Mechanisms of Ageing and Development, 11 (1979) 127-136

127

©Elsevier Sequoia S.A., Lausanne - Printed in the Netherlands

AGE-RELATED CHANGES IN IMMUNE FUNCTION OF RATS AND THE EFFECT OF LONG-TERM HYPOPHYSECTOMY

MITCHELL SCOTT* and ROBERT BOLLA** Department of Biology, University of Missouri - St. Louis, St. Louis, MO 63121 (U.S.A.)

W. DONNER DENCKLA NIAAA, 12501 WashingtonAvenue, Rockville, MD 20852 (U.S.A.)

(Received December 19, 1978; in revised form March 28, 1979)

SUMMARY The effect of age on the ability of rats to raise antibodies to sheep erythrocytes and on the number of IgM plaque-forming cells in the spleen was investigated. An age-related decline in both parameters was observed. Additionally, the possibility that long-term hypophysectomy coupled with minimal hormone replacement therapy might result in a delay and/or reversal of the age-related decline in immune function was studied. It was observed that long-term hypophysectomized rats responded better to immunization with sheep erythrocytes than did their age-matched unoperated littermates. The possible relationship of this to aging is discussed.

INTRODUCTION Several theories have been advanced to explain the mechanism underlying the physiological, biochemical and molecular changes that occur as a function of age in mammalian systems [1]. Among these theories is the hypothesis that failures in the immune system may be a primary cause of aging [2-8]. It has been demonstrated that with advancing age mice can no longer develop a significant humoral response to T cell dependent antigens [5, 6, 9 - 1 2 ] . This failure is apparently due to cellular and not environmental factors [6]. The direct plaque-forming cell (PFC) assay has therefore been used to demonstrate that the number of antibody-producing cells decreases with age but that the amount of antibody produced per cell apparently remains constant [6, 9]. A possible cause for the decrease of antibody-producing cells in aged animals could be a decrease in

*Present address: Department of Microbiology and Immunology, Washington University, St. Louis, MO, U.S.A. **To whom correspondence should be addressed.

128 the number of T helper cells [10]. It has been demonstrated that in mice there is an agerelated decrease in the responsiveness of T cells to the T cell specific mitogen phytohemagglutinin [13, 14]. Investigations on the effect of age on cell mediated immunity, such as graft versus host reactions, allograft rejections, and lymphocyte mediated cytotoxicity, have also shown that T cell mediated immunity declines with age [7]. Age-related failure of the immune system has, therefore, been attributed to the loss of proliferative capacity of T cells that begins after puberty and corresponds to the onset of thymic involution and atrophy [15, 16]. Makinodan and Adler [10] have suggested that the imbalance of T and B cells that results from this loss of T cell proliferative capacity, while the B cell number remains relatively unchanged, could lead to all the deficiencies of the immune system associated with aging. Bilder and Denckla [17] demonstrated that the ability of rats to reject xenografts decreased with increasing age. This decline in cellular immune response could be delayed or reversed by long-term hypophysectomy of the rat [17]. That is, 16-month-old rats hypophysectomized at 6 to 8 months of age rejected the xenograft in a time period more closely related to 3- to 6-month-old unoperated rats than to that of unoperated agematched controls; however, graft rejection by 16-month-old rats hypophysectomized at 14 months of age was comparable to that of age-matched controls [17]. Based on these and other observations on the effect of long-term hypophysectomy on several physiological and biochemical parameters [17-20], Denckla has suggested that aging might be partially "regulated" by a factor released from the pituitary. He has proposed that the effect of this factor is long lived, and that this factor mediates some of its effects by causing end organ resistance to thyroid hormones. It is known that maintenance of T cell proliferation and function depends on thyroid hormones [21-24]. To investigate further the hypothesis that aging results from failure in the immune system, we have investigated the effect of age on T cell dependent immune function and on the size of the IgM plaque-forming cell population in the spleen of aging rats. Additionally, we examined the effect of long-term hypophysectomy of rats on these parameters. The present paper reports the results of these experiments.

