Enhanced interferon production from chick embryo cells aged in in vitro

SUMMARYThe presence of a low level of interferon (IFN) activity was demonstrated in aged culture medium of primary chick embryo (CE) cells. The endogenous cytoplasmic level of 2',5'-oligoadenylate (2',5'-A) synthetase activity also increased during cell ageing. This increase was suppressed by addition of antiserum against chick IFN. In contrast, anti-chick IFN serum did not inhibit the ageing effect, i.e. the enhanced production of IFN upon induction after a prolonged preincubation of CE cells. This result indicates that the occurrence of the ageing effect is not mediated by the IFN system which had been expressed spontaneously.It has been reported that interferon (IFN) production by primary chick embryo (CE) cells is enhanced during ageing of the cell cultures in vitro (Carver & Marcus, 1968). As well as this, we have observed increased levels of endogenous 2',5'-oligoadenylate (2',5'-A) synthetase in aged CE cells. Kato & Eggers (1969) reported the presence of a humoral factor, aged medium factor (AMF), in aged culture media which could enhance IFN production by 1-to 2-day-old primary CE cells. However, experiments to confirm the occurrence of AMF have so far given equivocal results (M. Kohase et al., unpublished data).It has been suggested that the ageing effect is the result of priming (Stewart et al., 1971) of CE cells by a low level of IFN spontaneously released during ageing (Stewart, 1979). We therefore attempted to determine whether IFN activity is present in the aged culture media and, if so, whether the ageing effect can be inhibited by anti-chick IFN serum.Anti-chick IFN serum was prepared as follows. Primary CE cells prepared from 11-to 12-dayold hens' eggs (Kohno et al., 1968) were cultured in Eagle's minimum essential medium (MEM) containing 2~ calf serum (CS), for 10 days at 37 °C. IFN was induced by inoculation of u.v.-irradiated Newcastle disease virus (NDV; m.o.i. = 10) as described previously (Kohno et al., 1968) and the medium (Eagle's MEM without CS) was collected at 24 h after induction. IFN was semi-purified from this crude sample (10000 IU/mg protein) to a specific activity of about 3 x 106 IU/mg protein by a combination of adsorption to and elution from Controlled Pore Glass (CPG) and Blue Sepharose as described previously for the purification of mouse L cell IFN (Fujita & Kohno, 1981). Protein concentrations were measured by the method of Lowry et al. (1951). Rabbits were immunized with semi-purified IFN samples. The sera obtained were exhaustively absorbed with CE cells. The antiserum when diluted 1:10 neutralized about 6000 IU of CE IFN to <5 IU.To examine the presence of IFN activity in the aged culture medium, CPG column chromatography was used as indicated above. The CPG-bound materials were eluted by 50~ ethylene glycol in 1 M-NaCI. The eluate was placed in dialysis tubing, further concentrated by polyvinylpyrrolidone powder and dialysed against phosphate buffer. The volume of the final sample (aged culture medium-CPG; AM-CPG) was 1/300th of the original aged culture medium. The ...

Section: Discussionsupporting

confidence: 61%

supporting

confidence: 61%

Interferon Production Associated with the Ageing of Primary Chick Embryo Cells Is Not Caused by Priming

Kohase

Saito

Iizuka

et al. 1983

“…As has been reported by Kowal & Youngner (1978) with primed CE cells, our ts mutants were also capable of inducing IFN at 41 °C in CE cells when the cells had been either primed with 20 units of chick IFN overnight, or aged (Carver & Marcus, 1967) in vitro for 10 days (Table 2). However, when these cells were induced for IFN production by inoculating with ts mutants at lower m.o.i.…”

Section: Cellular State and Ifn Inductionsupporting

confidence: 52%

Temperature-sensitive Mutants of Newcastle Disease Virus Affecting Interferon Induction

Kohase

Kohno

1983

SUMMARYTemperature-sensitive (ts) mutants of Newcastle disease virus (NDV) were isolated and studied for interferon (IFN) induction in primary chick embryo (CE) cells. At the non-permissive temperature (41 °C), there was no viral RNA synthesis or IFN induction by u.v.-treated virions except for ts-3 (RNA+), which did synthesize RNA at 41 °C, and whose u.v.-treated virions did induce IFN at this temperature. Another mutant (ts-4) induced IFN without irradiation, at the permissive temperature (37 °C). The minimum u.v. target size for IFN inducibility was unaffected by the mutation and corresponded to about 5 ~ of the genome required for the expression of infectivity. These results support the hypothesis that the appearance of NDV RNA immediately after infection (primary transcription) plays a key role in IFN induction.

“…As reported previously (Carver & Marcus, 1967;Sekellick & Marcus, 1985), primary chick embryo cells aged in vitro for several days are capable of producing such high levels of IFN that viruses which share the attributes of being both good inducers of IFN and sensitive to its action fail to develop plaques on these host cells. This characteristic feature of aged chick embryo cells can be abolished by including 2-AP (10 mM) in the agar overlay : plaque formation then proceeds normally (Fig.…”

Section: Discussionmentioning

confidence: 81%

Interferon Induction by Viruses. XVI. 2-Aminopurine Blocks Selectively and Reversibly an Early Stage in Interferon Induction

Marcus

Sekellick

1988

SUMMARYA purine analogue, 2-aminopurine, reported to act as an inhibitor of protein kinase, selectively, reversibly and in a dose-dependent manner blocked a very early stage in interferon induction. With chick embryo cells and mouse L cells as hosts, and different viral inducers of interferon, maximal effects of 2-aminopurine were observed during the first 4 h of induction. At 10 mM-2-aminopurine there was a 20-fold reduction in the yield of interferon from both cell types. 2-Aminopurine and actinomycin D both prevented interferon induction with the same time course, indicating a transcriptional block to induction; however, only the action of the former was reversed upon removal of the drug. Addition of 2-aminopurine to an agarose overlay resulted in high efficiency plaque formation by vesicular stomatitis virus New Jersey (Hazelhurst) under conditions where endogenous induction of interferon and its feedback action on aged chick embryo cells normally prevented plaque formation. Two other inducible systems, representing genes involved in interferon action (both its development and activation), and those of heat shock, were not affected by 2-aminopurine. A model is presented implicating the interferon-inducible dsRNA-dependent protein kinase as an interferon induction receptor which, on interaction with dsRNA, generates an amplified signal via phosphorylation that ultimately derepresses the interferon gene(s).