Mechanism of stimulation of natural killer-cell cytotoxicity by interferon and an interferon-inducer in the rat.

The interferon-induced Newcastle disease virus (NDV) was shown to augment cytotoxicity attributable to natural killer (NK) cells in all of the major lymphoid organs of W/Fu rats except the thymus. The levels of interferon isolated from the spleen following NDV inoculation correlated with the increase in splenic cytotoxicity from the same spleen. Spleen-derived interferon was shown to augment splenic cytotoxicity following intravenous inoculation, and to augment spleen cell cytotoxicity in vitro. Three major peaks of interferon type I were found in spleen homogenates corresponding to mol. wt of greater than 100,000, 29-33,000 and 19-23,000. All these fractions stimulated spleen cell cytotoxicity when tested in vitro. The rapid drop in splenic cytotoxicity 24 hr after NDV inoculation was associated with a rapid fall in interferon levels in vivo. The need for the continued presence of interferon for the stimulation of cytotoxicity was demonstrated when spleen cells pretreated with interferon for 4 hr in vitro lost their augmented cytotoxicity upon culturing for a further 20 hr in the absence of interferon. Although splenic cytotoxicity returned to control levels within 24 hr of a single 10(7.3) EID50 dose of NDV, repeated doses of NDV maintained augmented cytotoxicity over a longer period. Spleen cells either taken from rats injected with NDV or pretreated in vitro with interferon showed a two-fold increase in the number of cytotoxic cells bound to W/FuG-1 target cells, with no change in the target binding-cell numbers. However, only the cells pretreated with interferon showed an increase in lytic efficiency.     

Induction and properties of guinea pig serum interferon. Preliminary report

Guinea pigs, 250-350 g body weight, both sexes, were injected with 5X10(8.5) EID50 NDV (Radom strain) intracardially and intraperitoneally simultaneously. The animals were bled by cardiac puncture 0, 3, 6, 12, 24 and 48 hours after injection. After virus inactivation, serum interferon titration was performed in cultures of guinea pig embryo kidney cells with 50 percent plaque inhibition test using VSV. The highest interferon titer (64 u./ml) was found after 6 hours of inductor injection. Interferon titer decreased quickly and after 12 hours it was lower than 16 u./ml. Guinea pig serum interferon induced by NDV was resistant to pH 2 and 56 degrees C during 1 hour. Interferon was inactivated by trypsin. The decribed interferon did not protect heterologous species cells (swine) against Teschen Disease Virus infection. Other properties of this interferon are being studied.     

Suppression of interferon production in mouse spleen cells by cytochalasin D.

Cytochalasin D is thought to impair microfilament function. The present study has investigated its effects on four different systems in which interferon is formed, namely (1) mouse fibroblasts induced with virus (2) mouse spleen cells induced with virus, or (3) with endotoxin or (4) by allogeneic stimulation. Cytochalasin D did not suppress formation of interferon by fibroblasts (L cells) or spleen cells stimulated with either HVJ or NDV. However it did suppress production of interferon by spleen cells in response to endotoxin or an allogeneic stimulation; here its action was apparently not on the secretion of interferon, but on some earlier event. It also suppressed the production of interferon by mouse spleen cells induced with HVJ if this had been u.v. irradiated for more than 15 min: this suggests that cytochalasin D sensitive structures do play some role in interferon production by mouse spleen cells when stimulated with HVJ, as well as when they are stimulated with endotoxin or an allogeneic stimulus.     

The enhancement of interferon production by endotoxin

Summary Pretreatment of bovine leukocyte cultures with endotoxin enhanced interferon production by Newcastle disease virus (NDV) as evidenced by an early and high rate of interferon production. This enhancing effect was greatest when NDV was inoculated at three hours after endotoxin treatment. It was blocked by actinomycin D both before and after the addition of endotoxin. Propagation of NDV was less marked in endotoxin-treated cultures than in untreated control cultures. Pretreatment with endotoxin interfered with the cytopathic effect of the NDV on the cultured cells.     

