Interferon Induction with Statolon in the Intact Animal'

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1 BACTERIOLOGICAL REVIEWS, June 1967, p Vol. 31, No. 2 Copyright 1967 American Society for Microbiology Printed in U.S.A. Interferon Induction with Statolon in the Intact Animal' W. J. KLEINSCHMIDT AND E. B. MURPHY The Lilly Research Laboratories, Eli Lilly and Company, Indianapolis, Indiana 4626 INTRODUCTION APPEARANCE AND DECAY OF STATOLON-INDUCED INTERFERON wrrh TIME DOSAGE RESPONSE OF STATOLON CORRELATION OF PROTECTION WITH INTERFERON LEVELS EFFEcT OF REPEATED ADMINISTRATION OF STATOLON ON INTERFERON PRODUC- TION IN CELLS EFFECT OF PRIOR INJECTION OF STATOLON ON INTERFERON RESPONSE OF MICE TO SECOND INJECTION DURATION OF STATOLON ACTIVITY IN MICE STATOLON-INDUCED INTERFERON IN THE MONKEY CONCLUSIONS SUMMARY LITERATURE CITED INTRODUCTION The discovery of interferon in 1957 by Isaacs and Lindenmann (6) offered an explanation of the phenomenon of viral interference that had been studied for 2 years previously. Only viruses or viral components were thought to be capable of inducing interferon production in cells. Rotem, Cox, and Isaacs (13), however, reported in 1963 that heterologous nucleic acids were capable of stimulating the production of interferon. Doubt may exist today that nucleic acids are actually able to function as interferon inducers, but it was this report that prompted us to examine our antiviral agent, statolon, as an inducer of interferon. Two years ago, we reported that statolon did indeed induce cells to produce interferon and, thereby, exerted its antiviral action (7; W. J. Kleinschmidt, J. C. Cline, and E. B. Murphy, Federation Proc. 2:57, 1964). Soon, other nonviral agents were reported capable of inducing interferon production, including certain bacteria (17), bacterial endotoxin (4, 16), rickettsia (H. E. Hopps et al., Bacteriol. Proc., p. 115, 1964), and phytohemagglutinin (19). Statolon, a polysaccharide material produced by the mold Penicillium stoloniferum (9), stimulates the production of interferon in the animal (8) and in tissue culture (7). The properties of the 1 A contribution to the symposium on "Interferon," held at the Annual Meeting of the American Society for Microbiology, Los Angeles, Calif., 2 May 1966, under the sponsorship of the Division of Virology with Warren Stinebring as convener. statolon-induced inhibitor have been found to be consistent with the known properties of virally induced interferon, including species specificity, lack of viral specificity, ph stability, nonsedimentability at 15, x g for 9 min, sensitivity to proteinases, and its characteristic heat sensitivity. In collaborative studies with T. C. Merigan on statolon-induced interferon samples (11), some inconstancy in molecular weight was revealed. Interferon produced in tissue culture and in spleens of mice treated with statolon was similar in molecular weight to virally induced interferon, although a higher molecular weight species is found in the serum. These molecular weight relationships are fully discussed by Merigan (1). APPEARANCE AND DECAY OF STATOLON- INDUCED INTERFERON WITH TIME Earlier studies in our laboratories with statolon (12) demonstrated that optimal protection against infective viruses in mice was obtained when statolon was administered intraperitoneally 24 hr prior to virus challenge. This became the standard procedure for assay of statolon in mice. In an attempt to define this system further, the time pattern of peak-level production of interferon, its disappearance, and the relationship of interferon level to protection was determined. A group of 15 mice (weighing 14 g each) were injected intraperitoneally with statolon, and at appropriate times the sera from 15 mice were obtained and pooled. The interferon level of each lot of sera was measured by plaque inhibition of 132 Downloaded from on April 28, 218 by guest

2 VOL. 31, 1967 INTERFERON INDUCTION WITH STATOLON 133 vesicular stomatitis virus on confluent monolayers of MCN cells. [MCN cells were from Friedrich Deinhardt, Presbyterian-St. Luke's Hospital, Chicago, Ill. Immunological and karyological evidence identified them as mouse cells, probably the L strain (3).] Interferon titers are expressed in units. A unit of interferon is defined as the reciprocal of the dilution of the original material required to reduce the plaque count to 5% of that of controls. A maximal interferon level was attained at 12 hr (Fig. 1). The level remained relatively high for another 12 hr. By 72 hr, however, interferon decreased, not to the original base, but to a plateau of about 15 units where it remained for at least another 4 days. This residual interferon may