Effect of Temperature on the Induction of Interferons by Endotoxin and Virus

Transcription

1 JOURNAL OF BACTERIOLOGY, Mar., 1966 Copyright 1966 American Society for Microbiology Vol. 91, No. 3 Printed in U.S.A. Effect of Temperature on the Induction of Interferons by Endotoxin and Virus BOSKO POSTIC, CATHERINE DEANGELIS, MARY K. BREINIG, AND MONTO HO Department of Epidemiology and Microbiology, Graduate School of Public Health, University of Pittsburgh, Pittsburgh, Pennsylvania Received for publication 4 October 1965 ABSTRACT POSTIC, BOSKO (University of Pittsburgh, Pittsburgh, Pa.), CATHERINE DEANGELIS, MARY K. BREINIG, AND MoNTo Ho. Effect of temperature on the induction of interferons by endotoxin and virus. J. Bacteriol. 91: The effect of ambient and body temperature on interferon formation in rabbits injected intravenously with virus differed from that seen after injection of endotoxin. Newcastle disease virus-induced interferon (VII) was elevated by increasing ambient temperature to 35 C, whereas cooling of the rabbit at 4 C resulted in low VII levels. Neither of these conditions affected the titers of endotoxin-induced interferon (EII). However, a significant enhancement of EIl levels was found in sera of shorn rabbits, in which the body temperature was lower than in unshorn animals by 1.0 to 1.5 F and the pyrogenic response to endotoxin was less by about 2 F. This enhancement of EII by relatively low body temperatures was also in striking contrast to the reported enhancing effect of high body temperature of the rabbit on the lethal action of endotoxin. It is suggested that the temperature optimum for formation of EII is lower than for formation of VII. Gledhil (5) first showed that bacterial endotoxin induced in the serum of mice a factor which he suggested might be related to interferon (6, 7). Stinebring and Youngner (13) reported that interferon was indeed induced by endotoxin in chickens and mice; Ho (8) demonstrated independently that endotoxin induced an interferonlike viral inhibitor in rabbits. Since then, there has been some interest in whether (i) endotoxininduced interferon (EII) is the same as virusinduced interferon (VII), and (ii) whether the process of their induction is the same (9, 1 la; Hallum et al., private communication; Youngner et al., private communication). (All private communications cited were obtained through the Information Exchange Group no. 6.) As a new approach to this problem, we decided to study the effect of ambient and body temperature of rabbits on the formation of EII and VII in order to differentiate further the interferon responses to endotoxin and virus in an intact animal. It was thought that if these responses were metabolically different, they might 1277 have different temperature optima. In addition, such a study has some intrinsic value in itself. Since it is known that elevation of body temperature markedly enhances the lethal action of endotoxin (11), it was considered of interest to determine what effect such elevation might have on another biological function of endotoxin, i.e., the induction of EII. MATERIALS AND METHODS Rabbits, endotoxin, viruses, sera and assay. As in previous studies (8), rabbits used weighed around 1 kg. Endotoxin from Escherichia coli 0113 (courtesy of A. I. Braude), extracted according to the Boivin method, was inoculated intravenously in the marginal ear vein in 10-,ug amounts unless otherwise specified. For induction of VII, the method of Baron and Buckler (3) was used. A 0.5-ml amount (3 X 109 plaqueforming units) of Newcastle disease virus (NDV, Herts strain, kindly supplied by Samuel Baron), propagated in the allantoic cavity of 11-day-old chicken embryos, was inoculated intravenously into each rabbit. Sera from rabbits inoculated with endotoxin or virus were obtained at indicated intervals by cardiac puncture. Those from the virus-inoculated groups were heated at 56 C for 15 min to assure inactivation of residual virus, which even before such treatment was usually undetectable by plaquing on chick embryo cell cultures. VII and EII were assayed as follows (10). Serial twofold dilutions of sera were made and 0.3 ml was incubated with duplicate rabbit embryo cell cultures for 18 hr, after which the cultures