MATERIALS AND METHODS Experimental animals All rats were female Sprague-Dawley CD strain derived rats obtained from Zivic Miller Laboratories, Pittsburgh, Pennsylvania. Hypophysectomized rats were littermates of the unoperated rats used in this study. These animals were hypophysectomized by Zivic Miller. Upon receipt these animals were maintained on a salt solution containing per liter 0.045 M Na3C6HsOT"2H20, 0.018 M KC1, 0.04 M Mg(C2H302)2, 3.5 mM CaC12, 0.3 M NaCI, 9 mM NaH2PO4, 1 mg corticosterone, 0.067 mg deoxycorticosterone, 0.15 mg thyroxine (salt solution A plus hormones) and fed Purina Lab Chow ad libitum. Seven days prior to initial immunization, these rats were injected daily with hormones as described in Table I. This daily injection

129 TABLE I HORMONE THERAPY GIVEN TO HYPOPHYSECTOMIZED AND CONTROL RATS Hormone*

t~g per kg body wt. per day* * .

Triiodothyronine Thyroxine Corticosterone Deoxycorticosterone Growth hormone* * *

8.3 25.0 300.0 20.0 100.0

*Hormones were given as a suspension in Planters Peanut Off and were injected subcutaneously using an 18-gauge needle. **Injections were begun 7 days before initial immunization and were continued on a daffy basis for the duration of the experiments. ***Growth hormone was prepared by one of the authors (W.D.D.) from bovine pituitary by isoelectric focusing. The preparation was homogeneous, as determined by polyacrylamide electrophoresis. schedule was maintained throughout the course o f experimentation. During hormone injection therapy the rats were maintained on salt solution A without hormones. Parallel treatment of unoperated, control, age-matched rats was carried out as described for the hypophysectomized rats. Unoperated rats which received no hormone treatment were maintained on distilled water and Purina Lab Chow. All rats were housed two per cage in filter-top cages. To avoid any variations due to diurnal rhythms, all hormone injections, immunizations and bleedings were performed between 6 and 8 p.m. H e m a g g l u t i n a t i o n assay

Rats were immunized with sheep erythrocytes (SRBC) and hemagglutination titers determined by methods modified from Kishimoto and Yamahura [5, 9]. Initial immunization was by intraperitoneal (i.p.) injection o f 0.8 ml o f a 10% suspension of SRBC. The cells were washed in phosphate buffered saline (0.9% NaC1 in 50 mM phosphate buffer, pH 7.2) before injection. Following immunization, the rats were bled under mild ether anesthesia from the lateral tail vein and the serum was collected. Hemagglutination titers were determined using standard microtiter methods. Serial dilution of immune serum to a 1024-fold dilution was used in all experiments. Standard methods were used to determine the ability o f serum from different aged and treated rats immunized to SRBC to agglutinate 25/al of a 1% suspension of SRBC in microtiter test plates following 2 h incubation at 25 °C.

P l a q u e - f o r m i n g cell assay

The number o f splenic lymphocytes capable of producing IgM coinplement fixing antibodies against SRBC was determined by the direct plaque-forming assay of Jerne e t al. [25] as modified by Kishimoto and Yamahura [9].

130 Rats were immunized with SRBC as described in the hemagglutination assay. Rats were killed by decapitation seven days after either primary or secondary immunization, since PFC response was maximum at day 6-8 (data not shown), and the spleen removed using aseptic technique. The isolated spleens were homogenized by gentle teasing on wire mesh in Media 199, Hanks base (Microbiological Associates, Bethesda, MD). The resulting cell suspensions were washed in Media 199 and the cells suspended gently with a Dounce homogenizer. Cell viability was determined by Trypan blue exclusion. Viability averaged 80%. Cell number was determined using a hemocytometer and a suspension containing 1 X 107 spleen cells was made. These cells were suspended in 0.7% Difco Agar containing 2 X 107 SRBC and this suspension layered over a solid 1.5% agar base. Following incubation for 1 h at 37 °C, the plates were overlaid with a 1:5 dilution of complement (Gibco, Grand Island, NY) and incubated for 1 h at 37 °C. The plates were then observed for plaques representing lysis caused by anti-SRBC antibodies produced by a single plasma cell. Plaques were scored and corrections were made for dilution of spleen cells. Only animals that appeared to be healthy and disease-free were used for these studies. No incidence of respiratory disease was identified in routine pathological examination of randomly selected animals at the Department of Veterinary Medicine, University of Missouri at Columbia. RESULTS

