificity, did not affect the Venezuelan equine encephalomyelitis virus and interferon antiviral activity. Unlike the repressor, the FSIP proved to be thermolabile and pH-sensitive in acid media. The half lives of the repressor and stimulator were about 10 and 5 hours respectively.