2 1278 POSTIC ET AL. J. BACTERIOL. were drained and challenged with about 1,000 TCD50 of vesicular stomatitis virus (VSV). Inhibitory titers were expressed as the highest serum dilution capable of inhibiting the cytopathic effect in 50% of the culture tubes, as determined by the Reed-Muench method. Shearing and temperatures. Rabbits were sheared from head to tail with electric clippers. They were placed at the particular ambient temperature at least 24 hr prior to being inoculated, with the exception of those placed at 4 C. The latter group was placed at 4 C 1 hr prior to observations. Rabbit temperatures were taken hourly, starting from the time of inoculation by use of a rectal thermometer and were recorded as degrees F. Each temperature reported (Fig. 1) represents the mean obtained from five to seven rabbits. Environmental conditions used. Endotoxin or virus was administered to animals placed under the following four conditions determined by whether or not they were shorn, and the ambient temperature (recorded as degreesc): (A) unshom, 35 C; (B) unshorn, 25 C; (C) shorn, 25 C; and (D) shorn, 4 C. These temperatures varied at most 3 C as monitored during these experiments. The ambient temperature of 25 C was the normal room temperature of a completely air-conditioned building. For controls, uninoculated rabbits were used. This choice was based on results of the following preliminary experiments. Intravenous inoculation of 0.5 ml of normal allantoic fluid into rabbits maintained at 35 C did not result in induction of viral inhibitor and did not raise their mean body temperature more than 0.5 F during 8 hr of observation. The procedures of placing the animals in a 35 C environment (condition A) for 24 hr or shearing (condition B), by themselves, did not result in the induction of a circulating viral inhibitor, as revealed by testing serum samples obtained 2, 4, 20, and 24 hr after these procedures were undertaken. Normal rabbit serum obtained from animals kept at room temperature had previously been shown to be free from any interferon-like activity (8). RESULTS Temperature studies in rabbits. First we attempted to ascertain the effects on body temperature by (i) the four different environmental conditions, and (ii) the administration of endotoxin or virus to animals (Fig. 1). The mean body temperatures of uninoculated rabbits subjected to conditions A, B, C, and D show significant differences. An ambient temperature of 35 C (condition A) for 24 hr raised the body temperature by 2.0 F, i.e., from to F with little variation (105.6 to F) over a 7-hr period of observation, recorded in Fig. 1. At room temperature (25 C, condition B), the body temperature of unshorn rabbits was to F. Shearing and exposure to room temperature for 24 hr (condition C) reduced the body temperature from to F and it IiJ 105 Z 104 Qt 103 M 102 I0I L 100 : 99 L 35 C 25 C 2_525C /rv S-4C 35'C 35c 25WC S 25 C _/0_S 4 C UNINOCULArEo AFTER NDV 95J.. X,I O HOURS OF OBSERVATION FIG. 1. Effects of endotoxin and virus on body temperatures (as degrees F) ofrabbits subjected to environmental conditions: (A) 35 C; (B) 25 C; (C) S (shorn), 25 C; and (D) S, 4 C. was somewhat labile (101.0 to F) during the 7-hr period of observation. The lowest body temperatures (100.1 to F) were recorded in animals shorn and placed at 4 C for 1 hr prior to the observations shown in Fig. 1 (condition D). The inoculation of endotoxin into rabbits affected profoundly their body temperatures, as judged by the differences from observations before inoculation (0 hr) and subsequent hourly determinations (Fig. 1). At normal room temperature of 25 C (condition B), animals responded with the characteristic biphasic fever (1), with a peak at F. A similar but lower febrile response was obtained after shearing (condition C). Animals placed in a heated room at 35 C (condition A) responded with a monophasic hyperthermia which reached 108 F after endotoxin. Shorn animals placed at 4 C (condition D) responded with marked hypothermia. This type of response to endotoxin in cooled rabbits was previously noted by Kass et al. (11). The administration of virus to animals at room temperature (condition B), as shown originally by Wagner et al. (15) and Bennett et al. (4), had a pyrogenic effect (Fig. 1). This was also true of animals placed in a heated environment (condition A). In shorn animals (condition C), the injection of NDV produced a generally lower febrile response. Little or no elevation of body temperature was elicited by NDV under the severe cold stress (condition D). We next obtained blood samples from animals kept under these same four conditions after inoculation of endotoxin or virus.

