Adrenalectomy, and the Administratioil of

Transcription

1 INFECTiON AND IMMUNITY, May 1972, p Copyright 1972 American Society for Microbiology Vol. 5, No. 5 Printed in U.S.A Differential Susceptibility of Male and Female Mice to Encephalomyocarditis Virus: Effects of Castration, Adrenalectomy, and the Administratioil of Sex Hormones STANFORD B. FRIEDMAN, LEE J. GROTA, AND LOWELL A. GLASGOW' Departments of Pediatrics, Psychiatry, atid Microbiology. University of Rochester School of Medicine anld Denttistry, Rochester, New York Received for publication 14 January 1972 Male mice have been noted to be more susceptible to encephalomyocarditis (EMC) virus than female animals. Castration of male mice abolished this difference and resulted in enhanced resistance similar to that of female mice. This increase in resistance in castrated males could be eliminated by adrenalectomy. Adrenalectomy alone increased susceptibility in male mice but not in female mice. The administration of a long-acting preparation of testosterone markedly increased susceptibility both in castrated male and female mice, as well as in intact females. Testosterone also had a similar effect in immature mice of both sexes. On the other hand, estradiol, but not progesterone, increased susceptibility to EMC virus in castrated mice of both sexes. These data indicate that testicular, ovarian, and adrenal hormones may influence resistance to EMC virus in the mouse and may be critical determinants of the clinical outcome of infection with this virus. Sex differences in susceptibility to infectious agents have been repeatedly described in man (4, 25, 26, 30, 44). In general, males are more prone to develop an infectious disease than females, though the mechanisms responsible for this difference have never been completely delineated. It has been proposed that the increased susceptibility of males to infection may be related to genetic differences in immunologic responses (44). It is evident that such immunologic variations between the sexes probably are not entirely dependent upon sex hormones in that these differences in resistance also may be demonstrated in the sexually immature host. On the other hand, clinical evidence that resistance in women varies as a function of the menstrual cycle and during pregnancy (6, 45) suggests that susceptibility to viral agents is influenced by sex hormones. In acute poliomyelitis, males are more susceptible than females in the younger age group, but this difference in resistance had been noted to be reversed in adults 25 to 29 years of age even when the increased susceptibility noted during pregnancy is eliminated (1). More recently, Ainbender et al. (2) have shown differ- I Present address: Department of Microbiology, College of Medicine, University of Utah, Salt Lake City ences in the immunoglobulin pattern of the polioantibody in the serum of the adult male and the adult female. Evidence from animal experimentation also substantiates the concept of a sex difference in susceptibility. Studies have included comparison of resistance and immune response of males and females to various infectious agents (15, 20, 29, 35, 36, 39), the effects of administering sex hormones (16, 17, 37, 38, 40-43, 47), changes during pregnancy (19, 24), and the influence of castration (3, 33). Nicol and co-workers (31, 32) have emphasized the role of the estrogen hormones, concluding that "our results to date suggest that estrogen is the principal natural stimulant of body defense in both the male and the female..." (34). On the other hand, others have shown that testosterone, as well as estradiol, may influence serum complement (5). A sex difference in susceptibility to encephalomyocarditis (EMC) virus has been documented (46; L. A. Glasgow, and S. B. Friedman, Abstr., Pediat. Res. 1:319, 1967) as one of a number of factors which may modify host resistance to this agent. In our laboratory X-irradiation (28), immunosuppressive therapy (27), housing (10), and pregnancy (7) have all been shown to influence 637

2 638 FRIEDMAN, GROTA, AND GLASGOW INFECT. IMMUNITY susceptibility. Giron and co-workers (12, 13) have recently reported that estrone treatment results in an increased mortality to MM virus, a member of the EMC group of viruses, whereas progesterone or testosterone had no effect in the dosages used. Weinstein and Chang (46) have also observed increased EMC virus multiplication in muscle tissue from female mice as compared to tissue derived from male animals. They, as well as Murphy and Syverton (29), point out the importance of adjusting dosage in demonstrating sex differences in susceptibility. The studies to be reported here have been directed toward delineating the physiological basis for the observed sex difference in resistance to EMC virus in mice. The effects upon susceptibility to EMC virus of castration, castration combined with adrenalectomy, and adrenalectomy alone, as well as the administration of sex hormones, have been described. MATERIALS AND METHODS Animals. Randomly bred CD-1 mice were received