be of significance in the length of duration of protection against viral infection afforded by a single injection of statolon. DOSAGE RESPONSE OF STATOLON The relationship of statolon dosage to interferon production in vivo was studied. Nine groups of 15 mice (14 g) were injected intraperitoneally with statolon, ranging in dosage from 7 to 2,145,ug per mouse. At 16 hr after statolon administration, the mice were bled, and the sera from each group were pooled and then assayed for interferon activity. The level of circulating interferon increased linearly with increased dosage of statolon in the lower dose range (Fig. 2). An end point occurred at about 175,ug of statolon per mouse. Increasing the dosage beyond this point did not result in further increase in 1, F O 1 = I=_ Z 1_ HOURS AFTER INJECTION OF STATOLON FIG. 1. Appearance and decay of statolon-induced interferon with time. o- M.U I- 4 k 35k 3 p 25 k 2 F ,, S pg STATOLON INJECTED PER MOUSE FIG. 2. Dosage response of statolon in mice. production of interferon, but in a plateau at about 3,5 units of interferon. CORRELATION OF PROTECTION WITH INTERFERON LEVELS The dose response of statolon on interferon production allowed us to determine whether circulating interferon levels correlated with protection against viral infection. Five groups of 1 mice were injected intraperitoneally with the same quantities of statolon used in the experiment above in the range of 36 to 715,ug of statolon. After 16 hr, these mice plus 1 control mice not injected with statolon were infected subcutaneously with a 32-LD5 dose of MM virus. (MM virus was obtained by our laboratories from C. W. Jungeblut in It has been passaged many times in mice by subcutaneous inoculation. Stock virus is prepared as a 1% homogenate suspension of infected mouse brains and stored at -9 C.) Protection measured by survival was demonstrated in mice injected with 72,ug of statolon or more (Table 1). Injection of this quantity of statolon produced a response of 1,7 units of interferon. More than 76 units of interferon was required for complete protection, as shown by the death of five mice in the group receiving 36 Mg of statolon. None of the control mice which did not receive statolon survived. EFFECT OF REPEATED ADMINISTRATION OF STATOLON ON INTERFERON PRoDucnION IN CELLS The effect of repeated daily application of statolon on interferon production by cells in tissue culture was studied (8). Statolon was added Downloaded from on April 28, 218 by guest

3 1346 KLEINSCHMIDT AND MURPHY BACTERIOL. REV. TABLE 1. Amt of statolon injected per mouse mg Control Correlation of statolon-induced interferon levels with antiviral protectiona Amt of active statolon 1, interferon 3,5 3,15 3,7 3,2 1, Observed protection 7, SSSS 6, 7, 7, 8, 9, 5, 5, 5, 5, 5 6, 6, 6 6, 8 a The figures in the column on the left represent the quantity of our statolon preparation injected. This preparation contained considerable quantities of glucose (58%) and (NH4)2CO3 (16.2%) for stability and solubility purposes. The second column indicates the quantity of active statolon as determined by nondialyable solids. The volume of serum to which the units of interferon is referred is 2.5 ml. Numbers in the column on the right refer to the day of death of the mice after injection. The letter "S" refers to mice that survived 1 days, the termination time of the experiment. to monolayers of chick embryo cells and left in contact for 24 hr, after which the fluids were harvested and pooled. This procedure was repeated daily for 1 days. The daily pooled fluids were freed from statolon by lowering the ph to 4.5 and centrifuging at 15, x g for 9 min. The fluids were again brought to ph 7.5 and then assayed for interferon. It was found that interferon production continued, but that it decreased continuously after day I until day 7, after which it began to increase (Fig. 3). These data suggest that the presence of interferon in cells limits the production of more interferon; the cells become resistant to production of higher levels of interferon until its concentration has decreased to minimal amounts. mtection of inducer, enduring for as long as 5 to 6 days, have been reported by Youngner and Stinebring (18) and by Ho, Kono, and Breinig (5), who performed experiments in animals in which both virus and endotoxin were employed as inducers a DAYS Production ofinterferon in chick embryo cells FIG. 3. on repeated administration of statolon. cc is, U. MU w 4-2- I.- 1, EFFECT OF PRIOR INJECTION OF STATOLON ON INTERFERON RESPONSE OF MICE TO SECOND INJECTION HOURS AFTER INITIAL INJECTION Similar resistance to further stimulation of FIG. 4. Influence of prior injection of statolon on interferon production or hyporeactivity by prior interferon response in mice L_ SECOND INJECTION I INITIAL INJECTION Downloaded from on April 28, 218 by guest

