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Lack of correlation between serum corticosterone level and immune functions in undernourished mice
NUTRITION RESEARCH, Vol. 13, pp. 801-813, 1993 0271-5317/93 $6.00 + .00 Printed in the USA. Copyright (c) 1993 Pergamon Press Ltd. All rights reserved.
LACK OF CORRELATION BETWEEN SERUM CORTICOSTERONE LEVEL AND IMMUNE FUNCTIONS IN UNDERNOURISHED MICE S. M. Filteau, Ph.D., T. J. Kaido, Ph.D., Maureen P. O'Grady, Ph.D., Robert A. Menzies, Ph.D. and Nicholas R. S. Hall, Ph.D. Department of Psychiatry and Behavioral Medicine, University of South Florida College of Medicine, Tampa, Florida, USA
ABSTRACT Two models were developed in order to investigate the importance of elevated serum glucocorticoid levels in the immune dysfunction associated with protein energy malnutrition. Weight loss was induced in mice by restricting intake of a nutritionally complete diet. in one model, serum corticosterone levels of these mice were manipulated by adrenalectomy with or without a minimal maintenance level of corticosterone supplementation. This hormone supplementation was included in some animals in an effort to reduce mortality. In the second model, the amount of diet given was adjusted to produce groups of mice which were equivalently undernourished but which were either losing, maintaining or gaining weight at the time of sacrifice. Elevated serum corticosterone levels were seen only in the animals which were losing weight. Using these models, there was no consistent inverse relationship between serum corticosterone levels and blastogenic responses to mitogens, or in vivo antibody response to sheep red blood cells. The results suggest that elevated glucocorticoids may not be of major importance in the immunodepression of a chronic stress condition such as undernutrition. Key Words."
Protein energy malnutrition, Stress, Corticosterone, Immunity INTRODUCTION
In its severe forms, protein-energy malnutrition (PEM) is associated with impairments in immune function (1). The underlying cause of this immunodepression has Corresponding Author: S. M. Filteau, Ph.D. Department of International Child Health Institute of Child Health 30 Guilford Street, London, WCIN IEH, U.K. 071-242-9789
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usually been implied to be a decreased availability of amino acids or energy substrates (2) or a lack of micronutrients, such as zinc (3, 4). An additional mechanism of immunodepression, resulting from adaptive hormonal changes associated with PEM, has also been hypothesized (5, 7-9). PEM can be considered as an example of a stressor. Elevated glucocorticoid levels have been implicated in stress-induced immunodepression (10) and have frequently been cited as contributing to the immunodepression of PEM (7-9). However, direct supporting evidence for the importance of glucocorticoids in malnutrition-induced immune system changes is limited to effects on lymphoid organ size and lymphocyte numbers. Adrenalectomized rodents had less of a decrease in blood lymphocyte numbers (11, 12) and lymphoid organ weights (13) when undernourished, that is, feedrestricted (12), or protein-deprived (11, 13), than did sham-operated controls. The effect of prevention of increased serum glucocorticoid levels on immune functions and disease resistance of malnourished animals has not been extensively studied. However, work from our laboratory (14, 15) has indicated no correlation between serum corticosterone and several immune functions in undernourished mice, and similar results have been observed in acutely feed-restricted mice (16). Part of the difficulty in obtaining such information is that mortality in adrenalectomized, PEM rodents is very high (11, 12). Increased glucocorticoid levels are needed to mobilize muscle and adipose tissue to provide amino acids and energy substrates during undernutrition. An inability to mobilize these stores may be fatal. Therefore, the present studies were conducted in order to develop more successful models for the study of glucocorticoid involvement in immune changes of malnourished animals. Appropriate models for the study of such interactions among physiological systems are not always clear. It may be necessary to combine a variety of approaches since any perturbation of one neuroendocrine circuit will also affect others. Two models designed to separate undemutrJtion from elevated serum corticosterone levels were developed. In the first, adrenalectomized mice were implanted with corticosterone-containing pellets in an effort to provide enough of the hormone to ensure survival during severe feed restriction. In the second model, mice were fed restricted quantities of food in order to induce loss of 20-30% of their initial weight. Immune functions were assayed during the phase of weight loss, after the low weight had been maintained for two weeks, or during recovery from undernutrition. The rationale for this indirect dietary method of influencing serum cortioosterone level was that, since the hormone is needed for tissue catabolism, animals which have lost weight but are weight stable or regaining weight should have lower serum corticosterone levels than equivalently undernourished animals which are in the process of losing weight. These two models were used to test the hypothesis that elevated serum corticosterone levels contribute to the impaired immune function in undernourished mice. The immune functions chosen were lymphoblastogenesis since the effects of giucocorticoids on this response are well-documented (10), and an in vivo antibody response as an overall measure of the ability of mice to respond effectively to antigen challenge. METHODS AND MATERIALS Two experiments were conducted using young (4-5 weeks old) female, outbred ICR mice (Harlan, Indianapolis, IN). Mice were individually housed in hanging wire cages in a room maintained at 24~ with a 12/12 light-dark cycle (lights on 6:00 a.m.). For 1-2 weeks after arrival in the facility, the animals were acclimated to the powdered semipurified diet (AIN-76A with 50% starch, 50% glucose as the carbohydrate source - U.S.
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Biochemicals, Cleveland, OH). Sample sizes varied among experimental groups and are indicated in tables and figures. Experiment 1. Three to four days before the end of the acclimation period, mice were anesthetized with Penthrane (Abbott Laboratories, North Chicago, IL), and subjected to either bilateral adrenalectomy through a dorsal incision or sham operation. Half of the animals from each of these groups were implanted subcutaneously in the incision with a pellet weighing approximately 10 mg containing 30% corticosterone in cholesterol and the other half received cholesterol (vehicle) pellets (17). Preliminary work indicated that this level of corticosterone in the pellet would restore serum corticosterone to approximately normal levels in adrenalectomized mice, although without the normal circadian variation. All adrenalectomized mice were given 1% NaCI in their drinking water. At the end of the acclimation period, these four groups of mice were further subdivided into two dietary groups, one fed the control AIN-76A diet ad libitum (group C) and the other fed the same diet in restricted amounts such that they lost 15% of their initial bocly weight over a 10-day feeding period. The short feeding period was chosen since preliminary work indicated that a high proportion (about 75%) of adrenalectomized mice, undernourished to induce this rate of weight loss, died after about 14 days food restriction. Mouse weights and food intakes were measured daily. Feed-restricted animals were given their daily dietary allotment at 5:00 p.m., shortly before the beginning of the dark period, in order to minimize changes in circadian rhythms due to imposition of meal-feeding. Feeding at the beginning of the dark period is reported to increase the amplitude but not disrupt the periodicity of the cycle of serum corticosterone levels (18). At the end of the 10-day experimental period, at 8:00 a.m., mice were rapidly killed by decapitation in a separate room (within 20 seconds of disturbing a cage). All 15-20 animals sacrificed on a given day, were killed within 30 minutes in an effort to minimize both circadian changes and stress-induced increases in serum corticosterone levels. At this time of day, corticosterone levels in animals with an intact hypothalamic-pituitaryadrenal (HPA) axis would have been near their minimum, whereas mice with corticosterone pellets would presumably have had a constant hormone level throughout the day. Although efforts were made to enter the animal room at random times throughout the experimental period, there was still the likelihood that mice had learned to associate the entrance of research personnel into the room with feeding, which might have caused increases in corticosterone. Experiment 2. This experiment was designed to investigate the effect of the direction of change of nutritional status on immune system measures and serum corticosterone levels. Mice were fed the AIN-76A diet for 4 weeks according to one of four protocols: ad libitum (group C); to maintain initial weight for 2 weeks and then lose 20% of their weight over the following 2 weeks (group MR); to lose 20% of their weight for 2 weeks and then maintain this low weight for 2 weeks (group RM); and to lose 30% of their initial weight over 3 weeks and for the last week to regain weight up to 20% below initial weight (group RG). This degree of weight loss was chosen as the maximum likely to be achieved without inducing a high mortality rate, particularly in group RG. Other procedures were as for Experiment 1. Measures of Nutritional Status and Serum Corticosterone Levels. Serum albumin was measured by the bromcresol green method using a commercial kit (Sigma, St. Louis, MO). Carcass lipid composition was determined in a proportion of the animals in Experiment 2. Dry matter content was calculated after lyophilization and oven-drying of carcasses. After drying, percent lipid was determined by the method of Bligh and Dyer (19). Serum corticosterone levels in trunk blood samples were measured using an RIA kit from ICN (Carson, CA) with 1251-1abelledcorticosterone as the tracer.
