ANTIVIRAL ACTION OF INTERFERON IN EMBRYONIC CELLS

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ANTIVIRAL ACTION OF INTERFERON IN EMBRYONIC CELLS ALICK ISAACS SAMUEL BARON * M.D. Glasg. M.D. New York From the National Institute for Medical Research, Mill Hill, London, N.W.7

Ho and Enders (1959) made the very interesting observation that Hela cells (a cancerous human cell-line) could be induced to make interferon, which, however, could not be assayed in Hela cells, but only in noncancerous human cells. More recently, Chany (1960) found similarly that interferon produced in KB cells (of cancerous origin) showed antiviral action in normal cells but only a weak action in KB cells. It is known that interferon-treated cells show increased glycolysis (Isaacs 1960); one possible explanation for this increased glycolysis would be that interferon causes an uncoupling action on oxidative phosphorylation, thus limiting the amount of adenosine triphosphate (A.T.P.) available for viral synthesis. Indirect evidence for this suggestion comes from the observation (Isaacs, Klemperer, and Hitchcock 1960) of a number of points of resemblance between the actions of interferon and those of substances known to uncouple oxidative phosphorylation (e.g., 2, 4: dinitrophenol). The lack of antiviral action of interferon in cancerous cells would then be in line with Warburg’s (1956) evidence that cancerous cells can form by anaerobic processes all the A.T.P. they require for growth. If this line of reasoning is correct embryonic cells, which, resemble cancer cells in showing a high rate of anaerobic glycolysis (Warburg 1956), should show a similar resistance to the antiviral action of interferon during an early stage of their development, and should later assume the behaviour characteristic of cells from adult tissues. The

Experiments

This possibility was investigated by testing the antiviral action of interferon on the growth of irifluenza virus in pieces of chorioallantoic membrane taken from chick embryos at different stages of their development. The tissues were treated with interferon in different concentrations and then infected with the PR8 strain of influenza virus. After incubation in a roller-drum the virus yield was measured by the hxmagglutinin titre, and the degree of viral inhibition, or interference, is expressed as the difference between the virus yield in interferon-treated tissues and that of control tissue from embryos of the same age. It was found that the virus grew equally well in control tissues from embryos of 6, 7, 8, 10, 11, 13, or 15 days. There ,

striking differences, however, in the sensitivity to interembryos of different ages. The results of one experiment (see accompanying figure) show that 15-day chorioallantoic membrane pieces are very sensitive to the action of interferon, and significant interference was found with a 1/480 dilution of the interferon preparation used. On the other hand, less viral inhibition was produced by a 16-times greater concentration of the same interferon preparation in 6-day tissues. The 8- and 10-day chorioallaritoic membrane pieces showed intermediate sensitivity to interferon, and in this and other experiments there was a gradual, rather than a sudden, acquiring of sensitivity. Similar experiments with minced embryonic tissue from

were

feron of tissues from

chick

of the

age-group and infected with the the Nws influenza viruses and parainfluenza (Sendai) incubated with shaking gave essentially similar results. A similar result was also found in an experiment with minced embryonic tissue from mouse embryos of 7 and 18 days; in this

embroys

i

*

Degree of interference produced by different amounts of interferon in tissues front chick embryos of 6, 8, 10, and 15 days.

experiment encephalomyocarditis virus and interferon prepared in

mouse

tissues

were

used.

In these experiments the tissues from very young embryos resembled cancer cells in their insensitivity to interferon, but unlike cancer cells, early embryonic cells were found to produce only very low yields of interferon. This was shown by conipating the yield of interferon from 6- and 11- or 13-day chorioallaritoic membranes inoculated in vitro with ultraviolet irradiated influenza virus. The 6-day membranes, however, are much smaller than the older membranes, and in order to compensate for these differences pieces of membrane from 6- and 11-day embryos were compared in their ability to produce both virus and interferon. To do this, one group of membranes of each age-group was infected with fully infective influenza virus and the degree of virus multiplication was observed. The second group was infected with ultraviolet irradiated influenza virus and the yield of interferon was measured. The table shows that the 11-day membranes produced twice as much virus but 20 times as much interferon as the 6-day membranes. (The figure of 20 was obtained by finding that the 11-day interferon had to be diluted about 20 times to give the same degree of inhibition as the 6-day

membranes.) Conclusions

Very young embryos therefore differ from older embryos in being much less sensitive to the antiviral action of interferon, and in producing less interferon on stimulation with ultraviolet-irradiated influenza virus. We have recently found (Isaacs and Hitchcock 1960) that interferon YIELD OF VIRUS AND INTERFERON FROM TISSUES

6-

AND

11-DAY EMBRYONATED

same

or

Senior Surgeon, U.S. Public Health Service, at present attached to the National Institute for Medical Research.

6-day and 11-day embryo pieces were inoculated in vitro with (a) influenza virus or (b) ultraviolet irradiated virus. After 48 hours’ incubation at 35°C the virus yield was titrated in (a) and the yield of interferon in (b). The interferon titre is expressed as the difference between the mean log, yield of virus from untreated chick chorioallantoic membrane pieces and from pieces treated with the interferon preparation under test.