4 VOL. 31,1967 INTERFERON INDUCTION WITH STATOLON 135 We found that prior injections of statolon in mice also produced a period of hyporeactivity to a second injection (Fig. 4). The thin line curve shows the response produced by a single initial injection of statolon and the decay of interferon with time. The heavier line shows the response of the second injection of statolon. Since it takes 12 hr for the peak response of interferon production to occur, the points on the second injection curve refer to the time the mice were bled after receiving the second injection of statolon 12 hr previously. Complete resistance was observed at 48 hr. On the 4th day, a slight increased response was observed. This second response increased until the 6th day when it reached its highest level. The second response, however, did not attain the level obtained with the initial injection, but remained at a lower plateau. Note, too, that the interferon level obtained with the initial injection also reaches a plateau at 15 units in this experiment (cf. Fig. 1) rather than decreasing to the initial base level. Both these observations may be due to residual statolon activity in certain cells of the animal, producing a constant limited response which in turn limits response of the second injection. DuRATIoN OF STATOLON AcTIvITY IN MICE In early investigations on viral interference, Schlesinger et al. (14, 15) and Duffy and Morgan (2) reported that a single inoculation in animals of a nonfatal encephalitis virus imparted protection for 2 weeks against antigenically heterologous encephalitis viruses. The duration of protection that is produced in the mouse by a single injection of statolon against a subsequent fatal MM virus inoculation was investigated. Mice were injected with statolon (715,ug per 12- to 14-g mouse) and were periodically challenged with MM virus after the statolon treatment. In an initial experiment, TABLE 2. Duration of statolon activity in mice Time between treatment and infection with MM virus days Total Treated with statolon Survivors/ total 9/1 5/6 8/1 6/9 7/9 3/1 7/1 5/1 5/74 Per cent survival Controls Survivors/ total 1/6 /5 /6 /4 /6 /5 /6 2/6 3/44 Per cent survival a single injection of statolon afforded protection for 2 weeks, at which time the study was terminated (Table 2). In a second study, protection was still demonstrable when the experiment was terminated at the end of 3 days. In the initial experiment, animals were challenged with virus TABLE 3. Time between treatment and infection days Total Duration of statolon activity in mice Treated with statolona Survivors/total 12/15 9/15 1/15 11/15 14/15 12/15 14/15 14/15 15/15 15/15 1/225 a Of 75 control mice, 28 (37%) survived. 1 _ 9 - Z 8-7 LIIw Ix 6- LU. I.- Z 5 - I 4 LIn I- 3- D 2-1 IPer cent survival T I HOURS AFTER STATOLON ADMINISTRATION FIG. 5. Statolon-induced interferon in the monkey. Downloaded from on April 28, 218 by guest

5 136 KLEINSCHMIDT AND MURPHY BACTERIOL. REV. from a different ampoule each time. This could have produced some variation in infectivity. The mice also grew during the 2-week test, and an adjustment in quantity of virus injected had to be made. These variations were eliminated in the second experiment. Mice were injected with 5 mg of active statolon per kg of body weight on different days, and after 29 days all were infected on the same day. The mice were weighed prior to statolon treatment, and the quantity of statolon was adjusted to compensate for weight gain. The experiment suffered somewhat from the handicap that larger mice are less susceptible to MM virus. The number of control mice was increased to diminish the error due to their lower susceptibility. One injection of statolon increased significantly the survival of mice compared with those not treated (Table 3). STATOLON-INDUCED INTERFERON IN THE MONKEY The efficacy of statolon as an interferon inducer was also investigated in a primate. The response obtained with two African green monkeys injected subcutaneously with statolon is shown in Fig. 5. Injection of 19.3,4g of statolon per kg of body weight produced in one monkey a titer of 1, units of circulating interferon, assayed by use of the BSC1 green monkey cell-vesicular stomatitis virus system. The peak level occurred at 24 hr; by 48 to 72 hr, interferon again deteriorated to low levels. We have not been successful as yet in setting up a satisfactory protection test in the green monkey with a nonlethal virus. Cochran et al. (1) have, however, already demonstrated protection in cynomolgus monkeys injected