at 3 to 5 weeks of age from Charles River Laboratory (Wilmington, Mass.) and housed 4 to 6 per cage with food and water available ad libitum. All surgery was performed by our supplier under ether anesthesia, and adrenalectomized mice were maintained on normal saline in place of water. Room temperature was approximately 22 C, and a lighting cycle from 7 AM to 7 PM was maintained. Virus. The EMC virus was originally obtained from K. K. Takemoto at the National Institutes of Health. The stock preparations were grown and assayed in L cells. The virus stock used for the majority of experiments had a titer of 1.4 X 106 plaque-forming units (PFU) per ml with a ratio of approximately 30:1 of small to large plaque variants (pool I). A second pool (pool II) had a titer of 108 PFU/ml. These experiments were carried out over a 2 to 3 year period. During that time we observed a decrease in the susceptibility of experimental animals. At these times, the 50% lethal dose (LD5o0) of stock preparation was redetermined and the inoculum was adjusted to obtain approximately a 25 to 35% mortality in control female animals. The virus inoculum utilized in these experiments was calculated to kill 30 to 40% of female mice, based on the determination of the LD,,o. All inoculations were given by the intraperitoneal route in 0.2-ml amounts. During the inoculation procedure, the virus was kept iced, and cages of animals were injected alternately from all experimental groups. 'The mice were weighed the day prior to inoculation. After inoculation with EMC virus, each cage was examined daily for deaths. An experiment was terminated only after a minimum of 5 days had elapsed without any deaths and all surviving mice appeared healthy. Hormone treatment. All hormones were administered intramuscularly as "long-acting" preparations 3 to 4 days prior to inoculation with EMC virus. Dose (per animal) -of testosterone enanthate was 250 to 500,g/0.025 ml; estradiol valerate, 500,g; and hydroxyprogesterone caproate, 12.5 mg/0.05 ml. All dilutions of hormones were made with sesame oil. Analysis of data. Differences in susceptibility, reflected by death rate, were analyzed in two ways. First, comparisons were made of the frequency of death at the termination of a given experiment by chi square. Second, comparisons of the rate of death of those animals that died were performed by the Mann- Whitney technique. All probabilities of significance were based on two-tailed tables. RESULTS Mortality as a function of dose. The studies which show a sex difference in susceptibility to EMC virus were dependent upon the inoculum of EMC virus. In our experience, significant differences in susceptibility rarely occur when the frequency of female mice dying exceeds 50% or, conversely, falls below 20%C. For instance, in one representative experiment, using groups of 10 mice, 6 dilutions of virus (pool I) were used, ranging from 0.3 PFU to 3 x 104 PFU. The inoculum of 3 X 101 PFU (4 dilution) was the only dose that clearly demonstrated a difference in resistance. A larger inoculum resulted in a high mortality in both sexes, whereas 3 PFU or less produced a lethal infection in too few animals to obtain a statistically significant difference. These results illustrate the importance of the inoculum size when attempting to establish differences in susceptibility among different groups of animals (9, 29, 46). Effects of castration, adrenalectomy, and both procedures. At approximately 4 weeks of age, 5 groups of 30 male and female mice were subjected to: (i) castration, (ii) sham operation (sham castration), (iii) adrenalectomy, (iv) adrenalectomy and castration, or (v) were left intact by our animal supplier. Four days after surgery, the animals were shipped and housed 5 to 6 per cage for 1 month. At 62 days of age, the groups of 25 to 28 male mice had mean body weights of 31.4 to 36.8 g with those in the control group having a mean body weight of 33.4 g. Similar size groups of female mice had mean body weights of 26.5 to 28.4 g; the control group of female mice had a mean body weight of 26.5 g. On the following day, all mice were inoculated with 30 PFU of EMC virus (pool I). The effects of castration upon susceptibility to EMC virus may be seen by comparing the mortality rate of the castrated mice to that of the control and sham-operated animals. As shown in Fig. 1, the six curves of cumulative percentage of deaths show two distinct mortality patterns. The intact and sham-operated male mice were more susceptible to EMC virus than the other four