Induction of interferon by temperature-sensitive mutants of Newcastle disease virus

Spontaneously-selected and mutagen-induced temperature-sensitive (ts) mutants of Newcastle disease virus (NDV) were used to study interferon induction in chick embryo (CE) cells at temperatures permissive (37°) and nonpermissive (42°) for virus replication. Both infectious and UV-irradiated virus were tested for interferon-inducing ability in cells pretreated or not pretreated with homologous interferon. At 37°, only UV-irradiated NDV was capable of inducing interferon in cells not treated with interferon before infection. In cells pretreated with interferon, on the other hand, both unirradiated and UV-irradiated virus stimulated the production of interferon. At 42°, the interferon-inducing phenotype for some UV-irradiated ts mutants was dependent on whether or not cells were pretreated with interferon. For example, out of 10 mutants examined, one UV-irradiated ts mutant induced interferon in both untreated and interferon pretreated cells; 7 mutants failed to induce in untreated cells but induced from 25–100% of the wild-type level of interferon in cells pretreated with interferon; and two mutants failed to induce interferon in both types of cells. In addition, one mutant (NDV₀ts-100) induced low or undetectable levels of interferon at both 37° and 42°, conditions under which wild-type virus (NDV₀) produced significant levels of interferon. Co-infection of cells with UV-irradiated ts-100 and a preparation of NDV₀ exposed to prolonged irradiation resulted in considerable production of interferon. These results suggest the possibility that more than one virus function may be involved in interferon induction by NDV in CE cells.     

Comparative study of the interferon-inducing capacity of Newcastle disease and Sendai viruses in human and animal bone marrow cells

Sendai and Newcastle disease virus (NDV) induced high and approximately similar interferon production in bone marrow cells of man, rats, and mice. In contrast to NDV, Sendai virus induced no interferon production in chick bone marrow cells. Interferon production by the bone marrow cells did not differ in its time course and requirements from production of leukocyte interferon.     

Interferon induction by viruses and polynucleotides: a differential effect of comptothecin.

The plant alkaloid comptothecin inhibits interferon production induced by Newcastle disease virus (NDV) or ultraviolet-irradiated NDV in chick and human cells, and by Sindbis virus in chick cells. It has no effect on interferon production induced by poly (rI).poly(rC) in chick and human cells. No effect of comptothecin could be detected on the multiplication of NDV, and it is concluded that the inhibition reflects a difference between interferon induction by viruses and by polynucleotides.     

Modulation of natural killer cell activity in mice after interferon induction: depression of activity and depression of in vitro enhancement by interferon.

Splenic natural killer (NK) cell activity of BALB/c and C3H mice was assayed after administration of the interferon inducers Escherichia coli endotoxin or Newcastle disease virus (NDV). As expected, the NK cell activity rose early in response to the interferon inducers. At 1 to 3 days after an injection of endotoxin, NK activity was hyperesponsive to interferon stimulation. At 5 to 9 days after injection of either endotoxin or NDV, splenic NK activity was depressed, and the spleen cells showed a relative refractoriness to in vitro interferon stimulation. It is postulated that this phenomenon may be related to hyporeactivity, the inability to reinduce interferon after an initial period of interferon production.     

Neutralizing antibodies to human gamma-interferon

Spleen lymphocytes from normal subjects who suddenly died were used as cells producing gamma-interferon. One spleen yielded (3-8) X 10(8) viable cells which made it possible to prepare from 3 to 81 of splenocyte suspension culture. Staphylococcal enterotoxin A (SEA) and B (SEB) were used as gamma-interferon inducers. Stimulation of splenocyte suspension culture with SEA and SEB resulted in production of gamma-interferon with an average activity of 640-2560 units/ml. A partially purified interferon preparation with an activity of 2 X 10(4) units/ml was obtained by sorption of gamma-interferon on porous glass CPG-200-240 followed by elution with a buffer containing 50% ethylene glycol and Sephadex G-25 gel chromatography. As a result of 11 successive intramuscular immunizations of rabbits at 2-week intervals with a partially purified and concentrated preparation of gamma-interferon with Freund's complete adjuvant, blood serum was obtained which was capable of neutralizing 32 units of gamma-interferon up to a dilution of 1:128. The serum was highly specific: it showed no specific interaction with antigenic determinants of either natural alpha- and beta- or plasmid alpha-F and alpha-F/D human interferons.     

Distinguishing characteristics of interferon induction with poly(I)-poly(C) and Newcastle disease virus in human cells.