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Lymphocyte Blastogenic Transformation. Splenocyte suspensions were obtained by mechanical agitation of individual spleens in 10 ml of RPMI-1640 (Gibco, Grand Island, NY) using a Stomacher blender (Tekmar, Cincinnati, OH). The resultant cell preparation was washed twice with RPMI-1640. Spleen cells were seeded at 2.5x105 cells/well in RPMI-1640 containing 10% fetal bovine serum, 100 units/ml penicillin, 100 l.tg/ml streptomycin, and 5x10-5M 2-mercaptoethanol, (all from Gibco), in 96-well fiat-bottomed microtiter plates. Concanavalin A (Con A) or phytohemagglutinin (PHA) (Sigma, St. Louis, MO) was added to a final concentration of 0, 2.5, 5.0, or 10.0 I~g/ml. Tritiated thymidine (0.5 ~Ci/well) was added to the wells for the final 6 hours of a 72-hour incubation at 37~ and 5% CO2. Cells were harvested and thymidine incorporation was measured by liquid scintillation. Three replicates were tested for each sample at each of the different mitogen concentrations. In Vivo Antibody Response to Sheep Red Blood Cells (SRBC). A proportion of the mice were immunized five days before the end of the feeding period with an intraperitoneal injection of 4x10 s SRBC in 0.1 ml saline. Serum primary hemagglutinin titres were determined by standard methods and results are presented as the reciprocal of the base 2 log of the maximum dilution exhibiting spreading of SRBC. Statistics. Each experiment was run in three separate blocks and the block was included as a significant covariate in the analyses of variance. Where necessary, logarithmic or square root transformations were performed on data before analysis of variance in order to normalize residuals. Where data could not be so normalized, the Kruskal-Wallis non-parametric test was used. Data from Experiment 1 was analyzed by 4 X 2 ANOVA followed by comparison of group least squares means by t-test (20). Only a oriori comparisons were made, that is between sham-operated, cholesterol pellet-implanted animals of the two dietary groups, between sham-operated and adrenalectomized animals within each dietary group, and between animals with corticosterone and cholesterol pellets within each dietary and surgical operation group. Experiment 2 was analyzed by one-way ANOVA and group means were compared by Duncan's multiple range test. A 5% level of significance was used for all comparisons. Blastogenic responses were analyzed using a repeated measures test (20) since it was considered appropriate to consider all concentrations of a mitogen together. Differences between dietary groups within a mitogen level were determined by t-test. RESULTS Experiment 1 Animal Performance. Table 1 shows body and organ weights, food intakes and serum corticosterone levels of mice in Experiment 1. Initial body weight of adrenalectomized plus cholesterol animals was slightly larger than the others, presumably because the few animals who died as a result of the operation were smaller than most. Final body weight was decreased by food restriction and there was also a significant effect of HPA axis treatment, reflecting the differences in initial weights. Food intake over the 10-day feeding period was not affected by HPA manipulation. Spleen and thymus weights were expressed per gram body weight in order to facilitate comparisons between groups of animals of differing body weights. Spleen and thymus weight per gram body weight were significantly affected by HPA axis but not dietary treatment. Adrenalectomy increased weights of both organs in both dietary groups. Compared to cholesterol pellet implantation, 30% corticosterone pellets tended to lower thymus and spleen weights in both sham-operated and adrenalectomized mice.
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TABLE 1 Experiment 1: Body and Organ Weights, Food Intakes and Serum Corticosterone Levels Group*
N
Initial Weight (g)
C, sham, chol C, sham, cort C, adx, chol C, adx, cort R, sham, chol R, sham, cort R, adx, chol R, adx, cort
11 10 8 11 11 10 12 11
22.2 21.3 23.4 21.8 20.6 21.8 23.6 b 22.2
Error mean square
+ # a b c
3.6601
Final Weight (g)
Food Soleen Wt. Thvmus Wt. Intake Body Wt. Body Wt. (g/10 days) (mg/g) (mg/g)
24.1 23.6 25.4 24.4 17.9 a 18.6 20.5 b 19.0 4.5063
37.9 37.9 39.9 41.2 19.3 a 20.6 19.0 21.0 c
3.06 2.23 c 4.79 b 2.24 c 2.87 2.21 4.48 b 2.51 c
0.00239 + 0.5753
2.83 2.20 4.24 b 2.25 c 2.43 1.64c 4.07 b 2.01 c 0.7723
Serum Corticosterone (ng/ml) 120 124 34 b 116 c 141 200 29 b 123 c 19.713 #
C=ad libitum fed; R=restricted diet; sham=sham-operated; adx=adrenalectomized; chol=cholesterol pellet; cort=30% corticosterone pellet. From ANOVA using log transformed data. Values in table are antilogs of log means. From ANOVA using square root transformed data. Values in table are squares of square root means. R, sham, chol different from C, sham, chol. Adx, chol different from sham, chol of same dietary group. Different from cholesterol supplemented of same dietary and HPA axis treatment group.