3 VOL. 5, 1972 SUSCEPTIBILITY OF MALE AND FEMALE MICE TO EMC VIRUS F] CONTROL MJALES A-A CAST MALES 0-0 ADRENALX MALES *-O ADRENALX CAST MALES t4j t 40 KLJ~ (.0 ** A" CONTROL MALES O.O CONTROL FEMALES /* * -00 SHAM MALES * -O--O. SHAM FEMALES CASTRATED MALES -, CASTRATED FEMALES FIG. 1. Effects of castrationi upon2 susceptibility of adult mice to EMC virus. Groups conitaint 25 to 28 aniimals. groups, with 68%,, and 71 of the animals dying, respectively. The frequency of death of the control females (30%ci) was not significantly different from the mortality of the sham-operated animals (18c/%), the castrated females (24%/C), or the castrated male animals (34%). The greater frequency of death was, therefore, associated with intact males and presumably with testicular function. The role of the adrenocortical hormones in host resistance has been well documented, and cortisone has been shown to increase susceptibility to EMC virus (7). The question therefore arose whether the marked decreased susceptibility to EMC virus of castrated male mice was dependent upon a functioning adrenal gland. In Fig. 2, mortality rates of control and castrated male mice were compared with those of male mice subjected either to adrenalectomy or to adrenalectomy and castration. As presented in Fig. 1, the castrated males were more resistant and died at a frequency similar to female mice (34%), in contrast to 56%, and 72% of the mice in the other groups. When only the mice that died were analyzed by the Mann-Whitney test, the rate of death of the adrenalectomized males was greater than that of intact mice (P < 0.01), although the final mortality was similar. This rate did not differ significantly from that of mice which were both adrenalectomized and castrated. Male mice subjected to both procedures did differ, however, as "'I FIG. 2. Inifluenice of adrenialectomy (Adrenaix) in intact anid castrated male mice u(poni su3sceptibility to EMC viruis. Groups conitain2 25 to 28 aniimals. measured by rate of death, from both castrated (P < 0.01) and control males (P < 0.01). In summary, these data demonstrate that the marked decrease in susceptibility to EMC virus produced by castration of male mice does not occur in adrenalectomized animals. Further, the rate of death in adrenalectomized male mice, with and without castration, is greater than in intact control animals. The mortality rates of intact and castrated female mice were next compared to female animals adrenalectomized or adrenalectomized and castrated (Fig. 3). Examination of the rate of death of those animals that died (Mann-Whitney test) shows that adrenalectomized female mice did not differ significantly from controls, nor did they differ from mice subjected to both adrenalectomy and castration. Female mice adrenalectomized and castrated did differ in terms of rate of death from both castrated and intact female mice (P < 0.01 in both cases). In female mice, castration again did not significantly decrease susceptibility to EMC virus. In summary, in contrast to males, neither castration or adrenalectomy alone in female animals significantly modified susceptibility. [In a subsequent series of experiments, adrenalectomized female mice were administered testosterone, and susceptibility to EMC virus was compared to adrenalectomized female mice not given testosterone and to intact female mice. In two such experiments, adrenalectomy alone was 16

4 640 FRIEDMAN, GROTA, AND GLASGOW INFECT. IMMUNITY ;50- q rs D O CONTROL FEMALES A-A CAST. FEMALES 0-0 ADRENALX FEMALES *-0 ADRENALX a CAST. FEMALES */~~~~~~*,. A.- i 50- (f J ;4z k. 30- ( lo 0-C CONTROL MALES O O CONTROL FEMALES 0-O CASTRATED TESTOSTERONE MALES O OCASTRATED TESTOSTERONE FEMALES & A TESTOSTERONE FEMALES I I *-T FIG. 3. Influence of adrenalectomy (Adrenalx) in intact and castrated female mice upon susceptibility to EMC virus. Groups contain 25 to 28 animals. now noted to markedly increase susceptibility, with 88 to 90% of the adrenalectomized animals dying as compared to 56% of the control animals (intact females). We are unable to explain this difference in susceptibility in adrenalectomized female mice noted in these two series of experiments.] Combining the procedures, as in males, increased susceptibility to EMC virus, but the effect was not marked. At this point in the study, the data indicated that castration enhanced resistance of male mice to EMC virus and that this effect was not observed in adrenalectomized animals. The next series of experiments was designed to evaluate further the role of testicular function by administering testosterone to male, female, and castrated mice. Testosterone administration to intact and castrated mice. Intact and castrated mice 4 weeks of age were received from the supplier and housed 4 to 6 per cage until 9 weeks of age. Mean body weight for the castrated males was 35.3 g and for the castrated females was 32.1 g; intact animals weighed 37.6 g (males) and 28.6 g (females). All castrated mice and a group of intact female mice were injected with 500 Mug of long-acting testosterone. Four days later, all groups, including male and female control animals, were inoculated with 3 X 101 PFU of EMC virus (pool I). Twenty to 31 mice were in each group. As seen in Fig. 4, approximately 80% of the castrated mice and 85% Ị., DY 9 1AI FIG. 4. Effect of testosterone uponi susceptibility to EMC virus in castrated male and.female mice and in intact females. Groups contain 20 to 31 mice. of the intact female mice that received testosterone died. Seventy-one per cent of the untreated intact males