3% WIWWHIHIWNHIWWWWNI‘HWIHIWI ’1}.r‘x 2' IIII/II/II/ll/IIl/lI/I/I/l/llll Ill/IIII/ll/lll/I/l/l/II/l/lll 3 1293 10379 6425 This is to certify that the thesis entitled THE ROLE OF CORTICOSTERONE IN THE LOSS OF IMMUNE FUNCTION IN THE ZINC DEFICIENT A/J MOUSE presented by PAULA DEPASQUALE-JARDIEU has been accepted towards fulfillment of the requirements for MASTER OF S.C.I£N£E__degree in .BIOLHEMISIRY Date 2/ 20/80 0-7639 OVERDUE PINES ARE 25¢ PER DAY PER ITEM Return to book drop to remove this checkout from your record. \I M.U THE ROLE OF CORTICOSTERONE IN THE LOSS OF IMMUNE FUNCTION IN THE ZINC DEFICIENT A/J MOUSE By Paula DePasquale-Jardieu A Thesis Submitted to Michigan State University In partial fulfillment of the requirements for the degree of Master of Science Department of Biochemistry 1980 ABSTRACT THE ROLE OF CORTICOSTERONE IN THE LOSS OF IMMUNE FUNCTION IN THE ZINC DEFICIENT A/J MOUSE By Paula DePasquale-Jardieu To date, no explanation has been offered for the preferential involution of the thymus and subsequent immune dysfunction which occurs as a result of dietary deficiencies. It has been suggested that nutri- tional deficiencies constitute a stress on the animal leading to stimula- tion of the adrenal cortex and a rise in serum glucocorticoids, proven thymolytic hormones. The purpose of this research was to determine if the immune impairment resulting from zinc deficiency was stress induced. The data indicate that markedly elevated levels of plasma corticosteroids were present in the zinc deficient mouse during the final stages of zinc deficiency. Although 50% of the immune impairment occurred prior to corticosterone elevation, an additional significant reduction in T-cell function occurred shortly after the appearance of elevated levels of corticosterone. ”Removal of the steriod via adrenalectomy offered only a modest protection (20%) against this further dr0p in immune capacity. Thus, these data argue against a major role for corticosterone in the immunosuppression of zinc deficiency and further demonstrate the impor- tance of zinc to lymphocyte function. ACKNOWLEDGEMENTS The author wishes to express her sincerest appreciation to Dr. Pamela Fraker for her guidance, understanding, and friendship throughout these studies. Special thanks to the members of her guidance committee, Dr. S. Aust and Dr. G. Riegle, and to Dr. R. Luecke for his assistance with these experiments. She would also like to thank Mr. Craig Zwickl and Mr. Wayne Aldrich for the stimulating discussions, excellent technical assistance, and lasting friendship. Lastly, thanks to Pete and Pammie for their patience, encouragement and never-ending moral support. ii TABLE OF CONTENTS LIST OF TABLESOOOOOO0.0....0.I.OO...0.000000000000000000000000 LIST OF FIGURES..0.00...0.0...00......OOOOOOOOOOOOOOOOOOOO0.0. OBJECTIVEOCOOOOOOOOOOO0.0.0.0.0...0.0......OOOOOOOOOOOOOOOOOOO LITERATURE SURVEY.0.00......00.000.000.000...OOOOOOOOOOOOOOOOO CHAPTER I A POSSIBLE ROLE FOR CORTICOSTERONE IN THE IMMUNE DYSFUNCTION OF ZINC DEFICIENCY.OOOOOOOOOOOOOOOOOOOOOOOOOOIOOOOOOOOOOOOOOOO AbStraCtooooooooo00000000000000.0000.0.000000000000000... IntrOdUCtionooooo00000000000000.0000.00000000000000.0000. Materials and Methods.................................... Animals and diets................................... Zinc assay by atomic absorption spectrosc0py........ Collection of plasma for corticosteroid assay....... Corticosterone assay................................ Detection of antibody producing cells............... HiStOIOgyOOOOOOOOOOOOOOOOOOO0.000000000IOOOOOOOOOOOO Statistical methods................................. Results.................................................. Determination of corticosterone levels.............. Effect of corticosterone on l° response to SRBC..... Effect of zinc-deficiency on thymic integrity....... DiSCUSSion.0.0.000...00....0.0...OOOOOOOOOOOOIOIOCOOOOOOO CHAPTER II EFFECT OF ADRENALECTOMY ON THE IMMUNE DYSFUNCTION RESULTING FROM ZINC DEFICIENCY IN THE A/J FEMALE MOUSE.................. Abstract.OOOOOOOOOOOOOOOOOO0.0...OOOOOOOOOOOOOOOOOOOOOOOO IntrOdUCtionoooooooooooooooooooooooo0.0000000000000000... Materials and Methods.................................... Mice and diets...................................... Adrenalectomy....................................... Collection of plasma for zinc and corticosterone assayOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOI0.0.0.... Plasma zinc assay................................... Corticosterone assay................................ Detection of antibody producing cells............... Histo‘ogy.0.0000000000000COOOOOOOIOOOOOOOOOOOOOOOOOO iii Page vi 33 33 36 36 36 37 37 38 38 TABLE OF CONTENTS (Con'd) Resu‘ts.OIOOOOOOOOOOOOOOO00......OOOOOOOOOOOCOOOOOOOOOOO. Weight gain and food consumption.................... Assessment of plasma corticosterone levels.......... Determination of plasma zinc levels................. Determination of antibody mediated response......... Thymus integrityOOOOOOOOOOOO...OOOOOOOOOOOOOOOOOOOOO DISCUSSIO”.ooooooooooooooooooocoo.ooooooooooooooooooooooo SUMMARY AND CONCLUSIONSOOOOOOOOO00......OOOOOOOOOOOOOOOOOOOOOO LIST OF REFERENCES...0.0.0.000...0.0...OOOOOOOOOOOOOOOOOOOOOOO iv Table LIST OF TABLES Variation with time of body and thymus weight of A/J mice fed zinc-deficient or zinc-adequate diet.... Body weight and diet consumption of intact and adrenalectomized A/J mice maintained on zinc-adequate or dEfICTent dietSOOO...OOOOOOOOOOOOOO0.00.0.0000...O Plasma corticosterone levels of intact and adrenalectomized A/J mice maintained on zinc-adequate or defiCient dietSOOOOOO..00..OOOOOOOOOOOOOOOOOOOOOOO Plasma zinc concentrations of intact and adrenalectomized A/J mice maintained on zinc-adequate or dEfiCient diets.COO...0.0.0...OOOOOOOOOOOOOOOOO0.. Comparison of thymic weights and ratios of cortex to medullary area of adrenalectomized and intact mice maintained on zinc-deficient or adequate diets....... Page 28 4O 42 43 51 Figure LIST OF FIGURES Flow chart for determination of cortisol and corticosterone in blood plasma...................... Standard curve for fluorimetric determination of plasma corticosteronelOOOOOOO00.0.0000...00.......0. Determination of corticosterone levels in the A/J miceOOOOOOOOOIOOCOOOO0.0.0....COCOOOCOOOOOOOOOOOOOOO IgM PFC response of zinc-deficient and control A/J mice to SRBC as determined by Jerne plaque assay.... IgG PFC response of zinc-deficient and control A/J mice to SRBCOOOOOOOOOOOOOOCOO...OOOOOOOOOOCOOOOOOOOO Light micrographs of thin sections of representative thymuses of zinc-deficient and control A/J mice..... IgM PFC response of adrenalectomized and intact zinc-deficient and control A/J mice to SRBC......... lgG PFC response of adrenalectomized and intact zinc-deficient and control A/J mice to SRBC......... vi Page l4 l6 2] 24 26 3O 46 48 OBJECTIVE The primary goal of this research was to determine the cause of the severe thymic atrOphy and T-cell impairment brought about during zinc deficiency. Since thymus derived lymphocytes (T-cells) constitute the cell mediated arm of the immune system and also act in conjunction with B cells (as T-cell helpers) in most antibody mediated responses, any disruption of thymic integrity would result in a reduction in immune capacity. Thus, because of the significance of T-cells to immune func- tion, it is important to determine the mechanism whereby zinc deficiency causes rapid involution of the thymus and subsequent loss in immune function. Based on observations of adrenal hypertrophy in zinc-deficient animals, this study was undertaken to determine if zinc deficiency might constitute a chronic stress resulting in reactions mediated via the hypothalmus-pituitary-adrenocortical axis. If zinc deficiency activated this stress axis, it would result in a rise in serum glucocorticoids which could account for the observed decrease in immune capacity. LITERATURE SURVEY It is becoming increasingly more apparent that the ability to combat infection is greatly compromised by nutritional deficiencies. The partnership of infection and malnutrition is firmly established in many of the world's underdeveloped populations. A host of dietary deficiencies have been shown to cause preferential wasting of the lym- phatic organs compared to other body organs (1, 2). The thymus, the site of maturation of T lymphocytes, which are critical to both cellular and antibody mediated immunity, is often the most severely atrophied organ in deficient animals (3, 4). As a result, host defense mediated via the cellular immune mechanism is usually more severely impaired than that mediated through the humoral immune mechanism (5). It is not surprising then that diseases associated with depressed cellular immu- nity, such as measles, smallpox, and other viral infections, are leading causes of death in malnourished p0pulations (6). Based on these observations, a number of studies have been initiated to determine the effects of various dietary deficiencies on the immune response. Thus far, diets deficient in Specific vitamins (7), amino acids (8), trace elements (9), protein (10), or essential fatty acids (ll), have all been shown to impair immune function. This section will serve to review the results of these investigations. There seems to be little or no disagreement among investigators that protein calorie malnutrition (PCM) as seen in humans (12) and other animals (l3) alters cell mediated immunity but does not appear to adversely effect the humoral arm of the immune system (l4). A study 2 by Smythe (5) in 1971 examined the capacity of protein-calorie mal- nourished children to respond in the delayed type hypersensitivity reaction, a measure of cell mediated immunity. Seventeen children with PCM were tested against nineteen normal children for their ability to react to skin application of dinitrochlorobenzene (DCB). Twelve of the PCM children failed to react and none reacted severely. In contrast, all of the normal children were able to mount some response, while thirteen children deveIOped a severe reaction to DCB. In this same study, lymphocytes from thirty PCM infants and nine normal infants were tested for their ability to respond to the T-cell mitogen phytohemag- glutinin (PHA). Lymphocytes from normal children had a fourfold higher mitotic index than lymphocytes from PCM children. From these results, Smythe concluded that T-cell dependent cell mediated immunity was im- paired in PCM children. In another study by Gautam, (15), skin allograft survival, another measure of T-cell function, was increased from nine days to sixteen days in mice maintained on diets containing inadequate levels of protein. Neumann et al (16) investigated the effects of PCM on the humoral arm of the immune system of PCM children. Immunization with keyhole limpet hemocyanin (KLH) or pneumococcal polysaccharide, two B- cell dependent antigens, revealed no difference in immune responsiveness between PCM children and normal children. In addition, various tests of humoral responses of protein calorie deficient animals support these findings (l7). Other investigators have defined the effects of specific vitamin deficiencies on the immune response. Rats deficient in vitamin B6 had an increased success in skin transplants (l8). Vitamin 36 deficient guinea pigs had a reduced delayed type hypersensitivity re- sponse to purified protein derivative (l9). Jose (20) examined the effects of deficiencies of various essential amino acids on the immune response. Cell mediated immunity, as measured by the in vitro cytotoxic assay, was depressed when amino acid levels were decreased to 10% of required levels. However, a depression in humoral response was also detected in these studies. Several researchers have shown that metal deficiencies may result in depressed immunity. In a study of iron deficient children, Chandra (2l) found no difference in 196 levels or antibody titers to tetanus toxoid when the responses of these children were compared to normal children, but cell mediated immunity (CMI) as measured by delayed type hypersensitivity to Canida Albicans was impaired. Also, magnesium deficiency in rats depressed the hemolysin titer to sheep red blood cells (SRBC) (22). Studies designed to define the consequences of zinc deficiency on the immune system have suggested a similiar pattern of immune dys- function. In a major study on zinc deficiency in humans provided by Prasad (23), he found indications of lymphocytopenia, poor wound heal- ing, and increased susceptability to disease accompanied the deficiency. A genetic defect which results in decreased absorption of zinc has recently been identified. This disease, acrodermatitis entero- pathica, is transmitted as an autosomal recessive trait (24). Patients with this disease have low levels of immunoglobulin and death often results from infection with Canida Albicans. Post mortem studies on a child with this disease revealed no germinal centers in the spleen, lack of lymph nodes, and a thymus which consisted of epithelial cells with few thymocytes (25). Published reports on experimentally induced zinc deficiency have explored the relationship between immune function and the defi- ciency. Luecke had repeatedly observed that the thymus was the most severly atrOphied organ in zinc-deficient pigs (26) and mice (27). In an initial study by Fraker (28) on A/J female mice made zinc deficient for 4 weeks, then immunized with sheep red blood cells, the deficient mice produced only 10% as many plaque forming cells (PFC) as controls. Reconstitution of the deficient mice with thymocytes from normal mice, prior to immunization with SRBC, restored the response to near normal levels. This indicated a preferential impairment of T-cell function. These observations have been confirmed by Fernandas (29) using A/J males. In addition, he showed a defective development of T-killer lymphocytes, after in vitro sensitization with tumor cells, as well as a defective devel0pment of natural killer (NK) lymphocytes in zinc-deficient mice. Pekerak, et al, (30) showed that the antibody response to live Francisella vaccine was intact in the zinc-deficient rat if the animals were immunized prior to being made zinc deficient. This agrees with Frakers (27) studies which revealed that mice primed with a hapten- carrier prior to being made zinc deficient were able to respond as well as zinc-adequate mice when given a second injection of the antigen. Thus, mature antigen activated lymphocytes appear to be more resistant to the absence of zinc than are virgin lymphocytes. Fraker (3l) also demonstrated that T-cells involved in the delayed type hypersensitivity reaction were also depressed in zinc-deficient mice. Thus, it appears that zinc deficiency, as well as other nutri- tional deficiencies, produces a rapid and seemingly preferential wasting of the thymus as well as greatly impaired T-cell dependent processes. The question arose as to whether or not some common mechanism was respon- sible for these findings which is only indirectly related to the nutri- tional element in question. One possible explanation hinges on the observation of Luecke (32) and Quarterman (33) that the adrenal gland was enlarged in zinc-deficient animals. This finding has led to the suggestion that zinc deficiency might constitute a chronic stress on the animal resulting in adrenal hypertrOphy (33, 34, 35). Hypertrophy of the adrenal gland could result in increased synthesis and secretion of the adrenal steroids, including the glucocorticoids. Since the inju- rious effects of glucocorticoids on thymocytes have been well documented (36), a rise in serum glucocorticoids could account for the loss in immune capacity characterized in zinc-deficient animals. This hypothesis is not without precedent since it has been established that a variety of stressful stimuli can induce reactions that are mediated by the hypothalmus-pituitary-adrenocorticoid system which ultimately result in increased levels of adrenal steroids. In the early 50's Selye (37) noted what he termed a "general alarm syndrome“ in response to a variety of systemic stresses. Using a number of physical, as well as dietary stresses, he noted two general characteristics in the experimental animals: (a) adrenal hypertrophy and (b) thymic atr0phy. More recent work indicates that stress, in whatever form, causes a discharge of the hypothalmic releasing factor which in turn invokes pituitary adrenocorticotrOpic hormone (ACTH) secretion. ACTH acts on the adrenal cortex via adenylate cyclase to promote synthesis and secretion of the glucocorticoids. The release of glucocorticoids normally facilitate adaptive changes which allow the organism to survive noxious stimuli, possibly by diminishing the peripheral uptake of glu- cose and amino acids which can then be conserved for use by the vital organs. It is this function of the glucocorticoid which merits further emphasis. Clearly, histological (38) and immunological evidence (39) show that elevated levels of serum glucocorticoids cause lysis of im- mature cortical T-cells and wasting of the thymic cortex with the mature, cortical lymphocytes being more resistant. By destroying immature thymocytes, glucocorticoids interfere with the normal process of replen- ishing the peripheral lymphocyte population. The mechanism whereby the glucocorticoids destroy lymphocytes is open to controversy. Gluco- corticoids have been shown to inhibit DNA, RNA, and protein synthesis (40). These effects are all believed to result from hormonal inhibition of glucose uptake. It has been suggested that the hormone promotes the synthesis of a protein that inhibits transport of glucose into the cell, thus imparing the ability of the carbohydrates to provide ATP (4]). The inhibition of glucose uptake and subsequent reduction of carbohydrate dependent ATP production may then be responsible for the lysis of im- mature T-cells. Further evidence of the effects of stress on immunological processes is provided by studies employing mechanically induced stresses. Gisler (42) has determined that exposure of mice to stresses, such as cold, ether, and noise, result in a loss in immune capacity as measured by in vitro responsiveness to sheep red blood cells. Noting the adrenal hypertrophy and rapid wasting of the thymus observed in zinc deficiency, it was not unreasonable to assume that zinc deficiency, and possibly other nutritional deficiencies, might constitute a chronic stress on the animal leading to manifestations of the alarm syndrome. Thus, this thesis, using zinc deficiency as a model, inves- tigated the possible role of glucocorticoids as the common mechanism responsible for the immune dysfunction resulting from nutritional defi- ciencies. Chapter I A POSSIBLE ROLE FOR CORTICOSTERONE IN THE IMMUNE DYSFUNCTION 0F ZINC DEFICIENCY Abstract Previous investigations from our laboratory have shown that zinc deficiency causes rapid atrophy of the thymus with subsequent loss of T-cell helper function in the young adult A/J mouse. The purpose of this investigation was to determine if zinc deficiency constituted a chronic stress on the mouse leading to the elevation of glucocorticoid levels which is known to destroy thymic lymphocytes. The results of these experiments indicated that zinc-deficient mice indeed have increased levels of plasma corticosterone (115 ug/100 ml plasma) compared to mice fed zinc-adequate diets (40 ug/lOO m1 plasma). A significant reduction in T-cell helper function, which occurred four days after the rise in steroid concentration, suggested that corti- costerone might contribute to the loss in immunity; however, about half of the total loss in T-cell helper function occurred prior to the in- crease in plasma corticosterone and appeared to be due to other factors associated with the lowered zinc levels. 10 Introduction While the immune impairment resulting from zinc deficiency had been well characterized by our laboratory (28,31), the actual destruc- tive mechanism(s) evoked during the deficient state remained to be defined. Besides the various known and unknown zinc-dependent bio— chemical processes that could be adversely effected by dietary zinc deficiency, there was also evidence to suggest that elevated levels of corticosteroid could be present in the zinc-deficient mouse. Results of the literature survey suggested that zinc deficiency might constitute a chronic stress resulting in stimulation of the adrenal cortex and a rise in serum glucocorticoids. If zinc deficiency activated the stress axis, it would result in a rise in the level of serum corticosteroids which might account for the observed loss in numbers of functional thymocytes. The purpose of this study was to determine if indeed zinc deficiency was stressful to the mouse, since the injurious effects of glucocorticoids on thymocytes and thymic integrity have been well documented both_yn vivo (43) and in vitro (44). ll Materials and Methods Animals and Diets Twenty-one i 2 day old A/J female mice (Jackson Laboratory, Bar Harbor, Maine) weighing 12.: .99 were used in this experiment. To minimize exposure to environmental zinc, and prevent reabsorption of zinc from body wastes, the mice were housed in stainless steel cages with mesh bottoms. Feed containers and water bottles were washed with 4N HCl and rinsed with deionized water to remove all residual zinc. The mice were fed ad libitum a biotin-fortified egg white diet which contained either deficient ((0.7 ug Zn/g) or adequate (>10 ug Zn/g) levels of zinc as determined from previous studies (45). Diet consump- tion was measured 3 times per week, and the mice were weighed at least once a week. All mice had free access to deionized-distilled water ((0.2 ug Zn/g). Zinc Assay by Atomic Absorption Spectroscopy A known weight of sample to be analyzed for zinc content was added to a preweighted acid washed flask. Twenty-five ml of 32N HNO3 and 5 ml of 11.7N HClO4 were added to the flask and the sample was slowly evaporated to near dryness. The residue remaining was diluted to a known weight with 10% HCl. Flasks containing only the acid reagents served as blanks. Zinc concentration of the digested samples was determined with a Varian AA-l75 atomic absorption spectrOphotometer (Varian Techtron, Springvale, Australia). Absorption values were determined at 214 nm. A 12 standard curve for zinc absorption was obtained by dilution of a commer- cial zinc standard solution in 10% HCl. Collection of Plasma for Corticosteroid Assay Twenty-one day-old A/J female mice were divided into twenty- four groups consisting of nine mice per group. Twelve groups were fed zinc-deficient diets (<1 ug Zn/g) and twelve were maintained on zinc- adequate diets (50 ug Zn/g) ad libitum. On day zero, twelve mice were bled to determine basal levels of corticosterone. Every other day, beginning with day seven, and every day, from day twelve to nineteen, nine zinc-deficient and nine control mice were randomly selected to be bled. Each mouse was bled only once during the course of the experiment. Mice were bled at 0900 hours each day when the level of corticosterone was at its lowest point in the diurnal cycle. (Data supporting this observation will not be presented in this thesis.) Entrance to the animal room was prohibited for a 3-hour period prior to the bleeding in order to maintain basal levels of corticosterone. Zinc-deficient and control mice were selected at random for bleeding and immediately ether- ized. Blood was collected from each mouse by retroorbital puncture within l-minute of removal of the mouse from its cage. Mice from the same cage were removed simultaneously. Approximately 0.15 ml of blood was taken from each mouse and collected in heparinized tubes. The plasma was separated for use in the corticosterone assay. Corticosterone Assay A spectrofluorometric method for measuring unconjugated corti- sol and corticosterone was adapted from the methods of Sweat (46), Click (47), and Martin and Martin (48) to allow quantitation on a micro 13 scale of the steroid present in the small amount of plasma recoverable from mice. The experimental protocol is outlined in Figure 1. All glassware used in this assay was washed in 16 N HNO3 overnight and rinsed with distilled H20 to minimize nonspecific fluores- cense. Fifty ul of heparinized plasma were extracted by shaking for 2 minutes with 0.6 ml methylene chloride. After centrifugation, the aqueous layer was discarded and the solvent layer washed with 0.1 ml of 0.lN NaOH. Following centrifugation, the alkaline phase was discarded and the extract was evaported to dryness using a nitrogen evaporator. Double distilled water (0.1 ml) was then added to the residue. The water layer was shaken for 2 minutes with 0.6 ml carbon tetrachloride to extract the corticosterone. Following centrifugation, the aqueous phase was transferred to a clean test tube and the CCl4 layer was set aside for determination of corticosterone. Cortisol was removed from the remaining aqueous layer by extraction with 0.6 ml methylene chloride for 2 minutes. After centrifugation, the aqueous layer was discarded and the solvent fraction was assayed for cortisol. Half ml aliquots of the solvent layer from each tube containing either cortisol or corticosterone were then pipetted into tubes containing 200 ul of the fluorescent mixture which consisted of 3 parts 32N H2504 to 1 part absolute ethanol. The tubes were shaken for 2 minutes to develop the fluorescence, centri- fuged, and the solvent layer was then removed by aSpiration. The acid layer from each tube was transferred to a microcuvette and after 30 min- utes the amount of fluorescence was determined at 475 nm (1°) and 525 nm (2°) on a spectrofluorimeter (American Instrument Co., Silver Springs, Maryland). Standards containing 0.05 to 0.l ug corticosterone and cortisol (Applied Science Laboratories, State College, Pennsylvania) 14 Extract 20-l00 ul plasma with Methylene Chloride Aqueous phase Solvent layer (discard) wash with NaOH Solvent layer Aqueous phase (discard) Evaporate to dryness Resuspend in distilled water Extract with CCl4 Aqueous phase Solvent layer Extract with Add acid-ethanol reagent Methylene Chloride Solvent layer Aqueous phase Measure fluorescence (discard) (corticosterone) Add acid-ethanol reagent Measure fluorescence (cortisol) Figure 1. Flow chart for determination of cortisol and corticosterone in blood plasma. 15 were carried through the same procedure. Water blanks were used to correct for background fluorescence. This method was found to give a linear relationship between fluorescence and concentration of standard over a range of 0.01 to 0.2 ug of corticosterone (Figure 2). Related steroids, such as pro- gesterone and ll-deoxycortisol (Pflatz and Bauer, Stanford, Connecticut), gave fluorescent readings of less than 1% that of corticosterone. The Martin and Martin (48) method of oxime formation was used to determine approximate values for nonspecific fluorescence generated by contam- inating fluorogens. Based on multiple trials, this method yielded fluorescent readings equal to 1-7 ug/100 ml of plasma. Determination in triplicate of standard amounts of corticosterone added to plasma of known corticosterone concentration revealed that 85% of the steroid was recovered by this method. Detection of Antibody Producing Cells A modification of the Jerne plague assay was used to examine the humoral response of the mice to SRBC. In this assay, 5 days after immunization, the splenic lymphocytes from each mouse were mixed with a p0pu1ation of SRBC in an agarose layer. Anti-SRBC antibody produced by the lymphocytes will bind to SRBC in the region surrounding the plasma- cyte. Upon addition of complement, the sensitized SRBC will lyse and produce a clear circular plaque in the agar which when counted will yield the number of antibody producing cells per mouse spleen. In this assay, SRBC stored at 10°C in Alsever solution (Gibco Diagnostics, Grand Island, New York), were washed and suspended in phosphate buffered saline at a concentration of 2 x 109 cells/ml. l6 .mcocmpmoowucoo msmmpq co cowpmcwELmumu owcpmewco:_w com m>c=u ncmvcopm :3}... 22323:: 222.325 m? “'1 F ‘ ‘ ‘ .N weaned :6— :m— lusmul cauaasaionu l7 Sterile minimal essential medium (MEM) containing Earles salts (Gibco Diagnostics, Grand Island, New York) and penicillin was used to maintain the lymphocytes. The mice were etherized and their spleens removed. After being cut into several pieces, the spleens were pressed through an 80 mesh stainless steel screen to produce a single cell suspension. The suspension was washed and resuspended in a 1 ml volumn. One-tenth ml of splenocytes and 0.1 ml SRBC were added to 0.8 m1 of MEM containing 0.6% agarose maintained at 53°C. This mixture was poured onto a 60 mm petri dish containing a bottom layer of 0.6% agarose-MEM. Each lymphocyte suspension was plated in duplicate. The petri dishes were incubated for 1.5 hours in a humidified 37° tissue culture chamber. Direct plaques, resulting from IgM producing plasmocytes, can be visualized by addition of 0.5 ml of nonhemolytic guinea pig complement. To develOp the indirect or IgG plaques, it was necessary to increase the complement fixation efficiency by a l/2 hour incubation with 0.5 ml of rabbit anti-mouse IgG prior to addition of complement. Unimmunized A/J female mice produced 30-60 plaques/spleen. Correction was made for these background plaques and for the small number of IgM plaques which appear on the IgG plates. All data is expressed as average plaque number produced per spleen. Previously, it had been determined that a 6-hour recuperation period prior to immunization was adequate to eliminate any effect of the stress of bleeding on the Jerne response. Histology Immediately following etherization, the thymuses of the zinc- deficient and control mice were removed, weighed, and placed in Bouins l8 fixative for 72 hours (49). The tissues were dehydrated in a series of dioxane-water washes (DioxanezHZO, 1:4, 1:2, 1:1, 2:1) and embedded in Paraplast (Sherwood Medical Industries, St. Louis, Missouri). Sections 5 u thick were mounted on glass slides and stained using the Masson- Trichrome procedure (49) which allowed for discrimination of the medulla from the cortex. Mid-sections of each thymus were examined by light- microsc0py. A grid was superimposed over micrographs of these sections to allow quantitation of medulla and cortical areas. Statictical Methods All data were examined by analysis of variance with statis- tical significance of treatment differences being determined by Student's t'tESt (50). 19 Results Determination of Corticosterone Levels The results of the fluorescent assay for plasma corticosterone levels are shown in Figure 3. A significant elevation in corticosterone occurred in the zinc-deficient mice on day 14 and increased through day 19. At the peak of the response, plasma from the zinc-deficient mice averaged 115 ug of corticosterone per 100 ml. The control mice maintained a basal level of approximately 40 ug corticosterone per 100 ml of plasma. In addition, the adrenal glands were enlarged (33%) relative to body weight in the zinc-deficient mice. Fluorescence in the cortisol fraction was also calculated for both dietary groups and found to equal 5-10 ug/100 m1 of plasma based on the fluorescence of the cortisol standard. [The question has recently arisen as to whether or not cortisol is produced by the rodent (51). The small amount of fluorescence detected in these fractions from what- ever source did not contribute significantly to the date. Henceforth, only data for corticosterone will be reported.] Effect of Corticosterone on 1° Response to SRBC It was necessary to determine what role, if any, this eleva- tion in steroid levels played in the impairment of T-cell helper function in the zinc-deficient mice. To resolve this question, it was essential to relate the elevation in corticosterone levels to a concomitant reduc- tion in immune capacity. To examine this, the antibody-mediated response of the control and the zinc-deficient mice was also assessed throughout .mpocucoo ou umchEou wows u:m_o_emuuocw~ to Fm>mp mcocmpmoo_pcoo c_ Ammmp co Po.ov u av cowpm>m_w pcmo_mvcmwm mg» mwpmowccw A+v 3occ< .mo_5 mcw: co 2mm.H cams on» mpcmmmcqmc ucwoa 56mm .Apxmp mom .uocpms coev A.nlll.v mo_E Focpcou use A.-----.v ucmwowwmuuocw~ wcp sore meson come on xmu comm empom—_oo mm: wasp mamm_a co mums mew: mpm>mp mcocmpmoopocoo mama—q co cowpmc_acmpmu o_cpmeoco:_d .mova a\< mcp cw m_m>m_ mcocmpmoowpcoo co co_om:wscmpmo 0 2 .m mczmwm .m weaned .o_o co «in: a. o- s- a. n. v_ n. a. __ a. m o w a m b b L P n p - r1? be» on 21 T.o~ [.cn T.ov fan .I on 1.9s rues r.cm [.99— 1 I mono-«cu II 10...“ emse|d slm ooi/aUOJOisoagiJoa an 22 the experiment by means of the Jerne plaque assay. The number of direct and indirect PFC/spleen are shown in Figures 4 and 5, respectively. The IgM response of the zinc-deficient mice remained almost constant throughout the first 19 days of the experiment (11,000 PFC/ spleen). At this time, the reSponse began to fall and by day 24 it had decreased to about 1/2 this value to an average of 6000 IgM PFC/spleen. It should be noted that the ability of the control mice to respond to antigen increased with age and reached a plateau of approximately 25,000 PFC/spleen by day 19 as is normal during development (52). Zinc defi- ciency apparently prevented further development of plaquing capacity. An additional decrease in the direct response was observed from day 19 to day 24. Thus, as the mice aged, the difference in plaquing ability of the zinc-deficient mice compared to the control mice broadened. This is best illustrated by expressing the direct response of the zinc- deficient mice as % of controls as shown in Figure 4. The indirect response of the control mice also showed a sim- ilar increase with age, escalating from an initial response of 10,000 PFC/ spleen to a mean value of 35,000 PFC/spleen (Figure 5). On day 14, prior to significant elevation in glucocorticoids, the IgG response of the zinc-deficient mice was 60% of the control response (p < 0.02). However, an additional 25% dr0p in the indirect response of the zinc- deficient mice occurred between days 17 and 19. This second major decrease occurred 4 days after a significant elevation in plasma corti- costerone levels. Throughout days 19 to 24, the indirect response continued to decline in the presence of high steroid levels reaching a value of 15% of the control response at the termination of this experi- ment. It should be noted that the serum zinc level remained constant .pmmpua m.u:mu:um mew xn cmpm_:u_mo mm mmm_ co _o.ov u a .mows _ocu:oo op cmcmano wows pcm_owemvuo:_~ ago we mamm_a ms» cw m_m>m_ mcocmpmooppcoo :P :o_wm>mpm pcoo_we:mwm mo mocmcmmaqw mmumo_u:_ Aav Zocc< .mowe mcw: co 2mm.fl came mgp mucmmmcamc can comm .umam wo_ x P gov: »_m:ow>mca mxmv m umnwczeaw more pcmwowemuuu:_N new m_ocpcoo mo mmcoqmmc com—am cma\uma xmmmm mzcm_q occwn xn umcwscmpmu mm ummm ow move w\< Focpcoo new pcmwo_cmvuo:_N co mmcoammc om; ZmH .q mczmwd 24 .e meamwu «0.6 C. nuOO «N ..u on .0. .o. a. a. a. Q. n. a. —. a. :.5 nHu 7.: mm“ D. .- .h D. a U30l‘8/‘O'J'd 25 on“ xn nmpwpzo_mo mm mmm_ co No.ov .pmmo-p m.p:wu:pm a ._o.ov u a .m_ocp:oo op emcmasoo more pcmwo_$munoch mcp km mamm_q ecu cw mcocmpmoowucou co m—m>mp umpm>m_m mo mocccmmqam mmumovucw A.v zocc< .mo_a w:_: mo 2mm + cows ecu mpcmmmcamc can 50mm .mows p:m_o_wmuuoc_N new Pocpcoo roe ammmm mzcmpq ocean mg“ xn nmcchmpmu mew: com_qm 0mm pomcwucw co Longs: one .ommm 0p wows w\< pocpcoo new pcm_o_wwuuo:_~ we mmcoqmmg on; me .m mczmwm 26 in: :2. :6: $0: 3.5 7v 5 fl 7. 5 § 3:— ... a...— .m mczmmd :53 :53 .30: I So..— I 23.3 1 03.90 I 39.: "DUNS/'3'!" 27 throughout this second dr0p in immune function at 35 ug Zn/100 m1 plasma compared to a control value of 96 ug Zn/100 ml plasma. Effect of Zinc Deficiency on Thymic Integrity By day 14, the wet weight of the thymuses of the zinc—deficient mice had decreased to 48% of the control mice (Table 1). This reduction occurred before a significant rise in glucocorticoid level was observed. For the remaining 10 days of the experiment, thymic involution continued with the thymus diminishing to 16% of control weight in the presence Of the high levels of corticosterone generated during this time period. Representative sections of the thymuses taken during the experiment are found in Figure 6. Figure 6d represents the thymus of a control mouse taken on day 13 of the experiment. This organ has a large, darkly stained cortex packed with lymphocytes surrounding a lighter staining medulla. Figure 6a represents a thymus taken from a zinc-deficient mouse on day 12 of the deficiency prior to elevation in corticosterone levels. The thymus had undergone considerable atr0phy with preferential in- volution of the cortex over the medulla. With elevation of corti- costerone (Figure 6b) the cortex continued to involute until on day 16 it appeared as a dark-staining ring surrounding the medulla. By this time, some atr0phy of the medulla had also occurred. At the termination of the experiment, the thymuses of the zinc-deficient mice were severely involuted (Figure 6c). The cortex was completely indistinguishable, and the medulla had also undergone considerable atr0phy with few thymocytes visible. 28 TABLE 1 Variation With Time of Body anlehymus Weight of A/J Mice Fed Zinc- Deficient or Zinc-Adequate Diet Experimental Dietary Day GrouP Body Weight Thymus Weight 5 -Zinc (95%)311.9:0.62 (89%)22.8:3.7 +Zinc 12.3:p.9 25.7:2.8 12 ~Zinc (87%) ll.5:0.6 (47%) 9.2:1.74 +Zinc l3.2:0.7 19.6:2.8 14 -Zinc (82%) 12.1:p.7 (48%) 9.711.04 +Zinc 14.7:0.8 19.7:1.8 l6 ~Zinc (75%) 11.910.84 (39%) 7.211.04 +Zinc 15.8:0.3 l9.9:0.8 18 -Zinc (71%) 11.41054 (31%) 7.23.94 +Zinc l6.0:p.6 21.1:2.8 20 -Zinc (78%) 11.6:0.6 (30%) 7.711.64 +Zinc 14.7:p.7 25.7:3.9 22 -Zinc (72%) ll.3:0.84 (17%) 4.8:7.o4 +Zinc 15.7:p.8 28.2:2.9 24 -Zinc (68%) 11-219-74 (16%) 4.8:1.o4 +Zinc l6.5:0.4 31.1:3.9 1 0n the day of each Jerne assay, the body and thymus weights of the control and zinc-deficient mice were determined to assess the effect of the deficiency on these parameters. 2Mean.:_SEM,n = 9. 3Ratios are a comparison of weight of the zinc-deficient mice (-Zinc) to control mice (+Zinc). 4P = (0.01 or less as calculated by Student's t-test. .Aomxv mEocco_Lp comma: .w-=nma mcwcwmpm cmpzmw_ asp mcwuczoccam mmpxoonqsx_ sow: umxomq xmocoo uwcwmum xpxcwu .mmcmp mzp mpoz .pcmewcqum mcp mo 03 NP xmu co :mxmp more Focucoo m mo mzsxcp .o .cmmco msp cw mc_:wmemc mmpxoosxzp so; guy; m_nm;mw:mcwpmwucw “2 cwncon xgmppsume Fmovucoo aucmvo_wmu ummcopoca sngunaocmwovwmu on“ we m_ xmc co mm39: ucmwo_wwvuo:wN m we msex5H .o .wu:_o>:w op emacwpcoo mm; xmycoounmcocmpmoowucou mo :o_pm>mpm pcmowm_:mwm mewm up awe co cmxmu mmzos pcmwowwmunucw~ m we mzsxzp .m .xmpcoo owexgy 6:» mo :o_p:_o>:w _wwp:mccmemcq asp mpoz .:o_pm>m_m mcocmpmoowpcoo op cowca pcms_cmaxm mzp co m_ Xmu co cmxmu mwzos “cowo_emuuucP~ a mo mzexgh .< .mowe w\< Focpcoo ecu ucm_u_emuuocv~ mo mwmaexcu m>_pmu:mmmcqmc co m:o_pomm :_;p we mzqmcmocowe pcqu .m mczmwd 30 31 Discussion The adrenal hypertrOphy observed in zinc-deficient mice suggested that adrenal steroids might be responsible for the rapid thymic atrOphy and subsequent reduction in immune capacity characterized in these animals. The results of these experiments indicate that zinc deficiency did indeed produce a significant elevation in glucocorticoid levels in the A/J female mouse. To examine the possible role of this rise in steroid level on T-cell functionality, the indirect Jerne response was measured since it is more dependent on T-cell helper function than the direct response (53). The observed decline in the indirect response of the zinc-deficient mice occurred in two separate phases. The first significant dr0p in response (60%) was seen prior to elevation in plasma corticosteroid levels and, therefore, was due to other factor(s) associated with the absence of zinc itself. However, the second reduction in the T-cell helper-dependent response occurred 4 days after a significant rise in the corticosterone level. Since the life span of a mature SRBC helper T-cell is estimated to be 4 days (54), such a decrease would be expected if indeed precursor T-cells were lysed by the corticosterone. While a direct relationship between the rise in glucocorticoids and the second decline in immunity remained to be firmly established, there was evidence to suggest that the two phenomena were indeed related, and that elevated levels of adrenal steroids produced during zinc defi- ciency might contribute to the observed loss in immune capacity. 32 Since modification of the immune response by stress is implied by the results of this first experiment, ways were sought to more quantitatively relate the amount of T-cell impairment caused by the depletion in zinc itself to that which may result from the presence of high levels of corticosterone. Chapter II EFFECT OF ADRENALECTOMY ON THE IMMUNE DYSFUNCTION RESULTING FROM ZINC DEFICIENCY IN THE A/J FEMALE MOUSE Abstract Previous studies from this laboratory have shown that zinc- deficient mice had significantly elevated levels of plasma corticosterone compared to mice fed zinc adequate diets. The purpose of this investiga- tion was to determine the relationship, if any, between the increase in glucocorticoid levels and the rapid thymic atr0phy and consequent loss in T-cell helper function observed in zinc-deficient mice. The results of experiments employing adrenalectomized mice indicate about 1/2 the loss in T-cell helper function in the zinc- deficient mice occurs prior to any elevations in serum corticosterone (12 ug/lOO ml) and is therefore due to other factors associated with the depletion in zinc. Nevertheless, a second reduction in T-cell helper function did occur shortly after the appearance of significant quantities of glucocorticoids in the serum of the intact or nonadrenalectomized mice (82 ug/100 ml). The adrenalectomized zinc-deficient mice which still had minimal levels of serum corticosterone (7 ug/100 ml plasma) were not significantly protected by this treatment having only marginally better humoral immune capacity (20%) than the intact deficient mice. Further, the loss in T-cell function in the adrenalectomized zinc- deficient mice occurred despite the absence of thymic involution. The data presented here argue against a major role for corticosterone in 33 34 the immune dysfunction observed in zinc-deficient mice and further demonstrate the importance of zinc to lymphocyte function. 35 Introduction Although data from the previous experiment suggested that corticosterone might contribute substantially to the decline in immune function resulting from zinc deficiency, a direct relationship between the two remained to be firmly established. To further quantitate and evaluate the role of elevated levels of corticosterone in the immune dysfunction observed in zinc-deficient mice, a series of experiments were attempted. Using a variety of means of exogenous administration of corticosterone to mice, it was not pos- sible to simulate with any kind of accuracy the proper physiological levels of corticosterone produced by the deficient mice. Injection of metopirone, an ll-B-hydroxylase inhibitor (55), caused a persistant loss of balance and led to a reduced feed intake in these mice which made meaningful dietary experiments impossible. Failure of these methods to produce an adequate system for study led to the initiation of a series of experiments using adrenalectomized mice. Adrenalectomy permitted examination of the effects of zinc deficiency on immunity in the absence of high levels of circulating glucocorticoids. The data to be reported here indicate that corticosterone appears to play only a minor role in the loss of immune capacity resulting from zinc deficiency. 36 Material and Methods Mice and Diets Since the death rate following adrenalectomy was prohibitably high with three week old mice, six week old A/J female mice were used in this experiment. Mice were housed in cages constructed of polycarbonate and stainless steel and maintained in a temperature and light regulated environment. Cages, feed jars, and water bottles were washed as previously outlined in Chapter I. Mice were fed ad libitum a biotin fortified egg white diet containing either deficient (< 0.8 u Zn/g) or adequate (> 50 ug Zn/g) levels of zinc. The zinc content of the diet was determined as described in Experiment I. Diet consumption was measured 3 times per week. All intact mice had free access to deionized distilled water (< 0.2 ug Zn/g). Adrenalectomized mice were provided with 1% NaCl in deionized distilled water for drinking (< 0.3 ug Zn/g). Adrenalectomy All surgical procedures were done under ether anesthesia using a nose cone. Bilateral adrenalectomies were performed using a dorsal approach. Skin and muscle incisions were made; the adrenals were located, grasped with forceps and moved to a position suitable for excision. Following removal of the gland, the fascia was closed with 6-0 Ethilon (Ethicon, Inc., Somerville, New Jersey) sutures while stain- less steel clamps were used to close the incision. All of the mice were given a 5-day stabilization period prior to use in the experiment. 37 Collection of Plasma for Zinc and Corticosterone Assay Zinc—deficient and control mice were selected for bleeding and immediately etherized. Blood was collected from each mouse by retro- .orbital punctures within one minute of initial cage disturbance with all mice from a cage being removed simultaneously. Approximately 0.20 ml of blood was taken from each mouse by insertion of acid washed capillary tubes into the eye socket. Blood was collected in acid washed, hepar- inized tubes. Plasma was separated and used for corticosterone and zinc determinations. Mice were bled at 0900 hours when the corticosterone level is at its lowest point in the diurnal cycle (56). Entrance to the animal room was prohibited for a 3 hour period prior to bleeding in order to maintain basal levels of corticosterone in the control mice. In addi- tion, mice from each group were bled only once during the experiment to prevent measurement of erroneously high corticosterone levels resulting from the stress of multiple bleedings. Plasma Zinc Assay Plasma zinc levels were determined by flameless atomic absorp- tion spectrophotometry. Twenty ul of plasma were diluted in 10 mM HNO3 and analyzed using a Varian 175 Carbon Rod Atomizer. Concentrations were determined by comparison to standard zinc solution also prepared in HN03. Corticosterone Assay The spectrofluorometric assay for measuring unconjugated corticosterone in mouse plasma was discussed in detail in Chapter I. 38 Detection of Antibody Producing Cells Mice were immunized intraperitoneally with a l x 108 sheep red blood cells (SRBC) in sterile phosphate buffered saline. A modification of the Jerne plaque assay, previously described in Chapter I, was used to determine the total number of direct and indirect plaque forming cells (PFC) produced per mouse spleen. Histology At autopsy, the thymuses of all mice were removed, weighed, and prepared as described in Chapter I. 39 Results In the previous study of zinc deficiency using A/J female mice, it was determined that elevated levels of serum corticosterone were produced in the latter stages of the deficiency. Thus, to quanti- tate the possible effect of elevated glucocorticoid levels on the immune impairment resulting from zinc deficiency, the antibody response of zinc-deficient intact mice was compared to the antibody response of adrenalectomized zinc-deficient mice. To this end, 42.1.2 day old A/J female mice were divided into 4 groups; zinc-deficient adrenalectomized, zinc-deficient intact, zinc- adequate adrenalectomized, and zinc-adequate intact. Each of the four groups contained 36 mice. Body weight, diet consumption, plasma zinc and corticosterone levels, as well as humoral immune capacity of the mice, were assessed after 3, 4, and 6 weeks on the synthetic diets. Weight Gain and Food Consumption Throughout the experiment, adrenalectomized and intact zinc- deficient mice weighted significantly less than their respective con- trols (Table 2) with a 5 gram weight differential between zinc-deficient and zinc-adequate mice present by week 6. Conversely, there was no significant difference in body weight between the intact and adrenal- ectomized mice, thus indicating that adrenalectomy itself had little effect on weight gain. 0n the average, both groups of adrenalectomized mice consumed significantly less diet than the nonadrenalectomized mice [an observa- tion which is consistent with the literature on adrenalectomized 40 TABLE 2 Body Weight and Diet Consumption of Intact and Adrenalectomized A/J Mice Maintained on Zinc-Adequate 0r Deficient Diets Body Weight Zinc-Deficient Zinc-Deficient Zinc-Adequate Zinc-Adequate Weeks Adrenalectomized Intact Adrenalectomized Intact (9) (9) (9) (9) 0 15.7 1 0.2 + 16.11 0.6 15.8 1 0.4 17.11 0.4 3 16.7 1 2.06‘ 16.5 1 0.8b 19.3 1 0.8 18.6 1 0.8 4 16.9 1 0.8c 16.3 1 0.5d 20.11 0.5 20.0 1 0.4 6 15.811.0° 16.010.7d 20.511.0 21.010/9 Food Consumption g/mouse/day 1.8 1 0.1f 2.2 + 0.1 2.0 + 0.2b 2.3 + 0.1 + means :_SEM of 7 t0 9 mice a p < .05 as compared to zinc-adequate adrenalectomized mice b p < .05 as compared to zinc-adequate intact mice c p < .01 as compared to zinc-adequate adrenalectomized mice d p ( .001 as compared to zinc-adequate intact mice e p < .001 as compared to zinc-adequate adrenalectomized mice f p < .05 as compared to zinc-deficient intact mice 41 animals (57)]. However, as seen from the body weight data, this decrease in food consumption did not appear to interfere significantly with weight gain. Assessment of Plasma Corticosterone Levels At 3, 4, and 6 weeks, 9 mice from each of the 4 groups were bled and their plasma was assayed for corticosterone. The results of these determinations are shown in Table 3. After 3 weeks on the deficient diet, the nonadrenalectomized zinc-deficient mice showed no elevation in glucocorticoid levels; how- ever, by week 4 significant levels of corticosterone were present in the zinc-deficient intact mice (82 ug corticosterone/100 ml plasma). The zinc-adequate intact mice maintained a level of approximately 40 ug corticosterone/100 m1 plasma, while the adrenalectomized zinc-deficient and adrenalectomized zinc-adequate mice had a level of approximately 12 ug corticosterone/100 ml of plasma through weeks 3 and 4. By week 6 the corticosterone level of the intact zinc-deficient mice remained significantly elevated (101 ug/100 ml plasma). The plasma corti- costerone levels of the adrenalectomized zinc-deficient and adrenal- ectomized zinc-adequate mice remained at 10 ug/100 ml of plasma, while intact zinc-adequate mice had risen slightly to 55 ug/100 m1 of plasma by week 6. Determination of Plasma Zinc Levels The results of weekly determinations of plasma zinc levels of these mice are found in Table 4. Adrenalectomy appeared to have no effect on plasma zinc levels throughout the experimental period. No significant differences in plasma zinc was seen between zinc-deficient 42 TABLE 3 Plasma Corticosterone Levels of Intact and Adrenalectomized A/J Mice Maintained 0n Zinc-Adequate 0r Deficient Diets Zinc-Deficient Zinc-Deficient Zinc-Adequate Zinc-Adequate Weeks Adrenalectomized Intact Adrenalectomized Intact ug/100 ml ug/100 ml ug/lOO m1 ug/100 ml plasma plasma plasma plasma 3 7.013.6+ 12.013.6a 8.012.5 11.011.0 4 12.111.6 81.8 1 9.5C 13.0 1 4.0 43.3 _+_ 8.2 6 5.4 + 2.0 101.0 115.0c 5.2 11.8 55.11 9.0 Plasma Corticosterone were measured by spectrofluorometric assay (for method see text). + Means 1_SEM 0f 7 to 9 mice a NS as compared to zinc-deficient adrenalectomized level b NS as compared to zinc-adequate intact level c p < .01 or less as compared to zinc-adequate intact level w TABLE 4 Plasma Zinc Concentrations of Intact and Adrenalectomized A/J Mice Maintained 0n Zinc-Adequate 0r Deficient Diets Zinc-Deficient Zinc-Deficient Zinc-Adequate Zinc-Adequate Weeks Adrenalectomized Intact Adrenalectomized Intact ug/100 ml ug/100 ml ug/lOO ml ug/100 m1 plasma plasma plasma plasma 3 mm13m“ £815Jm° N614J° 6L7183 4 %316£a N8132m° nm13nd 8&21&0 6 mm1nma m2142m° n215m° 740132 Zinc determination (atomic absorption spectr0ph0t0metry) were made on plasma collected at 0900 hours from each experimental group. + Means : SEM of 7 t0 9 mice ap < .001 or less as compared to zinc-adequate adrenalectomized level bp < .001 or less as compared to zinc-adequate intact level CNS as compared to zinc-deficient adrenalectomized level dNS as compared to zinc-adequate intact level 44 adrenalectomized and zinc-deficient intact mice. Similarly, n0 differ- ence in plasma zinc levels were found between zinc-adequate adrenal- ectomized and zinc-adequate intact mice. However, the results do show that the plasma zinc levels of adrenalectomized and intact zinc-deficient mice were significantly depressed (30-38 ug Zn/100 ml plasma) compared to adrenalectomized and intact mice fed zinc-adequate diet (70-100 ug Zn/100 ml plasma) throughout the experiment. Determination of Antibody Mediated Response To determine the effects of the elevated levels of adrenal cortical steroids on the immune function of the deficient mice, the antibody mediated response of the adrenalectomized and intact mice to SRBC was assessed by the Jerne plaque assay. The numbers of direct and indirect PFC/spleen produced in response to immunization with SRBC are shown in Figures 7 and 8, respectively. At 3 and 4 weeks, both the direct and indirect reSponses 0f the adrenalectomized mice fed either adequate 0r zinc-deficient diet were higher than the antibody responses of the intact mice maintained on similar diets. Thus, for meaningful comparisons to be made between zinc-deficient adrenalectomized and zinc-deficient intact mice, their antibody responses should be expressed as a percentage of their respec— tive controls. Indeed when compared in this manner, at 3 and 4 weeks, the direct responses of the adrenalectomized zinc-deficient mice and intact zinc-deficient mice were not statistically different. Moreover, the direct response of the zinc-deficient adrenalectomized and intact mice did not differ significantly from their reSpective controls. By week 6, a significant depression in the direct reSponse was seen in both 45 con zoom _oo.vouoope poopcp A+V :N op oocooEoo poopcp A1v :N poo.vouoope oowpaopoopococoo A+v :N op ooeoosoo ooNPEopompococoo Auv cN "mxooz o mzumope poopcp A+v CN op oocoosoo poopcp Auv :N mzuoops ooNpEopooPocoLoo A+v :N op oocoosoo ooNpEopoopococoo Anv :N ”mxooz o + m .pmop-p m.p:oo=pm >8 oocpELopoo m6 mpmo .moppmppmpm .oops m op N co 2mm.u.cooa ocp mpcomocooc .ummm op oops o\< _ocpcoo oco pcopopwoouocpN poopcp oco ooNpEopoopococoo mo mmcoomoc umo zap .m mcompm 46 Mmdectomind lntocl 1mm“ I-Nntoct “Vt! ~3x:mmamxmmmaamnmnmmmmmmmaman :3:auuussannuuuwsaanancuvxss. 3.". 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MEN‘S/Odd 9’1 3 082. 49 the adrenalectomized and intact deficient mice with their responses dr0pping to 37% and 40% of their respective control group. In contrast, adrenalectomy appeared to delay, but not prevent, the loss of T-cell helper function. The indirect response of both intact and adrenalectomized zinc-deficient mice had dropped signif- icantly by week 3, decreasing to 56% of adrenalectomized controls in the case of the adrenalectomized zinc-deficient mice and to 58% of intact controls in the case of the intact deficient mice. By week 4, in the presence of elevated levels of corticosterone, the response of the intact zinc-deficient mice had dr0pped to 41% of the intact control response. In contrast, the adrenalectomized zinc-deficient mice were able to maintain an indirect response approximately equal to their response of the previous week (60%). However, by week 6 the response of the adrenalectomized zinc-deficient mice had declined, reaching a value of 38% 0f adrenalectomized controls, while the response of the non- adrenalectomized mice had dr0pped further to approximately 20% 0f the intact control response. Thymus Integrity The thymus weight (Table 5) 0f the intact zinc-deficient mice decreased at 3, 4, and 6 weeks to 60%, 50%, and 20%, respectively, of intact control weights. In contrast, the thymus weight of the adrenal- ectomized zinc-deficient mice remained at 90% 0f the adrenalectomized control thymus weight throughout the experiment. Since the thymuses 0f the adrenalectomized zinc-deficient mice did not undergo the large weight loss that was observed in the case of the nonadrenalectomized zinc-deficient mice, it was of interest to 50 examine these tissues histologically. At 3, 4, and 6 weeks, the thymuses from intact and adrenalectomized mice maintained on the zinc-adequate diet had a corticalzmedullary ratio of 2.0, while the zinc-deficient intact mice had a corticalzmedullary ratio of 1.0. This indicated preferential atr0phy of the thymic cortex. [These values are in agree- ment with previously reported values (58).] Interestingly, the thymuses of the zinc-deficient adrenalectomized mice showed no such preferential involution of the cortex at 3 weeks. However, by 4 weeks and continu- ing through 6 weeks, the cortica1:medullary thymic ratio of these mice was reduced to 1.0. This was the result of an apparent redistribution of lymphocytes which occurred in the absence of any thymic weight loss and paralleled the marginal increase in response seen at 4 weeks in the adrenalectomized zinc-deficient mice compared to intact zinc-deficient mice. There were no additional gross indications of abnormal pathology in thymuses from either group of deficient mice. 51 mops m mo 2mm.u.:oma .mopa m op N mo 2mm.H.=oms .4. oppoc opexsp omco xgoppoomE\—ooppcoo H mops pompcp pompopmmouocpN op omcooeoo mo mmmp co _oo. v o n mops poopcp mpooomoouocp~ op omcoosoo mo mmmp co poo. v oo mops poopcp mpooomoouocp~ op omcoosoo mo poo. v o a mops omNpEopompocmcoo mpooomoouocp~ op omcooEoo mo mzo + .Apxmp mmm oogpms Lomv coppocpaoxm poopmopopmp; Low omcoomco mom; mmommpp oco omcpacmpmo mcmz mpzmpmz washcp .xommo mccmo zoom wo Aoo mgp co N.o + o._ p.m + m.o~ N.o + _.N N._ + o.om _.o + o.~ o.¢_p.o.mN pompcp mpooomo