Exposure of a human diploid foreskin cell strain (FS-4) to polyinosinate-polycytidylate [poly(I)·poly(C)] resulted in an early rise of interferon production that peaked at about 4 hr after induction and decreased rapidly thereafter. Irradiation of cells with low to moderate doses of ultraviolet (uv) light immediately before induction with poly(I)·poly(C) increased the amount of interferon produced up to about tenfold. This enhancement was apparently due to interference with the shut-off process which in unirradiated cells leads to early termination of interferon production; in irradiated cells interferon production continued for much longer.Inoculation of FS-4 cells with Newcastle disease virus (NDV) resulted in interferon production that showed a slower rise and peaked only at about 10–15 hr after inoculation. Irradiation of cells at the time of induction with NDV resulted in a dose-dependent decrease of interferon production. However, a small fraction of the total amount of interferon produced in response to NDV, which appeared by about 5 hr after virus inoculation, was resistant to uv. This uv-resistant early peak of NDV-induced interferon was greatly enhanced in cells which 6 hr before virus inoculation had either been induced with poly(I)·poly(C) or incubated with interferon, while the appearance of the major, uv-sensitive peak of NDV-induced interferon was inhibited or delayed after the same treatments. In its characteristics the early peak of NDV-induced interferon resembled the poly(I)·poly(C)-induced interferon response. Poly(I)·poly(C)-induced, as well as the early and late NDV-induced interferons were all neutralized by an antiserum raised against poly(I)·poly(C)-induced interferon, suggesting that they represent products of the same structural gene(s). It is concluded that there may be more than one mechanism of interferon induction by a single virus.     

Participation of cytophilic antibody in enhancement of interferon production in macrophages by under-neutralized NDV

Summary Enhancement of interferon production in mouse peritoneal macrophages by Newcastle disease virus (NDV) under-neutralized with whole antiserum or its IgG fraction was inhibited in cells pretreated with iodoacetamide, sodium nitrite or formaldehyde, which blocked adsorption of cytophilic antibody to macrophages. Mouse embryo primary culture cells had no receptor for cytophilic antibody, and in such a cell culture, under-neutralized NDV did not enhance interferon production. Antiserum enhanced interferon production by NDV which had adsorbed to cell surfaces, but did not affect NDV that had penetrated the cells.     

Spleen depletion in neonatal sepsis and chorioamnionitis

Neonatal sepsis and chorioamnionitis induce morphologic modifications and shrinkage of the thymus. We show fetal and neonatal morphologic modifications of the spleen in the same autopsy subjects as previously used to describe thymus shrinkage, including 10 preterm or full-term neonates who died of proven sepsis within 48 hours after birth and 20 fetuses spontaneously aborted because of extensive ascending chorioamnionitis. Control subjects included 10 fetuses from induced termination of pregnancy and 10 neonates who died suddenly during the perinatal period without evidence of chorioamnionitis. Spleen cell populations were studied by means of immunohistochemical analysis. Neonatal sepsis occurred with severe spleen depletion, involving both B and T lymphocytes (P < .001). Fetuses with chorioamnionitis also showed spleen cell depletion. These observations, to our knowledge not described before, indicate that preterm and term neonates show an inflammatory reaction similar to that of adult patients and that severe chorioamnionitis is associated with a nonspecific inflammatory response comparable to that of sepsis.     

Effect of virus-induced interferon on mast cell resistance to the degranulating action of immune complexes

Regularities of endogenous interferon (IFN) induction with Newcastle disease virus (NDV) in rats and the effect of this process of the resistance of mast cells to the degranulating effect of immune complexes are described. Increased resistance of mast cells to the damaging effect of immune complexes for 72 hours after a single induction of endogenous IFN with virus was demonstrated in experiments in vivo. Inoculation of rats with NDV treated at pH = 2.0 did not induce the production of endogenous IFN, and the mast cells from these animals underwent degranulation to the same extent as those from intact animals. Protection of mast cells by IFN from the degranulating effect of immune complexes was also demonstrated in in vitro experiments.     

The effect of various inducers on interferon-synthesizing activity of leukocytes in patients with diabetes mellitus

The results of the study of interferon response of leukocytes in patients with diabetes mellitus (DM) with three inducers: Newcastle disease virus (NDV), poludan, and dipyridamole are presented. Different patterns of interferon production in patients with DM and normal subjects were shown. Dipyridamole and NDV induced high interferon levels in patients with DM which allow it to be recommended as an additional therapeutic means.     