Effect of age on production of antibodies to SRBC For these studies rats of various ages were bled on days 7, 14 and 21 after primary immunization with SRBC. On day 21 a challenge immunization of SRBC was given, and the rats were bled one week later to measure a secondary response. As can be seen in Table II, neither the primary nor the secondary response was greatly altered from 21 days to 12 months of age. There was a slight decrease in response in 18-month-old rats to the primary immunization. The secondary response also began to decline in 18-month-old rats and by 24 months of age the rats did not have a significant response to a challenge immunization. A significant decrease in the immune response to the primary immunization was observed in 22-month-old rats, and by 24 months of age the response was minimal. Effect of age on the number of splenic plaque-forming cells The plaque-forming assay [25], set up as described in Materials and Methods, was used to determine if the number of plaque-forming cells (PFC) in the rat spleen decreased with age. Rats aged 3, 6 and 28 months were used for these studies since the greatest change in immune response to SRBC, as determined by the hemagglutination assay, did not occur until 22-24 months of age (Table II). There was no significant difference in the number of PFCs in rats 3 and 6 months of age, but by 28 months of age the number of PFCs per spleen decreased approximately threefold (Table III).

131

T A B L E II SRBC HEMAGGLUTINATION TITERS BY SERA FROM IMMUNIZED RATS

Hg titer (log2 antisera dilution)***

Age**

Days post immunization."

21 days 5weeks 3months 5months 6months 12 months 18 months 22 months 24 months

7

14

21"

28

5 -+0 6 ~1 5 -~0 5 ±0 6 ~0 5 ~0.5 4.6±0.5 3.6 ± 0 1

6.5-+0.5 6.0±0 5 -+0 5 ±0 5 ±0 5 ±0.5 4.6±0.5 3.6 +- 0

7 +-0 6.3± 1 4.6-+0.5 6 -+0 5 ±0 6 ±0 4 ±0 3.6 -+ 0 1

7.6-+0.5 6.6±0.5 6.8±1.0 6 -+0 6.7±0.5 6.4±0.5 4 ±0 4 -+ 0 1

*After 21 days, the rats were given a challenge immunization with SRBC. **3-5 rats were used for each data point. ***Dilution titer was converted to log 2 for statistical analysis and results given as mean ± standard error of the mean. That is, 5 ± 0 --- 2 s = 32-fold dilution of serum, 6.5 ± 0.5 = 26.5 = 90.5 ± 1.4fold dilution of serum, and 7 -+ 0 = 27 = 128-fold serum dilution. Difference significant at p < 0.001 as determined by Student's t-Test. T A B L E III DIRECT PLAQUE-FORMING CELL ASSAY FOLLOWING PRIMARY RESPONSE IN RATS TO SRBC

Age (months]

No. o f rats

Cells per spleen

PFC per 107 cells*

PFC per spleen

3 6 18

5 5 5

8.0 X 108 6.0 X 10 s 6.4 X 108

30.3 ± 2.8 38.2 ± 2.3 14.0 ± 0.2

2424 -* 224 2292 -+ 138 896 -+ 128

*Differences significant at p < 0.001 as determined by Student's t-Test. The p l a q u e - f o r m i n g assay detects only IgM-producing cells. It is possible, therefore, that the apparent age-related decrease in the n u m b e r o f PFCs in the spleen was a result o f an age-related delay in IgM p r o d u c t i o n b y the PFCs o f the spleen, rather than an actual cell loss. A second series o f e x p e r i m e n t s was done to investigate this possibility. F o r these e x p e r i m e n t s the initial i m m u n i z a t i o n with S R B C was followed 21 days later w i t h a challenge i m m u n i z a t i o n . The rats were killed 7 days later and the spleen cell population assayed for PFCs. Since the a n t i b o d y class p r o d u c e d in response to a secondary i m m u n i z a t i o n is primarily IgG, n o increase in the n u m b e r o f ' P F C s shown in Table III w o u l d be e x p e c t e d if actual cell loss occurred. As can be seen in Table IV, there was no significant increase in the n u m b e r o f splenic PFCs in response