3 VOL. 91) 1966 TEMPERATURE AND INTERFERON 1279 Production of ElI in four environments (Table 1). The serum ElI titers after an inoculation of 10,g of endotoxin did not change significantly, irrespective of whether the rabbits were held at room temperature, at 35 C, or were shorn and placed at 4 C. The range of titers obtained under these three conditions (A, B and D) was wide, but this variability, as well as the mean titers, were comparable to data obtained from rabbits held at room temperature in a previous study (9). In contrast to the above, the most surprising finding was that EII titers were significantly elevated in shorn animals held at room temperature (condition C). Since EII is more heat-labile than VII (8), it was considered possible that EII might have been inactivated in animals held at 35 C, which registered body temperatures as high as 108 F (42.2 C) by the time of the 4-hr sampling. An augmentation of EII might thus remain undetected. Accordingly, experiments were undertaken to determine the lability of ElI at 38, 41, 43, and 45.5 C. Samples of a standard preparation of EII were placed in four water baths regulated at these temperatures. Samples were titrated after 1, 2, and 4 hr. No significant decrease in titer of any sample was observed. It is therefore unlikely that EII was preferentially inactivated at any body temperature sustained in these experiments. Production of VII in four environments (Table 2). The pattern of the VII titers in rabbits receiving NDV under four different conditions was entirely different from that seen in rabbits receiving endotoxin. It appears that, generally, the higher the environmental and body temperatures, the higher the titers of VII. Thus, the highest titers were obtained with sera from animals placed in the 35 C room (condition A), and the lowest were from animals in condition D (shorn and at 4 C). Intermediary titers were obtained under conditions B and C. Although the individual groups of titers may not be statistically different from corresponding groups under condition B, the general trend of decreasing VII titers with decreasing environmental and body temperatures appears quite striking. No such variation was observed in the case of EII. One obvious possibility that might account for the elevated VII titers in rabbits held at higher temperatures may have been brisk viral replication, although this is unlikely in view of the similar pattern of appearance and disappearance of VII in animals under these different conditions TABLE 1. Effect of environmental temperature and shearing on endotoxin-induced interferon (EII) Environmental condition Time" EII titersb ac Geometric mean titer (A) 35 C, not shorn 2 4, 4, 6, 8, 8, 12, 16, 16, 24, 48, 64, > , 64, 64, 96 4 <2, <2, 2, 4, 4, 4, 8, 12, 12, 12, > , 16, 24, 24 (B) 25 C, not shorn 2 4, 4, 8, 8, 8, 8, 12, 12, 12, 16, 24, 16 24, 32, 64, <2, <2, 2,'2, 2, 4, 6, 8, 8, 12, 12, S 16, 16 7 <2, <2, <2,2, 3, 4 <2 (C) 25 C, shorn 2 4, 16, 16, 24, 24, 64, 96, 96, 128, 128, <0.001* , 256, 256, 256, , 6, 24, 24, 24, 32, 32, 32, 32, 32, <0.001* 29 36, 96, 96, , 3, 4, 8, 8, 12, 12, 16, 16, * 8 (D) 4 C, shorn 2 6, 24, 32, 64, 96 > , 8, 8, 64 > a Hour rabbits were bled after 10,ug of endotoxin was injected intravenously. b As reciprocals of serum dilutions; each value refers to a titer from a separate rabbit. c Probability that each group of titers is chosen at random from the same population as the corresponding group under condition B. For example, the 2-hr titers under each of the conditions A, C, and D are compared with the 2-hr titers under, B. * Represents significant -difference (P < 0.05) by the Rank sum test.