Serum Corticosterone Levels. Serum corticosterone level did not differ between well-nourished and undernourished sham-operated plus cholesterol pellet mice. Adrenalectomy lowered corticosterone level and this was significantly increased to within the normal range by 30% corticosterone pellets in adrenalectomized animals. The type of pellet had no effect on hormone levels of sham-operated mice. The levels of corticosterone found in both experiments were relatively high compared to those normally found in rodents early in the light period (21), suggesting that animals may have been stressed in spite of precautions taken to minimize stress during sacrifice (see Methods). However, in neither experiment was there a significant correlation within a treatment groups between serum corticosterone and the order animals were killed. Technical problems with the sacrifice procedure or corticosterone assay were unlikely to have caused the high values since in our laboratory using identical procedures, previously untouched Sprague-Dawley rats had normal, unstressed corticosterone levels (20 _ 14 mg/ml, mean :!: SD, n=8). We believe the high levels found regularly in our nutrition
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S.M. FILTEAU et al.
experiments result from the animals' learning to associate the entrance of research personnel into the animal room with meal times. Immune Function Measures. Neither dietary treatment nor HPA manipulation significantly affected the splenic proliferative response to PHA (Figure 1). For Con A, these effects were not significant in the analysis of variance although several individual group means were different by t-test. In particular, at the two higher Con A concentrations, adrenalectomy lowered the response of well-nourished mice, and corticosterone pellets increased the response of undernourished, sham-operated mice, and at 101~g/ml Con A, undernutrition decreased the response. Food restriction slightly but significantly lowered serum anti-SRBC hemaggtutinin titre when animals with all HPA manipulations were grouped but HPA axis treatment had no effect. Individual treatment group means did not differ significantly (Figure 2). There were no significant correlations between serum corticosterone level and any immune response within any group.
120000
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C,Adx,cort
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0 0
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FIG. 1. Experiment I. Splenic proliferative responses to mitogens. C=control diet, R=restricted diet, Adx=adrenalectomized, sham=shamoperated, chol=cholesterol pellet. Sample size is 6-12 per data point. Between group error mean squares from ANOVA on log-transformed data were: Con A, 1.8959; PHA, 0.8914. At Con A=5 or 10p.g/ml C, sham, chol was greater than C, Adx, chol and R, sham, cort was greater than R, sham, chol. At con A=101~g/ml only, C, sham, chol was greater than R, sham, chol. T-tests, p<0.05.
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12. 9
=__=
10, []
t~
sham,chol sham,cort adx,chol adx.cort
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FIG. 2. Experiment 1. Antibody responses to sheep red blood cells. Sham,chol=sham-operated + cholesterol; sham,cort=sham-operated + corticosterone; Adx,chol=adrenalectom ized + cholesterol; Adx,cort=adrenalectomized + corticosterone. Sample size is 8-12 per group. Analysis by Kruskal-Wallis non-parametric test indicated no significant differences. Experiment 2 Animal Performance. One mouse from group RG died during the feeding period and the occasional animal in groups MR and RG was found on autopsy to exhibit gastrointestinal bleeding. Figure 3 shows body weight changes and cumulative weekly food intakes over the course of the feeding period. There were no significant differences in final body weights of the three undernourished groups, although MR mice were slightly smaller than RM or RG animals. Cumulative food intakes of groups RM and RG were not different except after 3 weeks and were always lower than intakes of groups C and MR.
TABLE 2 Experiment 2: Organ Weights, Carcass Lipid Content, Serum Albumin and Corticosterone Levels* Dietary Group
C MR FIG RM
~ZIJ~.J3_VY_~ Body Weight (rag/g) 2.97a,b (22) 2.54 b (23) 3.50 a (24) 2.98a,b(23)
Thvmus Weight Carcass Bc)dy Weight Lipid (rag/g) (% of carcass wet weight) 2.41 a 1.25 b 1.40 b 1.52 b
(22) (23) (24) (22)
30.3 a 9.3 b 10.8 b 9.8b
(7) (9) (8) (9)
Serum Albumin (g/di)
Serum Corticosterone (ng/ml)
3.9 a 3.8 a 4.5 a 3.9a
184a 449 b 198 a 279a
(10) (10) (10) (10)
(22) (22) (24) (23)
0.9569 0.3537 24.462 0.6808 32903 Error Mean Square Mean (number of mice) a,b Values not followed by the same superscript are significantly different, Duncan's multiple range test or KruskaI-Wallis test (SRBC titres), p<0.05.
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S.M. FILTEAU et al.
3~l
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_
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~
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~
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Weight 15
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-~. FM mI.M~ X'RG
I
9
80
Cumulative 6 0 Food Intake 40 (g)
0 Xr 0
1 I I 7 14 21 Days on Experimental Diets
I 28
FIG. 3. Experiment 2. Body weights and cumulative food intakes. Sample size is 22-24 per data point. Error mean squares from the analyses for weight are: 3.732, 3.245, 3.376, 3.614 and 3.965, and for food intake are: --, 3.719, 10.68, 19.09 and 30.63 at days 0, 7, 14, 21 and 28, respectively. Other animal performance data is shown in Table 2. When spleen and thymus weights were expressed per gram body weight, thymic indices did not differ among malnourished groups and mean splenic index of MR mice was significantly lower than that of RG mice only. Percent carcass lipid was decreased by feed restriction but the three undernourished groups did not differ among themselves. There was no effect of dietary treatment on serum albumin levels. MR mice had higher serum corticosterone levels than did mice of the other three dietary groups which did not differ among themselves. Immune Function Measures. Serum antibody response to SRBC was lower in group RG than in the other three dietary groups (Figure 4). Blastogenic responses to Con A and PHA did not differ among groups (Figure 5). Within a dietary group, serum corticosterone level was not significantly correlated with any of the immune function m eas ures.
CORTICOSTERONE AND IMMUNITY
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12 10
c:A 8
:30
m~
4
C
IVR R3 Dietary Group
FM
FIG. 4. Experiment 2. Antibody responses to sheep red blood cells. Sample size is 11-12 per group. Group RG has significantly lower titre than the other groups by KruskaI-Wallis non-parametric test, p<0.05. 80000 70000 60000 A
" o ,.~ r
E*"
"~
50000
40000 3OO00 20000 -
4
6
8
Con A Concentration
12
1 (pg/ml)
C 56o00 "=-
4oooo,
MR RG RM
30000.
I--
20000,
lOOOO 9
o
, 2
= 4 PHA
,'
i
,
6 Concentration
= 8
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i 10
12
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FIG. 5. Experiment 2. Splenic proliferative responses to mitogens. Sample size is 17-22 per data point. Between group error mean squares from ANOVA on log-transformed data were: Con A, 1.011; PHA, .7997. There were no significant differences among dietary groups.
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S.M. FILTEAU et al. DISCUSSION
Results from the two models used suggest that elevated glucocorticoids are not a major contributing factor to the immunodepression of PEM. In no experimental group were there found significant correlations between immune function measures and serum corticosterone levels. In Experiment 2 involving the manipulation of the direction of change of nutritional status, animals which were losing weight at the time of sacrifice had higher serum corticosterone levels than equivalently undernourished mice which were either maintaining or regaining weight. However, an inverse relationship between group mean hormone levels and immune functions was not seen. All malnourished groups had normal blastogenic responses in spite of the elevation of serum corticosterone in group MR, and only group RG, which had normal serum corticosterone level, had a decreased antibody response. In Experiment 1, the significant effect of HPA axis treatment on serum corticosterone was not accompanied by reciprocal differences in any immune functions. HPA manipulations which significantly affected corticosterone levels did not influence the response to SRBC, and the few differences in Con A responses were opposite to those which would be expected if corticosterone were suppressive. Spleen and thymus weight decreases in undernutrition could be accounted for by the decreased body weight. The effects of adrenalectomy and corticosterone supplementation on these organ weights were similar in both well-nourished and undernourished mice, suggesting corticosterone influences on lymphoid organ sizes were not limited to the malnourished state. Previous work on the subject has not always included adrenalectomized, well-nourished animals (11, 13), but where such controls were included (12), similar effects of adrenalectomy on lymphocyte numbers in well-nourished and undernourished mice were seen. An important aim of this work was to develop models to separate nutritional status from serum corticosterone level. The degree of success of the two types of models used affects conclusions drawn about the involvement of corticosterone in the immune dysfunction associated with severe undernutrition. Manipulation of the direction of change of nutritional status resulted in groups of equivalently undernourished mice with different serum corticosterone levels. Based on body weight, carcass lipid, and serum albumin levels, nutritional status of RM, MR and RG mice in Experiment 2 did not differ. In preliminary unpublished work with a similar model, carcass protein content also did not differ among these malnourished groups. MR mice, which were currently losing weight and presumably dependent on elevated serum corticosterone levels for catabolism of muscle and adipose tissue, had higher hormone levels than did the other undernourished groups which were maintaining or regaining weight at the time of sacrifice. Serum corticosterone levels of RM and RG mice were not different from those of well-nourished controls. Of course, it is likely that many other differences, including differences in levels of other hormones as well as changes in the autonomic nervous system, exist among groups of undernourished mice which are losing, gaining or maintaining weight. Direct manipulation of serum corticosterone by adrenalectomy or hormone supplementation was less successful as a means of separating severe undernutrition from increased serum corticosterone level. In Experiment 1 a relatively short, moderate undemutrition protocol was followed since preliminary work indicated a high mortality when adrenalectomized mice were undernourished for a longer period. Furthermore, those adrenalectomized mice which survived the longer (3 week) undernutrition were found to have serum corticosterone levels not different from those of well-nourished sham-operated mice (unpublished results). Although incomplete adrenalectomy remains a possibility in some cases, adrenal tissue was not found upon visual inspection at autopsy. It is likely that the increase in serum corticosterone with time after operation reflects the ability of adrenalectomized rodents to develop extra-adrenal tissue capable of
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corticosterone synthesis (22, 23). Presumably those animals which died in the preliminary work were those which were unable to develop such tissue since it was noted they did not lose much weight even when severely food-restricted. The shorter feeding period designed to avoid these problems unfortunately induced neither immunodepression nor increased serum corticosterone. It appears that the need for glucocorticoids for tissue catabolism during undernutrition makes it difficult to use an adrenalectomy model to separate undernutrition from elevated glucocorticoids in order to study glucocorticoid involvement in the immunodepression of PEM. Investigation of other immune functions using these models would be important since those used in the present work suffer certain drawbacks. The in vitro blastogenic assays are subject to great variability making significant differences elusive. In addition, a period of culture in nutritionally complete medium might reverse any nutritional deficiencies the lymphocytes suffered within the animals and would also tend to abolish any hormonal differences that existed between the groups in vivo. In preliminary work a similar lack of group differences was found for in vitro interferon production. That the culture period might, in fact, allow reversal of immunodepression is suggested by the presence in both experiments of groups of animals exhibiting low in vivo responses to SRBC but normal in vitro blastogenic responses. Blastogenic responses thus cannot be considered to represent immune system potential and were included in the present work since they are known to vary inversely with the level of glucocorticoids (24) and the degree of stress (25). In other work, using an assay which involved only 3 hours of in vitro leukocyte culture, we have found no depression in peritoneal macrophage interleukin-1 and tumor necrosis factor production in undernourished mice exhibiting increased serum corticosterone levels (14). Although use of an in vivo antibody response circumvents the in vitro problems, interpretation of glucocorticoid effects on the antibody response to SRBC in the present work is difficult since serum corticosterone levels were not measured at the time of immunization and it is not clear that hormone levels at sacrifice 5 days later reflect those at relevant points during the in vivo response. In particular, the depressed antibody response of RG mice in Experiment 3 may be explained by the fact that at the time of immunization these animals generally were at their body weight nadir and had not started regaining weight. The elevated serum corticosterone levels in severely undernourished animals (Experiment 2) can be viewed as part of the general response to a stressor, as well as being important for tissue catabolism. Although acute stress has been shown to depress immune functions, for example, mitogen responses (10, 25), such depressions are not always seen in chronic stress (10). In addition, the inverse relationship between serum corticosterone and blastogenic response after acute stress was not seen with a chronic stress protocol (10). The undernutrition models in the present work can be considered as examples of chronic stress, which may explain the general lack of reciprocal relationship between glucocorticoid levels and immune functions. Possibly, adaptations to a chronic stressor occur at the level of tissue glucocorticoid receptors or in post-receptor processes. In conclusion, the present work shows that neuroendocrine function, particularly as it relates to glucocorticoids, can be altered in undernutrition by altering the direction of change of nutritional status. Undernourished animals which are currently either maintaining or regaining weight do not have elevated corticosterone levels. This procedure appears more promising for studying glucocorticoid involvement in immune function in undernutrition than the more direct method of adrenalectomy. In general, the results suggest that, contrary to what has been suggested (7-9), elevated glucocorticoids may not be a major factor in malnutrition-induced immune impairments. PEM appears to be a type of chronic stressor, in which, unlike acute stressors, immunosuppressive effects of glucocorticoids are not seen (10).
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S.M. FILTEAU et al. ACKNOWLEDGEMENTS
The authors are grateful for the excellent technical assistance provided by Angela Adams, Meg Davis, Vera Peacock and Rod Stokes; and for manuscript preparation by Cecilia Figueredo and Julie Anderson, This work was supported by the National Institute of Mental Health (RO3 MH45646-01), a grant from the Paul P. Dosberg Foundation, Inc., and a Medical Research Council of Canada postdoctoral fellowship awarded to S.M.F. Nicholas Hall is supported by a RSDA (DA00158). We also wish to acknowledge the James A. Haley Veterans Hospital, Tampa, Florida, for the use of its vivarium.
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Schonland MM, Shanley BC, Loening WEK, Parent MA, Coovadia HM. Plasma cortisol and immunosuppression in protein-calorie malnutrition. Lancet 1972; 2:435-436. Watson RR. Stress caused by dietary changes: corticosteroid production, a partial explanation for immunosuppression in the malnourished. In: R.R. Watson, (Ed.), Nutrition, Disease Resistance and Immune Function, pp. 273-283, Marcel Dekker: New York & Basel, 1984.
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12. Woodruff JF.
The influence of quantitated post-weaning undernutrition on coxsackievirus B3 infection of adult mice. I1. Alteration of host defense mechanisms. J Infect Dis 1970; 121: 164-181.
13. Bell RG, Hazell LA, Price P. Influence of dietary protein restriction on immune competence. I1. Effect on lymphoid tissue. Clin Exp Immunol 1976; 26:314-326. 14. Filteau SM, Hall NRS. Increased production of tumor necrosis factor and interleukin 1 by peritoneal macrophages from severely undernourished mice: Lack of correlation with serum corticosterone. Nutr Res 1991; 11:1001-1011. 15. Filteau SM, Menzies RA, Kaido TJ, O'Grady MP, Gelderd JB, Hall NRS. Effects of exercise on immune functions of undernourished mice. Life Sci 1992; 51: 564574. 16. Nakamura K, Aoike A, Hosokawa K, Rokutan K, Koyama K, Nishi Y, Yoshida A,
Kawai K. Effect of food-restriction stress on immune response in mice. Neuroimmunol 1990; 30:23-29.
J
17. Akana SF, Cascio CS, Shinsalco J, Dallman MF. Corticosterone: narrow range required for normal body and thymus weight and ACTH. Am J Physiol 1985; 249:R527-R532. 18. Nelson W. Food restriction, circadian disorder and longevity of rats and mice. J Nutr 1986; 118:284-289. 19. Bligh EG, Dyer WJ. A rapid method of total lipid extraction and purification. Biochem Physiol 1959; 37:911-917.
Can J
20. SAS Institute, Inc. SAS User's Guide: Statistics, 5th ed., SAS Institute: Cary, N.C., 1985. 21. Gomez-Sanchez C, Murry BA, Kern DC, Kaplan NM. A direct radioimmunoassay of corticosterone in rat serum. Endocrinol 1975; 96: 796-798. 22. Gaunt R. Adrenalectomy in the rat. Am J Physiol 1933; 103: 494-510. 23. Hummel KP. Accessory adrenal cortical nodules in the mouse. Anat Rec 1958; 132: 281-291. 24. Bowen DL, Fauci AS. Selective suppressive effects of glucocorticoids on the early events in the human B cell activation process. J Immunol 1984; 133: 1885-1890. 25. Keller SE, Weiss JM, Schleifer SJ, Miller NE, M Stein. Suppression of immunity by stress: Effect of a graded series of stressors on lymphocyte stimulation in the rat. Science 1981; 213: 1397-1400. Accepted for publication April 21, 1993.
LACK OF CORRELATION BETWEEN SERUM CORTICOSTERONE LEVEL AND IMMUNE FUNCTIONS IN UNDERNOURISHED MICE S. M. Filteau, Ph.D., T. J. Kaido, Ph.D., Maureen P. O'Grady, Ph.D., Robert A. Menzies, Ph.D. and Nicholas R. S. Hall, Ph.D. Department of Psychiatry and Behavioral Medicine, University of South Florida College of Medicine, Tampa, Florida, USA
ABSTRACT Two models were developed in order to investigate the importance of elevated serum glucocorticoid levels in the immune dysfunction associated with protein energy malnutrition. Weight loss was induced in mice by restricting intake of a nutritionally complete diet. in one model, serum corticosterone levels of these mice were manipulated by adrenalectomy with or without a minimal maintenance level of corticosterone supplementation. This hormone supplementation was included in some animals in an effort to reduce mortality. In the second model, the amount of diet given was adjusted to produce groups of mice which were equivalently undernourished but which were either losing, maintaining or gaining weight at the time of sacrifice. Elevated serum corticosterone levels were seen only in the animals which were losing weight. Using these models, there was no consistent inverse relationship between serum corticosterone levels and blastogenic responses to mitogens, or in vivo antibody response to sheep red blood cells. The results suggest that elevated glucocorticoids may not be of major importance in the immunodepression of a chronic stress condition such as undernutrition. Key Words."
Protein energy malnutrition, Stress, Corticosterone, Immunity INTRODUCTION
In its severe forms, protein-energy malnutrition (PEM) is associated with impairments in immune function (1). The underlying cause of this immunodepression has Corresponding Author: S. M. Filteau, Ph.D. Department of International Child Health Institute of Child Health 30 Guilford Street, London, WCIN IEH, U.K. 071-242-9789
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usually been implied to be a decreased availability of amino acids or energy substrates (2) or a lack of micronutrients, such as zinc (3, 4). An additional mechanism of immunodepression, resulting from adaptive hormonal changes associated with PEM, has also been hypothesized (5, 7-9). PEM can be considered as an example of a stressor. Elevated glucocorticoid levels have been implicated in stress-induced immunodepression (10) and have frequently been cited as contributing to the immunodepression of PEM (7-9). However, direct supporting evidence for the importance of glucocorticoids in malnutrition-induced immune system changes is limited to effects on lymphoid organ size and lymphocyte numbers. Adrenalectomized rodents had less of a decrease in blood lymphocyte numbers (11, 12) and lymphoid organ weights (13) when undernourished, that is, feedrestricted (12), or protein-deprived (11, 13), than did sham-operated controls. The effect of prevention of increased serum glucocorticoid levels on immune functions and disease resistance of malnourished animals has not been extensively studied. However, work from our laboratory (14, 15) has indicated no correlation between serum corticosterone and several immune functions in undernourished mice, and similar results have been observed in acutely feed-restricted mice (16). Part of the difficulty in obtaining such information is that mortality in adrenalectomized, PEM rodents is very high (11, 12). Increased glucocorticoid levels are needed to mobilize muscle and adipose tissue to provide amino acids and energy substrates during undernutrition. An inability to mobilize these stores may be fatal. Therefore, the present studies were conducted in order to develop more successful models for the study of glucocorticoid involvement in immune changes of malnourished animals. Appropriate models for the study of such interactions among physiological systems are not always clear. It may be necessary to combine a variety of approaches since any perturbation of one neuroendocrine circuit will also affect others. Two models designed to separate undemutrJtion from elevated serum corticosterone levels were developed. In the first, adrenalectomized mice were implanted with corticosterone-containing pellets in an effort to provide enough of the hormone to ensure survival during severe feed restriction. In the second model, mice were fed restricted quantities of food in order to induce loss of 20-30% of their initial weight. Immune functions were assayed during the phase of weight loss, after the low weight had been maintained for two weeks, or during recovery from undernutrition. The rationale for this indirect dietary method of influencing serum cortioosterone level was that, since the hormone is needed for tissue catabolism, animals which have lost weight but are weight stable or regaining weight should have lower serum corticosterone levels than equivalently undernourished animals which are in the process of losing weight. These two models were used to test the hypothesis that elevated serum corticosterone levels contribute to the impaired immune function in undernourished mice. The immune functions chosen were lymphoblastogenesis since the effects of giucocorticoids on this response are well-documented (10), and an in vivo antibody response as an overall measure of the ability of mice to respond effectively to antigen challenge. METHODS AND MATERIALS Two experiments were conducted using young (4-5 weeks old) female, outbred ICR mice (Harlan, Indianapolis, IN). Mice were individually housed in hanging wire cages in a room maintained at 24~ with a 12/12 light-dark cycle (lights on 6:00 a.m.). For 1-2 weeks after arrival in the facility, the animals were acclimated to the powdered semipurified diet (AIN-76A with 50% starch, 50% glucose as the carbohydrate source - U.S.
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Biochemicals, Cleveland, OH). Sample sizes varied among experimental groups and are indicated in tables and figures. Experiment 1. Three to four days before the end of the acclimation period, mice were anesthetized with Penthrane (Abbott Laboratories, North Chicago, IL), and subjected to either bilateral adrenalectomy through a dorsal incision or sham operation. Half of the animals from each of these groups were implanted subcutaneously in the incision with a pellet weighing approximately 10 mg containing 30% corticosterone in cholesterol and the other half received cholesterol (vehicle) pellets (17). Preliminary work indicated that this level of corticosterone in the pellet would restore serum corticosterone to approximately normal levels in adrenalectomized mice, although without the normal circadian variation. All adrenalectomized mice were given 1% NaCI in their drinking water. At the end of the acclimation period, these four groups of mice were further subdivided into two dietary groups, one fed the control AIN-76A diet ad libitum (group C) and the other fed the same diet in restricted amounts such that they lost 15% of their initial bocly weight over a 10-day feeding period. The short feeding period was chosen since preliminary work indicated that a high proportion (about 75%) of adrenalectomized mice, undernourished to induce this rate of weight loss, died after about 14 days food restriction. Mouse weights and food intakes were measured daily. Feed-restricted animals were given their daily dietary allotment at 5:00 p.m., shortly before the beginning of the dark period, in order to minimize changes in circadian rhythms due to imposition of meal-feeding. Feeding at the beginning of the dark period is reported to increase the amplitude but not disrupt the periodicity of the cycle of serum corticosterone levels (18). At the end of the 10-day experimental period, at 8:00 a.m., mice were rapidly killed by decapitation in a separate room (within 20 seconds of disturbing a cage). All 15-20 animals sacrificed on a given day, were killed within 30 minutes in an effort to minimize both circadian changes and stress-induced increases in serum corticosterone levels. At this time of day, corticosterone levels in animals with an intact hypothalamic-pituitaryadrenal (HPA) axis would have been near their minimum, whereas mice with corticosterone pellets would presumably have had a constant hormone level throughout the day. Although efforts were made to enter the animal room at random times throughout the experimental period, there was still the likelihood that mice had learned to associate the entrance of research personnel into the room with feeding, which might have caused increases in corticosterone. Experiment 2. This experiment was designed to investigate the effect of the direction of change of nutritional status on immune system measures and serum corticosterone levels. Mice were fed the AIN-76A diet for 4 weeks according to one of four protocols: ad libitum (group C); to maintain initial weight for 2 weeks and then lose 20% of their weight over the following 2 weeks (group MR); to lose 20% of their weight for 2 weeks and then maintain this low weight for 2 weeks (group RM); and to lose 30% of their initial weight over 3 weeks and for the last week to regain weight up to 20% below initial weight (group RG). This degree of weight loss was chosen as the maximum likely to be achieved without inducing a high mortality rate, particularly in group RG. Other procedures were as for Experiment 1. Measures of Nutritional Status and Serum Corticosterone Levels. Serum albumin was measured by the bromcresol green method using a commercial kit (Sigma, St. Louis, MO). Carcass lipid composition was determined in a proportion of the animals in Experiment 2. Dry matter content was calculated after lyophilization and oven-drying of carcasses. After drying, percent lipid was determined by the method of Bligh and Dyer (19). Serum corticosterone levels in trunk blood samples were measured using an RIA kit from ICN (Carson, CA) with 1251-1abelledcorticosterone as the tracer.
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Lymphocyte Blastogenic Transformation. Splenocyte suspensions were obtained by mechanical agitation of individual spleens in 10 ml of RPMI-1640 (Gibco, Grand Island, NY) using a Stomacher blender (Tekmar, Cincinnati, OH). The resultant cell preparation was washed twice with RPMI-1640. Spleen cells were seeded at 2.5x105 cells/well in RPMI-1640 containing 10% fetal bovine serum, 100 units/ml penicillin, 100 l.tg/ml streptomycin, and 5x10-5M 2-mercaptoethanol, (all from Gibco), in 96-well fiat-bottomed microtiter plates. Concanavalin A (Con A) or phytohemagglutinin (PHA) (Sigma, St. Louis, MO) was added to a final concentration of 0, 2.5, 5.0, or 10.0 I~g/ml. Tritiated thymidine (0.5 ~Ci/well) was added to the wells for the final 6 hours of a 72-hour incubation at 37~ and 5% CO2. Cells were harvested and thymidine incorporation was measured by liquid scintillation. Three replicates were tested for each sample at each of the different mitogen concentrations. In Vivo Antibody Response to Sheep Red Blood Cells (SRBC). A proportion of the mice were immunized five days before the end of the feeding period with an intraperitoneal injection of 4x10 s SRBC in 0.1 ml saline. Serum primary hemagglutinin titres were determined by standard methods and results are presented as the reciprocal of the base 2 log of the maximum dilution exhibiting spreading of SRBC. Statistics. Each experiment was run in three separate blocks and the block was included as a significant covariate in the analyses of variance. Where necessary, logarithmic or square root transformations were performed on data before analysis of variance in order to normalize residuals. Where data could not be so normalized, the Kruskal-Wallis non-parametric test was used. Data from Experiment 1 was analyzed by 4 X 2 ANOVA followed by comparison of group least squares means by t-test (20). Only a oriori comparisons were made, that is between sham-operated, cholesterol pellet-implanted animals of the two dietary groups, between sham-operated and adrenalectomized animals within each dietary group, and between animals with corticosterone and cholesterol pellets within each dietary and surgical operation group. Experiment 2 was analyzed by one-way ANOVA and group means were compared by Duncan's multiple range test. A 5% level of significance was used for all comparisons. Blastogenic responses were analyzed using a repeated measures test (20) since it was considered appropriate to consider all concentrations of a mitogen together. Differences between dietary groups within a mitogen level were determined by t-test. RESULTS Experiment 1 Animal Performance. Table 1 shows body and organ weights, food intakes and serum corticosterone levels of mice in Experiment 1. Initial body weight of adrenalectomized plus cholesterol animals was slightly larger than the others, presumably because the few animals who died as a result of the operation were smaller than most. Final body weight was decreased by food restriction and there was also a significant effect of HPA axis treatment, reflecting the differences in initial weights. Food intake over the 10-day feeding period was not affected by HPA manipulation. Spleen and thymus weights were expressed per gram body weight in order to facilitate comparisons between groups of animals of differing body weights. Spleen and thymus weight per gram body weight were significantly affected by HPA axis but not dietary treatment. Adrenalectomy increased weights of both organs in both dietary groups. Compared to cholesterol pellet implantation, 30% corticosterone pellets tended to lower thymus and spleen weights in both sham-operated and adrenalectomized mice.
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TABLE 1 Experiment 1: Body and Organ Weights, Food Intakes and Serum Corticosterone Levels Group*
N
Initial Weight (g)
C, sham, chol C, sham, cort C, adx, chol C, adx, cort R, sham, chol R, sham, cort R, adx, chol R, adx, cort
11 10 8 11 11 10 12 11
22.2 21.3 23.4 21.8 20.6 21.8 23.6 b 22.2
Error mean square
+ # a b c
3.6601
Final Weight (g)
Food Soleen Wt. Thvmus Wt. Intake Body Wt. Body Wt. (g/10 days) (mg/g) (mg/g)
24.1 23.6 25.4 24.4 17.9 a 18.6 20.5 b 19.0 4.5063
37.9 37.9 39.9 41.2 19.3 a 20.6 19.0 21.0 c
3.06 2.23 c 4.79 b 2.24 c 2.87 2.21 4.48 b 2.51 c
0.00239 + 0.5753
2.83 2.20 4.24 b 2.25 c 2.43 1.64c 4.07 b 2.01 c 0.7723
Serum Corticosterone (ng/ml) 120 124 34 b 116 c 141 200 29 b 123 c 19.713 #
C=ad libitum fed; R=restricted diet; sham=sham-operated; adx=adrenalectomized; chol=cholesterol pellet; cort=30% corticosterone pellet. From ANOVA using log transformed data. Values in table are antilogs of log means. From ANOVA using square root transformed data. Values in table are squares of square root means. R, sham, chol different from C, sham, chol. Adx, chol different from sham, chol of same dietary group. Different from cholesterol supplemented of same dietary and HPA axis treatment group.
Serum Corticosterone Levels. Serum corticosterone level did not differ between well-nourished and undernourished sham-operated plus cholesterol pellet mice. Adrenalectomy lowered corticosterone level and this was significantly increased to within the normal range by 30% corticosterone pellets in adrenalectomized animals. The type of pellet had no effect on hormone levels of sham-operated mice. The levels of corticosterone found in both experiments were relatively high compared to those normally found in rodents early in the light period (21), suggesting that animals may have been stressed in spite of precautions taken to minimize stress during sacrifice (see Methods). However, in neither experiment was there a significant correlation within a treatment groups between serum corticosterone and the order animals were killed. Technical problems with the sacrifice procedure or corticosterone assay were unlikely to have caused the high values since in our laboratory using identical procedures, previously untouched Sprague-Dawley rats had normal, unstressed corticosterone levels (20 _ 14 mg/ml, mean :!: SD, n=8). We believe the high levels found regularly in our nutrition
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experiments result from the animals' learning to associate the entrance of research personnel into the animal room with meal times. Immune Function Measures. Neither dietary treatment nor HPA manipulation significantly affected the splenic proliferative response to PHA (Figure 1). For Con A, these effects were not significant in the analysis of variance although several individual group means were different by t-test. In particular, at the two higher Con A concentrations, adrenalectomy lowered the response of well-nourished mice, and corticosterone pellets increased the response of undernourished, sham-operated mice, and at 101~g/ml Con A, undernutrition decreased the response. Food restriction slightly but significantly lowered serum anti-SRBC hemaggtutinin titre when animals with all HPA manipulations were grouped but HPA axis treatment had no effect. Individual treatment group means did not differ significantly (Figure 2). There were no significant correlations between serum corticosterone level and any immune response within any group.
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FIG. 1. Experiment I. Splenic proliferative responses to mitogens. C=control diet, R=restricted diet, Adx=adrenalectomized, sham=shamoperated, chol=cholesterol pellet. Sample size is 6-12 per data point. Between group error mean squares from ANOVA on log-transformed data were: Con A, 1.8959; PHA, 0.8914. At Con A=5 or 10p.g/ml C, sham, chol was greater than C, Adx, chol and R, sham, cort was greater than R, sham, chol. At con A=101~g/ml only, C, sham, chol was greater than R, sham, chol. T-tests, p<0.05.
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12. 9
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FIG. 2. Experiment 1. Antibody responses to sheep red blood cells. Sham,chol=sham-operated + cholesterol; sham,cort=sham-operated + corticosterone; Adx,chol=adrenalectom ized + cholesterol; Adx,cort=adrenalectomized + corticosterone. Sample size is 8-12 per group. Analysis by Kruskal-Wallis non-parametric test indicated no significant differences. Experiment 2 Animal Performance. One mouse from group RG died during the feeding period and the occasional animal in groups MR and RG was found on autopsy to exhibit gastrointestinal bleeding. Figure 3 shows body weight changes and cumulative weekly food intakes over the course of the feeding period. There were no significant differences in final body weights of the three undernourished groups, although MR mice were slightly smaller than RM or RG animals. Cumulative food intakes of groups RM and RG were not different except after 3 weeks and were always lower than intakes of groups C and MR.
TABLE 2 Experiment 2: Organ Weights, Carcass Lipid Content, Serum Albumin and Corticosterone Levels* Dietary Group
C MR FIG RM
~ZIJ~.J3_VY_~ Body Weight (rag/g) 2.97a,b (22) 2.54 b (23) 3.50 a (24) 2.98a,b(23)
Thvmus Weight Carcass Bc)dy Weight Lipid (rag/g) (% of carcass wet weight) 2.41 a 1.25 b 1.40 b 1.52 b
(22) (23) (24) (22)
30.3 a 9.3 b 10.8 b 9.8b
(7) (9) (8) (9)
Serum Albumin (g/di)
Serum Corticosterone (ng/ml)
3.9 a 3.8 a 4.5 a 3.9a
184a 449 b 198 a 279a
(10) (10) (10) (10)
(22) (22) (24) (23)
0.9569 0.3537 24.462 0.6808 32903 Error Mean Square Mean (number of mice) a,b Values not followed by the same superscript are significantly different, Duncan's multiple range test or KruskaI-Wallis test (SRBC titres), p<0.05.
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S.M. FILTEAU et al.
3~l
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FIG. 3. Experiment 2. Body weights and cumulative food intakes. Sample size is 22-24 per data point. Error mean squares from the analyses for weight are: 3.732, 3.245, 3.376, 3.614 and 3.965, and for food intake are: --, 3.719, 10.68, 19.09 and 30.63 at days 0, 7, 14, 21 and 28, respectively. Other animal performance data is shown in Table 2. When spleen and thymus weights were expressed per gram body weight, thymic indices did not differ among malnourished groups and mean splenic index of MR mice was significantly lower than that of RG mice only. Percent carcass lipid was decreased by feed restriction but the three undernourished groups did not differ among themselves. There was no effect of dietary treatment on serum albumin levels. MR mice had higher serum corticosterone levels than did mice of the other three dietary groups which did not differ among themselves. Immune Function Measures. Serum antibody response to SRBC was lower in group RG than in the other three dietary groups (Figure 4). Blastogenic responses to Con A and PHA did not differ among groups (Figure 5). Within a dietary group, serum corticosterone level was not significantly correlated with any of the immune function m eas ures.
CORTICOSTERONE AND IMMUNITY
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12 10
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FIG. 4. Experiment 2. Antibody responses to sheep red blood cells. Sample size is 11-12 per group. Group RG has significantly lower titre than the other groups by KruskaI-Wallis non-parametric test, p<0.05. 80000 70000 60000 A
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FIG. 5. Experiment 2. Splenic proliferative responses to mitogens. Sample size is 17-22 per data point. Between group error mean squares from ANOVA on log-transformed data were: Con A, 1.011; PHA, .7997. There were no significant differences among dietary groups.
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S.M. FILTEAU et al. DISCUSSION
Results from the two models used suggest that elevated glucocorticoids are not a major contributing factor to the immunodepression of PEM. In no experimental group were there found significant correlations between immune function measures and serum corticosterone levels. In Experiment 2 involving the manipulation of the direction of change of nutritional status, animals which were losing weight at the time of sacrifice had higher serum corticosterone levels than equivalently undernourished mice which were either maintaining or regaining weight. However, an inverse relationship between group mean hormone levels and immune functions was not seen. All malnourished groups had normal blastogenic responses in spite of the elevation of serum corticosterone in group MR, and only group RG, which had normal serum corticosterone level, had a decreased antibody response. In Experiment 1, the significant effect of HPA axis treatment on serum corticosterone was not accompanied by reciprocal differences in any immune functions. HPA manipulations which significantly affected corticosterone levels did not influence the response to SRBC, and the few differences in Con A responses were opposite to those which would be expected if corticosterone were suppressive. Spleen and thymus weight decreases in undernutrition could be accounted for by the decreased body weight. The effects of adrenalectomy and corticosterone supplementation on these organ weights were similar in both well-nourished and undernourished mice, suggesting corticosterone influences on lymphoid organ sizes were not limited to the malnourished state. Previous work on the subject has not always included adrenalectomized, well-nourished animals (11, 13), but where such controls were included (12), similar effects of adrenalectomy on lymphocyte numbers in well-nourished and undernourished mice were seen. An important aim of this work was to develop models to separate nutritional status from serum corticosterone level. The degree of success of the two types of models used affects conclusions drawn about the involvement of corticosterone in the immune dysfunction associated with severe undernutrition. Manipulation of the direction of change of nutritional status resulted in groups of equivalently undernourished mice with different serum corticosterone levels. Based on body weight, carcass lipid, and serum albumin levels, nutritional status of RM, MR and RG mice in Experiment 2 did not differ. In preliminary unpublished work with a similar model, carcass protein content also did not differ among these malnourished groups. MR mice, which were currently losing weight and presumably dependent on elevated serum corticosterone levels for catabolism of muscle and adipose tissue, had higher hormone levels than did the other undernourished groups which were maintaining or regaining weight at the time of sacrifice. Serum corticosterone levels of RM and RG mice were not different from those of well-nourished controls. Of course, it is likely that many other differences, including differences in levels of other hormones as well as changes in the autonomic nervous system, exist among groups of undernourished mice which are losing, gaining or maintaining weight. Direct manipulation of serum corticosterone by adrenalectomy or hormone supplementation was less successful as a means of separating severe undernutrition from increased serum corticosterone level. In Experiment 1 a relatively short, moderate undemutrition protocol was followed since preliminary work indicated a high mortality when adrenalectomized mice were undernourished for a longer period. Furthermore, those adrenalectomized mice which survived the longer (3 week) undernutrition were found to have serum corticosterone levels not different from those of well-nourished sham-operated mice (unpublished results). Although incomplete adrenalectomy remains a possibility in some cases, adrenal tissue was not found upon visual inspection at autopsy. It is likely that the increase in serum corticosterone with time after operation reflects the ability of adrenalectomized rodents to develop extra-adrenal tissue capable of
CORTICOSTERONE AND IMMUNITY
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corticosterone synthesis (22, 23). Presumably those animals which died in the preliminary work were those which were unable to develop such tissue since it was noted they did not lose much weight even when severely food-restricted. The shorter feeding period designed to avoid these problems unfortunately induced neither immunodepression nor increased serum corticosterone. It appears that the need for glucocorticoids for tissue catabolism during undernutrition makes it difficult to use an adrenalectomy model to separate undernutrition from elevated glucocorticoids in order to study glucocorticoid involvement in the immunodepression of PEM. Investigation of other immune functions using these models would be important since those used in the present work suffer certain drawbacks. The in vitro blastogenic assays are subject to great variability making significant differences elusive. In addition, a period of culture in nutritionally complete medium might reverse any nutritional deficiencies the lymphocytes suffered within the animals and would also tend to abolish any hormonal differences that existed between the groups in vivo. In preliminary work a similar lack of group differences was found for in vitro interferon production. That the culture period might, in fact, allow reversal of immunodepression is suggested by the presence in both experiments of groups of animals exhibiting low in vivo responses to SRBC but normal in vitro blastogenic responses. Blastogenic responses thus cannot be considered to represent immune system potential and were included in the present work since they are known to vary inversely with the level of glucocorticoids (24) and the degree of stress (25). In other work, using an assay which involved only 3 hours of in vitro leukocyte culture, we have found no depression in peritoneal macrophage interleukin-1 and tumor necrosis factor production in undernourished mice exhibiting increased serum corticosterone levels (14). Although use of an in vivo antibody response circumvents the in vitro problems, interpretation of glucocorticoid effects on the antibody response to SRBC in the present work is difficult since serum corticosterone levels were not measured at the time of immunization and it is not clear that hormone levels at sacrifice 5 days later reflect those at relevant points during the in vivo response. In particular, the depressed antibody response of RG mice in Experiment 3 may be explained by the fact that at the time of immunization these animals generally were at their body weight nadir and had not started regaining weight. The elevated serum corticosterone levels in severely undernourished animals (Experiment 2) can be viewed as part of the general response to a stressor, as well as being important for tissue catabolism. Although acute stress has been shown to depress immune functions, for example, mitogen responses (10, 25), such depressions are not always seen in chronic stress (10). In addition, the inverse relationship between serum corticosterone and blastogenic response after acute stress was not seen with a chronic stress protocol (10). The undernutrition models in the present work can be considered as examples of chronic stress, which may explain the general lack of reciprocal relationship between glucocorticoid levels and immune functions. Possibly, adaptations to a chronic stressor occur at the level of tissue glucocorticoid receptors or in post-receptor processes. In conclusion, the present work shows that neuroendocrine function, particularly as it relates to glucocorticoids, can be altered in undernutrition by altering the direction of change of nutritional status. Undernourished animals which are currently either maintaining or regaining weight do not have elevated corticosterone levels. This procedure appears more promising for studying glucocorticoid involvement in immune function in undernutrition than the more direct method of adrenalectomy. In general, the results suggest that, contrary to what has been suggested (7-9), elevated glucocorticoids may not be a major factor in malnutrition-induced immune impairments. PEM appears to be a type of chronic stressor, in which, unlike acute stressors, immunosuppressive effects of glucocorticoids are not seen (10).
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S.M. FILTEAU et al. ACKNOWLEDGEMENTS
The authors are grateful for the excellent technical assistance provided by Angela Adams, Meg Davis, Vera Peacock and Rod Stokes; and for manuscript preparation by Cecilia Figueredo and Julie Anderson, This work was supported by the National Institute of Mental Health (RO3 MH45646-01), a grant from the Paul P. Dosberg Foundation, Inc., and a Medical Research Council of Canada postdoctoral fellowship awarded to S.M.F. Nicholas Hall is supported by a RSDA (DA00158). We also wish to acknowledge the James A. Haley Veterans Hospital, Tampa, Florida, for the use of its vivarium.
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