to a challenge i m m u n i z a t i o n as c o m p a r e d to the n u m b e r

132 TABLE 1V EFFECT OF AGE ON THE NUMBER OF SPLENIC PLAQUE-FORMINGCELLS FOLLOWINGA SECONDARY CHALLENGEIMMUNIZATIONOF INTACTRATS WITH SHEEP ERYTHROCYTES Age* (months)

Cells per spleen

PFC per 10 7 cells* *

PFC per spleen

3 6 12 28

7 7 7 6.4

39.9-+ 5.3 46.2± 1.7 44.5 ± 11.1 16.0 ± 1.6

2798±379 3234± 119 3651 ± 227 1024 +-228

x 108 × 108 X 108 × 10a

*5 rats used at each data point. **Differences significant at p < 0.001 as determined by Student's t-Test. observed following the primary immunization (Table III). Additionally, the age-related decline in the number of PFCs remained unchanged with the challenge immunization. This would suggest, therefore, an actual age-related loss of PFCs, rather than an agerelated delay of IgM production by these cells. Effect o f long-term hypophysectomy and hormone therapy on immune response and splenic plaque-forming cells Since both the immune response of the rat to SRBC and the number of plasma cells in the spleen decreased with age, the possibility that hypophysectomy could delay or reverse the decline in immune function was investigated. Rats were hypophysectomized at 6-12 months of age and maintained as described in Materials and Methods. The effects of specific hormones on the parameters measured were determined by deleting specific hormones from the water and from the complete hormone therapy (Table I). As a control, intact unoperated rats were given the same hormone therapy. Administration of complete hormone therapy to unoperated 22-month-old rats immunized with SRBC had no effect on the SRBC hemagglutination titer of the sera (Table V). Hypophysectomy at 6-12 months followed by complete hormone therapy until the rats were 18-24 months of age resulted in a delay in and/or reversal of the agerelated decline in hemagglutination titer. That is, rats hypophysectomized at 6-12 months of age and given complete hormone therapy as described in Table I, when studied at 18, 22 or 24 months of age had serum hemagglutination titers for SRBC comparable to animals younger than 18 months of age (Tables II and V). No hemagglutination was observed with sera from non-immunized rats. To determine if this greater immune response to SRBC in hypophysectomized rats was accompanied by an increased number of PFCs in the spleen, the direct plaque. forming assay was done using 28-month-old rats hypophysectomized at 12 months of age and maintained as described in Materials and Methods. As can be seen in Table VI, administration of complete hormone therapy, thyroxine and triiodothyronine alone, growth hormone alone or corticosterone-deoxycorticosterone alone had no effect on the

133

TABLE V EFFECT OF HYPOPHYSECTOMY AND SUBSEQUENT HORMONE THERAPY ON SRBC HEMAGGLUTINAT1ON TITERS BY SERA FROM IMMUNIZED RATS

Hg titer (log2 an tisera dilution)**

Age*

Days post immunization:

18 months chronic hypophys. + hormones 22 months chronic hypophys. + hormones 24 months chronic hypophys. + hormones 22 months intact + hormones Normal rat serum

7

14

21

28

6.3 -+ 1 4.68 +- 0.47 3.6 + 0 1

7.3 -+ 1 5.0 -+ 0.81 2.4 -+ 0.5 -

6.55 +- 0.5 4.68 -+ 0.47 3.6 +- 0 1

7.58 5.75 6 3.6 1

+- 0.5 + 0.47 -+ 0 + 0.5

*3-5 rats were used at each data point. **Dilution titer was converted to logs for statistical analysis and the results reported as mean -+ standard error of the mean. See Table 11 for further explanation. Differences significant at p < 0.001 as determined by Student's t-Test. TABLE VI EFFECT OF LONG-TERM HYPOPHYSECTOMY ON THE CONCENTRATION OF SPLENIC PLAQUE-FORMING CELLS IN RATS RECEIVING VARIOUS HORMONE THERAPIES

Age (monthsJ 28 28 28 28 28 28 28 28 28 22 22

Hypophyseetomy*

1

m

b

+ + + + +

Treatment**

Cells per spleent

none T3-T4 corticosterone, deoxycorticosterone growth hormone complete complete corticosterone Ta-T 4 growth hormone complete complete

6.4 X 109 4.4 X 109 3.0 X 109 2.6 0.9 6.9 6.6 0.8 5.0 1.5 7.0

× X X × × X × X

109 109 108 108 10 a 10 s 109 10 a

PFC per spleen

896 -+ 128 880 + 96 390 +- 30 416 747 2311 726 1206 923 1316 3241

-+ 52 -+ 216 -+ 118 -+ 66 -+ 23 -+ 80 -+ 5 0 0 t t -+ 3751t

*+ indicates that rats were hypophysectomized at 6 - 8 months of age as described in Materials and Methods; - indicates hormone treatment of unoperated intact rats. **Hormone treatment as in Table I. When specific hormones were used, the other hormones were removed from the injection schedule. tDifferences significant at p < 0.001 as determined by Student's t-Test. t t l n d i c a t e s response to secondary challenge immunization with sheep erythrocytes. n u m b e r o f PFCs per spleen in i n t a c t u n o p e r a t e d 28-month~old rats. Quite t h e o p p o s i t e was true w h e n t h e s e h o r m o n e s , singly or in c o m b i n a t i o n , w e r e given t o the h y p o p h y s e c t o m i z e d rats. C o m p l e t e h o r m o n e t r e a t m e n t given these rats r e s u l t e d in an increase in splenic PFCs to a level c o m p a r a b l e t o t h a t o b s e r v e d in 3 - 6 - m o n t h - o l d ' i n t a c t rats receiving n o e x o g e n o u s h o r m o n e s (Tables III a n d VI). The n u m b e r o f P F C s in 2 8 - m o n t h - o l d h y p o -

134 physectomized rats given only thyroxine and triiodothyronine increased approximately twofold as compared to 28-month-old control rats. A lesser, but significant, increase was observed in hypophysectomized rats given growth hormone alone. Corticosterone administration alone to these rats had no effect on the number of splenic PFCs (Table VI). These results suggest, therefore, a nearly additive effect of thyroid hormones and of growth hormone in restoring the number of PFCs in the spleen to a value observed in rats younger than 12 months of age.

DISCUSSION Several workers have demonstrated an age-dependent decrease in immune response in several species including man, mice and hamster [2-15]. In many cases the basis for this decline has been shown to be due to a decrease in proliferative capacity and function of T cells [ 10-12, 15, 16]. Since T cells must interact both with antigen and with B cells in order to mount a humoral response to T-dependent antigens, a decrease in T cell function would lead to a decline of the humoral response. Further evidence that changes occur in T cells with age has been shown by an age-related decrease in proliferative capacity of 0 positive cells [16] and by an age-related decrease in the number of cells responsive to T specific mitogens [21]. An age-related decline in B cell function or number has not been demonstrated [4]. The results of the present study, using rats as a model system, are comparable to similar studies on the effect of age on the immune response reported for other species. That is, there is an age-related decline in the humoral immune response. Most interesting, however, is the fact that the decrease in the immune response of rats to SRBC does not begin until approximately 18 months of age, and by 24 months of age the rat is virtually unable to mount an immune response to SRBC. Coincident with the decrease in immune response to SRBC shown in this study, an age-related decrease in the number of IgM anti-SRBC producing plasma cells has been observed. This might suggest a decreased function of T cells with age but is not conclusive in this regard. To examine this question (namely, that the age-related decrease in humoral response to SRBC is due to a loss of T helper cells for the antibody-producing B cell population), the number of cells possessing the 0 antigen and the number of cells responsive to T specific mitogens must be determined. The observations of decreased immune function and of decreased T cell function have led Walford to hypothesize that failure in the immune system may be one of the primary causes of aging [2]. Following involution of the thymus subsequent to puberty, the T cell population becomes static. Therefore, many of the theories of aging, which suggest that aging results from the damage of cellular populations, could be applied to age-related changes in the immune system [ 15 ]. It could be argued, therefore, that either the loss of function of T cells or the actual loss of T ceils from the population without replacement could upset the maintenance of the organism within its environment and result in the loss of the ability to survive.

135 A complementary approach which suggests a mechanism for a decrease in function of the immune system is that proposed by Denckla [17-20]. Both growth hormone and thyroid hormone have been shown to be required for normal immune system function and further proliferation o f T cells [22-24, 26]. Denckla [20] has proposed, therefore, that the pituitary releases a factor which decreases the ability of cells to respond to thyroxine and thus affects several physiological functions [17-20]. If T cells are also under the control of this proposed factor, they would lose the ability to respond to thyroxine and thus to thyroxine-dependent functions. It might be suggested that if the age-related loss of T cell dependent immune function that has been observed [10-12] was due to the proposed pituitary factor, removal of the pituitary coupled with replacement therapy of thyroid hormones would restore the function of the T cells. The data presented in this paper tend to give some support to this hypothesis. Hypophysectomy, followed with hormone replacement therapy, resulted in a delay in the decline in, or the restoration of, the response to SRBC and the number of splenic PFCs. Thus, in the hemagglutination studies the long-term hypophysectomized rats possessed serum titers of antibodies to SRBC greater than those of their intact littermates and comparable to those of younger rats. Hormone therapy given to intact aged rats had no effect on th e hemagglutination titers, thus suggesting that the cells of the immune system in these rats could not respond to the hormones. Additional evidence for the possible involvement of the pituitary in the function of the rat immune system can be seen in the results of the direct plaque-forming assay. It was observed that long-term hypophysectomy and supplemental hormone therapy result in an increase in the number of PFCs in these rats at'28 months of age to a value found in rats of an age younger than that at the time of hypophysectomy. Hormone supplements given to intact rats had no effect on the number of plaque-forming cells, indicating again a possible loss of cellular response to exogenous hormones as a function of age. In addition, the results of the individual hormones given to the hypophysectomized rats indicate that both thyroxine and growth hormone are required for the observed response and that they act in an additive fashion. Similar additive effects of these hormones on biochemical function in aged hypophysectomized rats have been observed in the case of the activity of RNA poly~nerase (Bolla and Denckla, manuscript in preparation). It can be concluded from the studies presented in this paper that some aspects of immune function in rats decline with age and that these decreases are either delayed or reversed by long-term hypophysectomy and subsequent hormone treatment. Further studies are needed, however, to determine the involvement of long-term hypophysectomy and hormone treatment with the reported age-related changes in immune function.

ACKNOWLEDGEMENTS This research was supported in part by grants from the Glenn Foundation for Medical Research and from the University of Missouri Faculty Research Council.

136 REFERENCES M. S. Kanungo, A model for aging, J. Theor. Biol., 53 (1976) 253-261. R. L. Walford, The Immunologic Theory of Aging, Munksgaard, Copenhagen, 1969. R. L. Walford, Immunologic theory of aging: current status, Fed. Proc., 33 (1974) 2020-2027. T. Makinodan and E. Yunis (eds.), Immunology and Aging, Plenum Press, New York, 1977. S. Kishimoto, I. Tsauyuguchi and Y. Yamahura, Immune response in aged mice, Clin. Exp. Immunol., 5 (1969) 525-530. 6 A. A. Nordin and T. Makinodan, Humoral immunity in aging, Fed. Proc., 33 (1974) 2033-2035. 7 0 . Stutman, Cell-mediated immunity and aging, Fed. Proc., 33 (1974) 2028-2032. 8 R. Good and E. J. Yunis, Association of autoimmunity, immunodefieiency and aging in man, rabbits and mice, Fed. Proc., 33 (1974) 2040-2050. 9 S. Kishimoto and Y. Yamahura, Immune responses in aged mice: changes of antibody-forming cell precursors and antigen reactive cells with aging, Clin. Exp. Immunol., 8 (1971) 957-962. 10 T. Makinodan and W. Adler, The effects of aging on the differentiation and proliferation potentials of cells of the immune system, Fed. Proc., 34 (1975) 153-158. 11 T. Makinodan and W. J. Peterson, Relative antibody-forming capacity of spleen cells as a function of age, Proc. Nat. Acad. ScL U.S., 48 (1962) 234-238. 12 T. Makinodan, F. Chino, W. E. Lever and B. S. Brewen, The immune systems of mice reared in clean and dirty conventional laboratory farms. II. Primary antibody-forming activity of young and old mice with long life spans, J. Gerontol., 26 (1971) 508--514. 13 D. Amerding and D. Katz, Activation of T and B lymphocytes in vitro. I1. Biological and biochemical properties of an allogenic effect factor (AEF) active in triggering specific B lymphocytes, J. Exp. Med., 140 (1974) 19-37. 14 M. Mathies, L. Lipps, G. S. Smith and R. L. Watford, Age-related decline in response to phytohemagglutinin and pokeweed mitogen by spleen cells from hamsters and a long lived mouse strain, J. Gerontol., 28 (1973) 425-430. 15 K. Hirokawa and T. Makinodan, Thymic involution effect on T cell differentiation, J. Immunol, 114 (1975) 1659-1664. 16 M. Gerbase-De Lima, J. Wilkinson, G. S. Smith and R. L. Walford, Age-related decline in thymicindependent immune function in a long lived mouse strain, or. Gerontol., 29 (1974) 261-268. 17 G. E. Bilder and W. D. Denckla, Restoration of ability to reject xenografts and clear carbon after hypophysectomy of adult rats, Mech. Ageing Dev., 6 (1977) 153-163. 18 W. D. Denckla, Role of the pituitary and thyroid glands in the decline of minimal 02 consumption with age, J. Clin. Invest., 53 (1974) 572-581. 19 W. D. Denckla, A time to die, Life ScL, 16 (1975) 31-44. 20 W. D. Denckla, Interactions between age and the neuroendocrine and immune systems, Fed. Proc., 37 (1978) 1263-1267. 21 G. B. Price and T. Makinodan, Immunologic deficiencies in senescence I. Characterization of intrinsic deficiencies, J. Immunol., 108 (1972) 403-412. 22 P. M. Lundin, Anterior pituitary gland and lymphoid tissue growth, Acta Endocrinol., 40 (1958) suppl. 128. 23 U. Ernstrom and B. Larsson, Thymic and thoracic duct contribution to blood lymphocytes in normal and thyroxin treated guinea-pig, Acta Physiol. Scand., 66 (1966) 189-195. 24 N. Fabris, Immunodepression in thyroid-deprived animals, Clin. Exp. Immunol., 15 (1973) 601-611. 25 N. K. Jerne, C. Henry, A. A. Nordin, H. Fugi and I. Lefkovitz, Plaque forming cells-methodology and theory, Transplant. Res., 18 (1974) 130-191. 26 L. Piantanelli and N. Fabies, Hypopituitary dwarf and athymic mice in the study of relationship among thymus hormones and aging, in D. Bergsma and D. Harrison (eds.), Genetic Effects on Aging, Less, New York, 1978, pp. 315-334. 1 2 3 4 5

Age-related changes in immune function of rats and the effect of long-term hypophysectomy.

Mechanisms of Ageing and Development, 11 (1979) 127-136 127 ©Elsevier Sequoia S.A., Lausanne - Printed in the Netherlands AGE-RELATED CHANGES IN IM...
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