4 1280 POSTIC ET AL. J. BACTERIOL. TABLE 2. Effect of environmental temperature and shearing on virus-induced interferon (VII) Environmental condition Timea VII titersb ac metr (A) 35 C, not shorn 2 16, 32, 32, 256, 256, 384, 512, 768, 768, * , 64, 96, 1,536, 2,048, 2,048, 3,072, ,096, 8, , 128, 384, 512, 768, 1,536, 4,096, 8, * <2, <2, <2, 4,'4 <2 (B) 25 C, not shorn 2 8, 16, 24, 96, 96, 128, 128, 192, 192, , 96, 512, 1,024, 2,048, 3,072, 3,072, 776 4, , 32, 48, 64, 64, , 2, <2, <2, 2 <2 (C) 25 C, shorn 2 24, 24, 32, 48, 48, 48, 64, 128, 256, , 48, 64, 128, 192, 192, 334, 1,024, 1, * , 32, 48, 48, 64, 256, 1, (D) 4 C, shorn 2 6, 12, 12, 32, * , 12, 16, 128, * 30 Hour rabbits were bled after intravenous injection of 3 X 109 plaque-forming units of NDV. b As reciprocals of serum dilutions; each value refers to a titer from a separate rabbit. c Probability that each group of titers is chosen at random from the same population as the corresponding group under condition B. For example, the 2-hr titers under each of the conditions A, C, and D are compared with the 2-hr titers under B. * Represents significant difference (P < 0.05) by the Rank sum test. (Table 2). One would expect higher titers in samples taken later if viral replication had taken place. This point was nevertheless tested as follows. Two rabbits under condition A and two under condition B were killed 7, 24, and 48 hr after inoculation of virus. Two rabbits under condition C and two under condition D were also killed 7 hr after receiving NDV. Serum and 10% (w/v) extracts of lung, spleen, and liver were prepared from each of the killed animals and titrated for viral infectivity in chick cell monolayers. None was recovered. It appears then that NDV induced interferon in rabbits without replication. The ambient temperatures to which the animal was subjected affected the yield of interferon, but failed to initiate detectable viral replication. DISCUSSION It is of course hazardous to make a direct comparison between the inoculation of endotoxin and of virus under different environmental conditions if for no other reason than the profoundly different pharmocological effect of these two substances. Ideally, to study the effect of temperature, cell culture systems for producing= interferon are preferable. This, however, is unfortunately not yet possible since endotoxin does not inhibit viruses or induce interferon in cell culture (8, 13, 14). The more efficient formation of VII in cell culture at higher temperatures has been pointed out by Ruiz-Gomez and Isaacs (12). They found in chick embryo fibroblasts infected with NDV lower yields of interferon at 35 and 37 C than at 39 and 42 C. Similarly, an increased yield of interferon was recovered from embryonated chicken eggs infected with the Herts strains of NDV at higher (36 to 40 C) temperatures of incubation (2). Ruiz-Gomez and Sosa-Martinez (private communication) showed that interferon was depressed in mice infected with coxsackievirus B1 when they were placed at 4 C. More interferon was formed in animals held at a higher environmental temperature. The findings of this study relating to the potentiation of VII in rabbits experiencing high environmental and body temperatures are in general agreement with the above-cited reports. The inability of high temperatures to enhance EII titers cannot be easily reconciled with the picture obtained with regard to VII. Interestingly, the only significant influence on EII was achieved by shearing. It is a striking contrast that, whereas shorn rabbits released more EII, they produced less VII when compared with unshorn animals.