and 36% of the intact females died. These findings demonstrate that, in castrated mice of either sex and in intact adult females, testosterone markedly increases susceptibility to EMC virus. Effects of testosterone in sexually immature mice. Pregnant mice were obtained at approximately 14 days of gestation. The pups were weaned at 21 days of age, weighed, and housed five per cage. The mean body weight for males was 14.6 g and for females was 14.0 g. Thirty mice of each sex were injected with 250,ug of testosterone, and an equal number of mice of each sex served as controls. At 23 days of age, all mice were inoculated with 2 X 102 PFU of EMC virus (pool II). As shown in Fig. 5, 70% of the control males and 53 % of the control females died. This difference was not significant by chi square analysis, but of those animals that died, there was a significant difference in rate of death (P < 0.01). As in castrated animals, testosterone increased the rate of death in both sexes (P < 0.02 for males and P < for females). Combining the groups of testosterone-treated and control mice of each sex resulted in a significant difference between male and female animals in terms of the number of animals dying (x2 = 5.81; P < 0.05). Thus, testosterone increased susceptibility to EMC virus in both male and female weanlings; interestingly, however, the males treated with testosterone died at a faster rate

5 VOL. 5,1972 SUSCEPTIBILITY OF MALE AND FEMALE MICE TO EMC VIRUS '.4. M 50- i 40-4Q. gll 30- k 20-0 O.' p- O' 0-0 V o. * CONTROL MALES CONTROL FEMALES TESTOSTERONE MALES TESTOSTERONE FEMALES Ir FIG. 5. Effect of testosteronie onz susceptibility tc EMC virus in sexuially immature male anid female mice. Groups containi 29 to 30 mice. than the females injected with the same dose of hormones (P < 0.01). Testosterone, in the dosages administered, increased susceptibility to EMC virus in female, castrated, and immature mice. The two other major sex hormones, estrogen and progesterone, were now administered to mice to compare their influence upon susceptibility to that of testosterone. Effects of estradiol and progesterone. Male and female mice approximately 4 weeks old were castrated or left intact by the supplier, shipped to our laboratory, and housed five per cage until 9 weeks of age. The groups of castrated male mice had mean body weights of 32.2 to 34.8 g and the control males had a mean body weight of 31.4 g. Comparable weights for the groups of female mice were 29.8 to 32.0 g and 24.6 g for the controls. Groups of 25 mice were injected with either depot estradiol, progesterone, or estradiol and progesterone, or were left untreated. Three days later, all mice were challenged with EMC virus (pool II). In Fig. 6, the cumulative mortality of castrated male mice receiving estradiol, progesterone, or both hormones, is compared with intact male and female mice. In interpreting these data, it should ;E 50- ('.S : ;A 40- "R r CONTROL MALES C)... CONTROL FEMALES O-O PROGESTERONE dr V-V ESTRADIOL PROGESTERONE + ESTRADIOL Cf FIG. 6. Effects of long-acting preparations of estradiol, progesterone, and both hormones, upon susceptibility to EMC virus in castrated male mice. All groups conitain 25 animals. be remembered that castrated male mice manifest a similar degree of resistance to EMC virus as normal intact females (Fig. 2). The cumulative mortality curve for intact males is also included. Analysis by chi square demonstrated a significant difference in frequency of death between castrated males receiving estradiol and intact female animals (P < 0.05). Except for the usual sex difference between intact male and female animals, other differences in frequency of death were not significant. Of those animals that died, a difference in rate of death existed between castrated male mice treated with progesterone and intact females (P < 0.01). This difference was due to the delay in death observed in the progesteronetreated mice as compared to intact females, though no difference existed in number of mice that ultimately died in these two groups. The rate of death also differed in mice treated with progesterone and those treated with both hormones (P < 0.01). Thus, castrated males receiving estradiol clearly differed from intact females in resistance to EMC virus and died in a pattern analogous to intact males. The rate of death was initially retarded by progesterone treatment. The cumulative mortality curve of castrated female mice treated with estradiol, progesterone, or both hormones, is shown in Fig. 7 and compared to the death rate of intact female mice. In this instance, the frequency of deaths from EMC virus infection among mice treated with estradiol 16 I