with statolon against poliovirus infection. CONCLUSIONS A unique mode of protection against virus infection is apparently made available to animal cells through the production of interferon. Generally, the infecting virus stimulates sufficient interferon production to curb virus multiplication, thereby inhibiting the potentially lethal course of the virus. It is of interest that statolon, a nonviral material, can substitute for a viral stimulus of interferon. Thus it seems possible, eventually, to prevent virus infections from being established by prophylactic use of a more purified statolon or other interferon-inducing substance. The observation that a single injection of statolon provides significant protection of considerable duration in mice increases the rationale of the use of interferon inducers. SUMMARY Statolon, a nonviral material, is capable of inducing interferon in intact animals as well as in tissue culture. The peak level of production of circulating interferon in the mouse occurs at 12 hr after intraperitoneal injection of statolon. A dosage response is seen with statolon, and levels of interferon produced in response to varied doses of statolon correlate with protection against virus infection. Hyporeactivity to a second response by statolon endures for 5 to 6 days, a period similar to that observed with other interferon inducers. One injection of statolon affords significant protection in the mouse for at least 1 month against a lethal MM virus infection. Statolon also is capable of inducing circulating interferon in primates as demonstrated in the green monkey. ACKNOWLEDGMENTS The able assistance of E. L. Hayes in this study and the aid of F. D. Streightoff and associates in conducting the statolon assays in mice are gratefully acknowledged. L1mIERATuRE CITED 1. COCHRAN, K. W., C. G. BROWN, AND T. FRANCIS Antiviral action of a mold filtrate on experimental poliomyelitis in cynomolgus monkeys. Proc. Soc. Exptl. Biol. Med. 85: DUFFY, C. E., AND P. N. MORGAN Interval between inoculations as a factor in interference between neurotropic viruses. Proc. Soc. Exptl. Biol. Med. 84: DEFENDI, V., R. E. BILLINGHAM, W. K. SILVERS, AND P. MOORHEAD Immunological and karyological criteria for identification of cell lines. J. Natl. Cancer Inst. 25: Ho, M Interferon-like viral inhibitor in rabbits after intravenous administration of endotoxin. Science 146: Ho, M., Y. KoNo, AND M. K. BREINIG Tolerance to the induction of interferons by endotoxin and virus: role of a humoral factor. Proc. Soc. Exptl. Biol. Med. 119: ISAACS, A., AND J. LINDENMANN Virus interference. I. The interferon. Proc. Roy. Soc. (London) Ser. B 147 : KLEINSCHMIDT, W. J., J. C. CLINE, AND E. B. MURPHY Interferon production induced by statolon. Proc. Natl. Acad. Sci. U.S. 52: KLEINSCHMIDT, W. J., AND E. B. MURPHY Investigations on interferon induced by statolon. Virology 27: KLEINSCHMIDT, W. J., AND G. W. PROBST The nature of statolon, an antiviral agent. Antibiot. Chemotherapy 12: Downloaded from on April 28, 218 by guest

6 VOL. 31, 1967 INTERFERON INDUCTION WITH STATOLON MERIGAN, T. C Various molecular species of interferon induced by viral and nonviral agents. Bacteriol. Rev. 31: MERIGAN, T. C., Am W. J. KLEINSCHMIDT Different molecular species of mouse interferon induced by statolon. Nature 28: POWELL, H. M., C. G. CULBERTSON, J. M. MC- GUIRE, M. M. HolHN, ANm L. A. BAKER A filtrate with chemoprophylactic and chemotherapeutic action against MM and Semliki Forest viruses in mice. Antibiot. Chemotherapy 2: ROTEM, Z., R. A. Cox, AND A. ISAACS Inhibition of virus multiplication by foreign nucleic acid. Nature 197: SCHLESINGER, R. W., P. K. OLrrSKY, AND I. M. MORGAN Observations on acquired cellular resistance to equine encephalomyelitis virus. Proc. Soc. Exptl. Biol. Med. 54: SCHLESINGER, R. W., P. K. OLITSKY, AND I. M. MORGAN Induced resistance of the central nervous system to experimental infections with equine encephalomyelitis virus. III. Abortive infection with Western virus and subsequent interference with the action of heterologous viruses. J. Exptl. Med. 8: STINEBRING, W. R., AND J. S. YOUNGNER Patterns of interferon appearance in mice injected with bacteria or bacterial endotoxin. Nature 24: YOUNGNER, J. S., AND W. R. STINEBRING Interferon production in chickens injected with Brucella abortus. Science 144: YOUNGNER, J. S., AND W. R. STNEBRING Interferon appearance stimulated by endotoxin, bacteria, or viruses in mice pretreated with Escherichia coli endotoxin or infected with Mycobacterium tuberculosis. Nature 28: WHEELOCK, E Interferon-like virus inhibitor induced in human leucocytes by phytohemagglutinin. Science 149:31. Downloaded from on April 28, 218 by guest