Interferon induction with Newcastle disease virus in FS-4 cells: Effect of 5,6-dichloro-1-β-D-ribofuranosylbenzimidazole (DRB)

Summary DRB is an inhibitor of heterogeneous nuclear RNA (hnRNA) and messenger RNA (mRNA) synthesis. The effect of DRB on interferon production stimulated by Newcastle disease virus (NDV) in the human FS-4 cells was studied. Interferon production in cells primed by treatment with interferon was markedly enhanced (superinduced) in the presence of DRB. This superinduction was essentially due to an inhibition of the rapid decline (shutoff) of interferon production observed in primed cells not treated with DRB. Continuous presence of DRB was required for maximal superinduction. In this and other respects the interferon response induced by NDV in primed cells resembled poly(I) · poly(C)-induced interferon production. In contrast interferon production in cells not primed with interferon was virtually abolished by DRB treatment. Since neither virus specific RNA synthesis nor virus replication were significantly affected by DRB, the inhibition of interferon production is likely to result from the inhibitory action of DRB on a cellular, rather than viral, function. Apparently some differences exist in the synthesis or processing of the mRNAs for interferons in primed and unprimed cells and these determine the different sensitivities of these two responses to DRB.With 2 Figures     

Interferon-hyporesponsiveness in mice in connection with dose interval and site of administration of Newcastle disease virus. Reflexion in serum complement levels.

Intraperitoneal administration of Newcastle disease virus (NDV) resulted in enhanced serum levels of complement not accompanied by an increase of interferon levels, when measured at 24 hours' intervals. On the other hand, intravenous injection of NDV caused a drop of complement levels of short duration with an accompanying increase of interferon levels. Hyporeactivity to induction of serum interferon could not be achieved by intraperitoneal administration of NDV, but an incomplete hyporeactivity could be achieved by intravenous administration of NDV. It might be assumed that production of interferon in mice occurs in different separated compartments depending on the route of inoculation of the inducer.     

The interferon system in ducks infected with viral hepatitis B

The duck peripheral blood lymphocytes and spleen cells were shown to be relatively resistant to interferon induction by viral (NDV) and nonviral (dsRNA) inducers as well as to Con A induction of gamma-interferon. The concentration of the inducers had to be increased 4-5-fold to induce interferon production. Charry valley ducks produced more alpha-interferon than Peking ducks. The level of interferon production induced by NDV and dsRNA (Ridostin) was similar in DHBV-positive Peking ducks and in the control group, but in the infected Charry valley ducks interferon production was lower than in the controls (224 to 180 U/0.1 ml in NDV-induced and from 192 to 43.3 U/0.1 ml in dsRNA-induced). Early bursectomy brought down the interferon production in non-infected ducts, but in DHBV-positive bursectomized ducks (Y) the level of interferon was higher than in the control bursectomized ducks.     

Production of Interferon-β by Murine T-Cell Lines Induced by 10-Carboxymethyl-9-Acridanone

Besides the established T-cell property of producing gamma interferon (IFN-gamma), murine T cells additionally possess the ability to produce IFN-alpha and IFN-beta when appropriate inducers such as 10-carboxymethyl-9-acridanone (CMA) or Newcastle disease virus (NDV) are used. Interleukin 2 (IL-2)-dependent murine T-cell lines, but not purified resting splenic T cells, responded to CMA and NDV with production of IFN-alpha, beta. The IFN production by these T cells was not restricted to a special subset, since T cells expressing the Lyt 1+2- and the Lyt 1-2+ phenotype responded to these inducers with IFN production. After prolonged passaging of the T-cell lines in IL-2-containing medium, the ability to respond to CMA with production of antiviral activity was sustained longer than the ability for concanavalin A-induced IFN-gamma production. Whereas the NDV-induced T-cell supernates contained both IFN-alpha and IFN-beta, the induction with CMA resulted exclusively in the synthesis of IFN-beta by the T-cell lines.     

Interferon production by individual L cells

Using a protected centre technique in which agarose prevents the diffusion of interferon from individual producing cells, we have shown that essentially every cell in a monolayer of mouse L cells can be induced to produce interferon by infection with Newcastle disease virus (NDV). The amount of interferon produced by individual cells appeared to be highly variable, even when cloned cells and viruses were used. U.v.-irradiated virus lost its capacity to induce interferon in L cells and to infect chick embryo fibroblasts at the same rate. A small proportion of cells (1 X 10-6 to 10 X 10-6) appeared to produce interferon constitutively. This fraction was increased threefold by u.v. irradiation of the cells, and up to 10-fold by exposing cells to the mutagen ethyl methane sulphonate.