6 642 INFECT. IMMUNITY FIG. 7. estradiol, susceptibil FRIEDMAN, GROTA, AND GLASGOW All groupss contain 25 mice. current studies and not in the work of Giron and upon host were more susceptible to EMC virus than females (46), though data presented in this paper show this phenomenon to be dose-related. Castration in the male was observed to decrease susceptibility markedly, and the administration of both testosterone and estradiol was shown to increase susceptibility. The effects of castration in male mice upon resistance to EMC virus were also noted to be dependent upon the presence of the adrenal gland, and in castrated mice of both sexes, the superimposition of adrenalectomy increased r..0.. o susceptibility as compared to intact animals. The 0 administration of progesterone had little effect on susceptibility but tended to delay deaths due to EMC virus.,0 Nicol et al. (31-34) have emphasized the im- P0 Q* 0-fl0 CONTROL MALES 0 0 CONTROL FEMALES portant role of estrogen in host resistance, partic-.--o PROGESTERONE ularly the influence of this hormone upon the v v ESTRADIOL reticuloendothelial system, and that testosterone.~' A\ APROGESTERONE + has little or no effect. Testosterone does, however, o t ESTRADIOL?. U $appear to have a significant effect upon comple- 668lo ment in mice (5, 47). The observations of Giron and Allen (12) are of special interest in that they found that estrone, but not testosterone, increased Effects of lontg-acting preparationts of mortality to EMC (MM) virus. Differences in the progesteroie, and botlh hormon2es, upoi virus or in the strain of mice as well as the use of Fity to EMC virus in castrated female mice. a long-acting preparation of testosterone in the Allen might explain the discrepancy between their or estradliol and progesterone was significantly findings and ours. increased from the number of intact females dying The mode of action by which testosterone and (P <0.015). Of those animals that died, the rate estradiol increase susceptibility to EMC virus is of death of mice treated with both hormones not clear. Our data demonstrate that castration, differed from that of the mice treated with pro- which we assumed lowered testosterone levels, gesterone alone (P < 0.001) but not from that of reduced susceptibility only in the presence of the mice treated with estrogen. Progesterone signifi- adrenal gland. This does not, however, necessarily cantly mlodified the mortality rate in castrated mean that the primary action of testosterone in females a: s compared to intact females (P < 0.05). modifying susceptibility is mediated by changing Thus, iin castrated female mice, the administra- corticosterone secretion, plasma levels, metabotion of e-stradiol, either alone or with proges- lism, or protein binding. Further, we have asterone, re*sulted in a pattern of enhanced suscepti- sumed that the effect of adrenalectomy was due to bility sirnilar to that seen in intact males. the loss of the cortical portion of the gland, and, Progesterione alone appears to delay initial deaths though it would seem unlikely, the adrenal but not t] he ultimate number of mice dying from medulla may have a role in modifying the effects EMC virus; this phenomenon was noted in both of castration upon susceptibility to EMC virus. castrated males and females. Also in both sexes The interrelationships between the adrenocortical estradiol modifies susceptibility to EMC virus in hormones and the sex hormones are complex (8, a manner similar to testosterone. 11, 22, 23), though the evidence suggests that testosterone DISCUSSION tends to depress and estrogen to stimulate adrenocorticotropin (ACTH) secretion The inifluence of the adrenocortical hormones (21, 22). Both hormones, however, increased t susceptibility to infectious agents is well susceptibility to EMC virus. It is also possible, L8). Less attention has been directed to at least in male mice, that the increased suscepti- known (1 the physiological and pharmacological effects of bility produced by adrenalectomy masks the opormones upon host resistance, although posite effect upon susceptibility observed in the sex h4 the existence of a sex difference in susceptibility castrated animals. to infecti( cus disease is not a recent observation. Female mice generally have been found to have It had previously been noted that male mice higher plasma corticosterone levels than males

7 VOL. 5,1972 SUSCEPTIBILITY OF MALE AND FEMALE MICE TO EMC VIRUS 643 (14). For instance, in one study in our laboratory, female mice were noted at the peak of the adrenocortical rhythm to have a mean plasma corticosterone level of 15.7,ug/ml and males 5.8,g/ml. Comparable values at the trough of the cycle for females and males were 6.0 Ag/ml and 3.1 Ag/ml, respectively (unpublished data). However, the relevance of such differences in plasma corticosterone levels to susceptibility to EMC virus remains speculative. The question remains as to why, exposed to the identical dose of an infectious agent, some animals become clinically ill, or die, and others do not. The hormonal status of the animal is one factor in host resistance, but the data presented in this paper illustrate the complexity of defining the role of specific hormones. Even in discussing a single infectious agent, in this case EMC virus, host resistance appears to be the interaction of multiple factors. 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