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play an important role in recovery from virus infections. If these results hold true for infection of the human embryo the present results would suggest an explanation of the fact that maternal infection with rubella virus during the first 3 months of pregnancy often leads to congenital malformations (Gregg 1941, Swan 1949). Infection after the third month rarely causes congenital malformations. This could be explained by assuming that if the embryo is infected during the first third of the embryonic life, it produces very little interferon and is very insensitive to the antiviral action of interferon, just as occurs in the chick embryo. At a later stage of embryonic development it is presumed that the interferon mechanism comes into play and limits more effectively the viral infection. These results may have wider implications. We can speculate that the antiviral action of interferon has developed in the course of evolution as an adaptive response to superficial virus infections, such as respiratory virus infections, but that the original function of interferon may have been to play a part in controlling cellular division by controlling the supply of A.T.P. During the first third of embryonic development this mechanism may not become fully established but it may play a more important role thereafter. On this view, the cancer cell is one which has escaped from the control of interferon. These speculations have the advantage that they can be

may

subjected to experimental test. Summary Chorioallantoic membranes from 6-day-old chick embryos were much more resistant to the antiviral action of interferon than tissues from older embryos. Similar differences were found in whole chick and mouse embryo minced tissue, and in this respect the behaviour of early embryonic tissue resembles that of cancer cells. Unlike cancer cells, however, cells from 6-day-old chick embryos showed very weak production of interferon. We should like to thank Mr. V. G. Law and Mrs. V. Clay-Peters for their able technical assistance. REFERENCES

Chany, C. (1960) In preparation. Gregg, N. M. (1941) Trans. ophthal. Soc. Aust. 3, 35. Ho, M., Enders, J. F. (1959) Virology, 9, 446. Isaacs, A. (1960) ibid. 10, 144. — Hitchcock G. (1960) Lancet, ii, 69. Klemperer, H. G., Hitchcock, G. (1960) In preparation. Swan, C. (1949) J. Obstet. Gynœc. Brit. Emp. 56, 341. Warburg, O. (1956) Science, 124, 269.

TWO CASES OF SARCOIDOSIS TREATED WITH MEPACRINE NILS SÖDERSTRÖM M.D. From the Department of Internal Medicine, Karlstad Central Hospital, Karlstad, Sweden

problem. Though

Scandinavia, sarcoidosis is

lymphoma. She returned in February, 1953, complaining of headache and impaired nasal breathing. She now had numerous enlarged painless lymph-nodes. Biopsy of a cervical node showed small crowded tuberculoid granulomas typical of sarcoidosis. The nasal mucosa both on the conchx and on the septum showed a general, firm, nodular swelling which did not respond to decongestives. The findings were said to be typical of sarcoidosis of the nasal mucosa. During the following years there was a slow centrifugal spread of the cutaneous lesions and new lesions appeared on her right cheek and on her thighs. The nasal obstruction had become disabling. The only effective drugs were the corticosteroids which had to be administered more or less continuously from Nov. 1954 to Oct. 1958, (for most of the time, a maintenance dose of prednisone 5 mg. (three times daily). Every attempt to withdraw the steroids led to an intolerable recurrence of the nasal obstruction. During the steroid treatment, the glandular and skin lesions were suppressed but did not

disappear. Mepacrine treatment was started in Oct. 1958, the dose being 0.1 g. three times daily for the first two weeks, 0-1 g. twice daily for another two weeks, and 0-1 g. daily until the end of Jan. 1959. Mepacrine was not available after this, and the treatment was continued with chloroquine in a dose of 0-25 g. daily, which the patient is still receiving. The result was spectacular. When the patient returned after

—

IN certain parts of

both cheeks). There were similar papules in several places on her back and on her left knee, often arranged in rings around patches of atrophic skin. This rash had been present for more than ten years and had slowly but steadily spread in spite of various forms of treatment. The histological appearance of a biopsy specimen later proved typical of sarcoidosis, but meanwhile because of the superficial similarity of the exanthem to discoid lupus erythematosus tentative mepacrine treatment (0-1 g. three times daily) was begun. Two weeks later all the skin papules had become converted into atrophic maculas of a peculiar brownish colour and seemed to be in full regression. The lymph-nodes were smaller but still palpable. Mepacrine was continued, a dose of 0-1 g. twice daily being given for another fortnight, and 0-1 g. daily for the following two months. During this treatment the papules were replaced by atrophic brown scars: the brown pigmentation slowly disappeared towards the end of the treatment. When the mepacrine was withdrawn, no active manifestation of sarcoidosis could be detected. The patient did not return until August, 1958. Two new papules had then appeared on her back, but all the skin sarcoids seen in November, 1955, still seemed firmly scarred and inactive. Case 2.-A woman, aged 30, was admitted in May, 1952, with arthralgia, erythema nodosum, and bilateral hilar

a

major

of its symptoms are of little consequence to the patient, it may become a distressing and even fatal affection for which satisfactory therapy is lacking. I feel justified, therefore, in reporting some observations which may indicate a new approach to some

treatment.

Case-reports Case 1.-A woman, aged 51, first seen in the outpatient department in November, 1955. She complained of vague involutional symptoms and the routine clinical examination revealed little relevant, except for a peculiar rash, and moderately enlarged, firm, painless lymph-nodes on both sides of the neck. The rash consisted of groups of medium-sized, firm, brownish-red papules distributed over the face (nose, forehead, and

three weeks all the skin sarcoids had become tar-brown and atrophic, her nasal breathing was quite free, and the lymphnodes had disappeared. During the following months, the cutaneous scars contracted and the brown discoloration faded. The steroid dosage (in Oct. 1958, triamcinoline 8 mg. three times daily) was slowly reduced, and the drug was withdrawn in May, 1959, without there being any signs of a recurrence of the sarcoid granulomas. Discussion

introduced in 1932 as an antimalarial Mepacrine remedy; during the last war it was found to be a useful drug in the treatment of discoid lupus erythematosus (Prokoptchouk 1940). Since then it seems to have been abandoned for both these diseases and replaced by chloroquine and allied drugs, which probably have a similar action but are less toxic and do not give the same disagreeable pigmentation of the skin. It is known that mepacrine is taken up preferentially by cells in the lungs, in the liver, in the spleen, and even in was