5 VOL. 91, 1966 TEMPERATURE AND INTERFERON 1281 It is noteworthy that the body temperatures of shorn rabbits receiving endotoxin or virus were quite comparable. Since this was substantially lower than body temperatures of unshorn animals, or of those held at 35 C, it is tempting to suggest that the optimal temperature for the formation of ElI is lower than for forming VII. This may be supported by the fact that mice, which do not respond to endotoxin by fever, appear to produce circulating interferon of higher titers after an injection of endotoxin (13) then do rabbits (8). Another interesting point is that, while the cold environment (condition D) markedly inhibited VII in rabbits, it had relatively little effect on ElI. This is reminiscent of the reported inability of puromycin to inhibit the formation of ElI in intact animals in which protein synthesis and VII formation were effectively blocked (lla; Youngner et al., private communication), and is consistent with the hypothesis that the metabolic determinants of EII formation are different from those for VII (9). Finally, these experiments show that at least two different biological effects of endotoxin, i.e., its lethality and its interferon-inducing capacity, may be strikingly dissociated by alterations of the animal's environment. Kass et al. (1) found that the lethal action of endotoxin is potentiated by elevation of body temperature, whereas it was reduced by a decrease in body temperature brought about by shearing. For a i-kg rabbit, the LD5o of endotoxin was 2,000,g when the animal was shorn, and 4,ug when it was held at 37 C. This is exactly the reverse of the situation observed in this study, as far as induction of EII is concerned, which was not altered by fever, but was more efficient in shorn rabbits. ACKNOWLEDGMENTS This investigation was supported by Public Health Service grant AI from the National Institute of Allergy and Infectious Diseases. Gerard Monahan, Linda Elston, and Demetra Dordas provided excellent technical assistance. We thank Edward H. Kass, William McD. Hammon and J. A. Armstrong for discussion of this work and for their help in the preparation of the manuscript. LITERATURE CITED 1. ATKINS, E Pathogenesis of fever. Physiol. Rev. 40: BARON, S Relationship of interferon and temperature to virulence of Newcastle disease virus, p In R. P. Hanson [ed.], Newcastle disease virus. The Univ. Wisconsin Press, Madison. 3. BARON, S., AND C. E. BUCKLER Circulating interferon in mice after intravenous injection of virus. Science 141: BENNETT, I. L., JR., R. R. WAGNER, AND V. S. LEQUIRE The production of fever by influenzal viruses. II. Tolerance in rabbits to the pyrogenic effect of influenzal viruses. J. Exptl. Med. 90: GLEDHILL, A. W Sparing effect of serum of mice treated with endotoxin upon certain murine virus diseases. Nature 183: GLEDHILL, A. W The interference of mouse hepatitis virus with ectromelia in mice and a possible explanation of its mechanism. Brit. J. Exptl. Pathol. 40: GLEDHILL, A. W Influence of endotoxin upon pathogenesis of viral infections, p In M. Landy and W. Braun [ed.], Bacterial endotoxins, Rutgers Univ. Press, New Brunswick, N.J. 8. Ho, M Interferon-like viral inhibitor in rabbits after intravenous administration of endotoxin. Science 146: Ho, M., AND Y. KONO Effect of actinomycin D on virus and endotoxin-induced interferonlike inhibitors in rabbits. Proc. Natl. Acad. Sci. U.S. 53: 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: KASS, E. H., R. P. ATWOOD, AND P. J. PORTER Observations on the locus of lethal action of bacterial endotoxin, p In M. Landy and W. Braun [ed.], Bacterial endotoxins. Rutgers Univ. Press, New Brunswick, N.J. lla. KE, Y. H., S. H. SINGER, B. POSTIC, AND M. Ho Effect of puromycin on virus and endotoxin-induced interferonlike inhibitors in rabbits. Proc. Soc. Exptl. Biol. Med. 121: RuIz-GoMEz, J., AND A. IsAAcs Interferon production by different viruses. Virology 19: STINEBRING, W. R., AND J. S. YOUNGNER Patterns of interferon appearance in mice injected with bacteria or bacterial endotoxin. Nature 204: WAGNER, R. R Cytotoxic action of influenza virus. Failure to induce acquired resistance phenomenon in tissue culture. Proc. Soc. Exptl. Biol. Med. 90: WAGNER, R. R., I. L. BENNETT, JR., AND V. S. LEQUIRE The production of fever by influenzal viruses. I. Factors influencing the febrile response to single injections of virus. J. Exptl. Med. 90: