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Transplantation of pituitary grafts fail to restore immune function and to reconstitute the thymus glands of aged mice
Mechanisms of Ageing and Development, 56 (1990) 11--22 Elsevier Scientific Publishers Ireland Ltd.
11
TRANSPLANTATION OF PITUITARY GRAFTS FAIL TO RESTORE IMMUNE FUNCTION AND TO RECONSTITUTE THE THYMUS GLANDS OF AGED MICE
RICHARD J. CROSS j'*, JENNIFER L. CAMPBELL', WILLIAM R. MARKESBERYb and THOMAS L. ROSZMANj •Department of Microbiology and Immunology and bSanders-Brown Center on Aging and the Departments of Pathology and Neurology, University of Kentucky Medical Center, Lexington K Y 40536
(U.S.A.) (Received August 12th, 1989) (Revision received April 30th, 1990)
SUMMARY
There is evidence to indicate that the neuroendocrine and immune systems can interact. Thus, neuroendocrine hormones can modulate a variety of immune functions and there have been attempts to manipulate the neuroendocrine system of aged animals to enhance immune function. We have previously shown that the transplantation of a syngeneic pituitary gland under the kidney capsule of young adult mice elevates serum prolactin and enhances immune responsiveness. In the present study pituitary glands were transplanted under the kidney capsule of 22-month-old mice to determine if this maneuver can enhance a number of immunologic parameters. The results demonstrate that aged animals bearing transplanted pituitary grafts for 10 days did not exhibit any enhancement in their primary antibody response to sheep red blood cells, splenic T or B-cell mitogen responsiveness or restoration of thymic architecture. When these immunologic assessments are performed on animals bearing pituitary grafts for 28 days, the IgM and IgG primary antibody responses and splenic T-cell responsiveness are enhanced but repopulation of the thymus still does not occur. Importantly, this enhancement does not restore immunocompetence to levels observed in young mice.
Key words: Aging; Immune response; Prolactin; Pituitary graft; Thymus
*To whom aft correspondence should be addressed at: Department of Microbiology and Immunology, MS 401, University of Kentucky Medical Center, 800 Rose St., Lexington, KY 40536-0084, U.S.A. 0047-6374/90/$03.50 Printed and Published in Ireland
© 1990 Elsevier Scientific Publishers Ireland Ltd.
12
INTRODUCTION
It is well-documented that immune function declines with advancing age. This decline is characterized by thymic involution, impaired T-cell proliferation [1], diminished interleukin-2 (IL-2) synthesis [2], impaired humoral antibody production [31 and cell-mediated immune responsiveness [4]. These deficits may result from intrinsic defects within the specific cellular compartments of the immune response [5] or may be due to extrinsic changes which impinge on immunocompetent cells and thus impair function [6]. One such group of extrinsic factors are those derived from the neuroendocrine system. Pituitary hormones appear to be important not only in the modulation but also the development of the immune system [7,8], particularly thymic function. It is possible to successfully restore immunocompetence in hormonally deficient mice. Using hypopituitary dwarf mice which have abnormal thymic development and subsequent decreased immune function, Fabris et al. [8] have restored immune responsiveness by administering growth hormone (GH) and thyroxine or GH alone. Similarly, age-related impairment of immune responsiveness can be restored by two different manipulations of the neuroendocrine system. Utsuyoma and Hirokawa [91 gonadectomized old male and female mice and observed a regeneration of the thymus as well as an improvement in the humoral antibody response to sheep red blood cells (SRBC). Thymic regeneration was more pronounced in male mice while females showed the greatest enhancement of the humoral antibody response, lmmunofluorescence analysis showed an increase in the percentage of splenic T-cells and B-cells, although the response to SRBC in gonadectomized old mice never approached the response found in young mice. This enhancement of immune function may result from the observed decrease in secondary sex hormones (testosterone, estradiol) or a concomitant increase in luteinizing hormone as a result of the loss of negative feedback, although these levels were not measured. There also is evidence to suggest that neuroendocrine manipulation can improve the immune response status of aged animals. There are reports that restoration of Tcell function can be achieved by implanting G H 3 tumor cells which secrete both GH and prolactin into aged rats [10]. Two months after implantation, these 18-monthold rats had repopulated their thymus glands to near normal histology and the thymocytes from these animals had normal mitogen-induced proliferative responses and increased IL-2 synthesis compared to non-implanted 18-month-old rats. Similarly treated 24-month-old rats also exhibited restoration of thymus cellularity and thymocyte proliferative responses although the reconstitution of immune function in these animals was not as complete as that found in 18-month-old animals. These neuroendocrine effects on the immune response of aged mice were more directly examined by treating old rats with 1.5 mg of G H / d a y for 5 weeks [11]. This treatment enhanced both the proliferative response of spleen cells to mitogens as well as natural killer cell activity, but, reconstitution of thymus gland, or I1-2 pro-
13 duction did not occur following G H treatment. Because control groups using spleen cells from young rats were not included, it is difficult to determine if G H treatment induced a full recovery of immunocompetence. We have previously shown that elevation of serum prolactin by implanting syngeneic pituitary grafts under the kidney capsule enhances the humoral antibody response of young mice [12]. The present studies were performed to determine whether elevated serum prolactin resulting from grafting aged mice with pituitary glands could restore immune responsiveness. The data presented indicate that elevation of prolactin does not restore antibody responsiveness, splenic mitogen responsiveness nor thymus cellularity in 22-month-old mice ten days following pituitary gland transplantation. However, when pituitary grafts remain in place for 28 days antibody responsiveness is enhanced compared to sham grafted animals, but this enhancement is considerably less than response observed In young animals. Moreover, thymic regeneration does not occur. MATERIALSAND METHODS
Animals Female C57BL/6 22-month-old female mice were obtained from the National Institute on Aging. Young, 55--70-day-old C57BL/6 female controls were purchased from Charles River Laboratories, Wilmington, MA. The animals were maintained on sterile lab chow and water ad libitum and housed in Microisolator boxes. Reagents SRBC were obtained from one animal maintained by the Division of Laboratory Animal Resources, University of Kentucky. The mouse prolactin radioimmunoassay kit was provided by Dr. A.F. Parlow through the Pituitary Hormone and Antisera Center, Torrance, CA. Calculations of serum prolactin were made using the mouse prolactin standard provided with the kit as previously described [ 11 ]. Pituitary gland transplantation Pituitary gland transplantation was performed as described previously [12]. Briefly, mice were anesthetized with sodium pentobarbital, the skin and body wall excised and kidney exposed. A syngeneic pituitary gland was placed under the kidney capsule and the body wall and skin were closed with silk sutures. Surgical control groups (sham) were treated identically except for the implantation of the graft. Immunization and PFC assay Mice were immunized with 2 x 10s SRBC (i.p.) 14 or 28 days after pituitary gland transplantation. Mice were killed by cervical dislocation under metofane anesthesia. The spleens were teased into a single cell suspension, counted on a hemocytometer in 3°7o acetic acid to lyse erythrocytes and adjusted to the proper concentration
14
for the assay. Direct plaque forming cells (DPFC) which secrete lgM were enumerated using the method of Cunningham and Szenberg [13]. Indirect lgG secreting PFC were developed with rabbit anti-mouse IgG antibody at a final dilution of 1/50.
Mitogen-inducedlymphocyte blastogenesis Dispersed spleen cells were cultured in complete medium (Dulbecco's modified MEM supplemented with penicillin-streptomycin, glutamine, non-essential amino acids, vitamins, 10070 heat inactivated fetal calf serum and 50 mM 2-mercaptoethanol) with the T-cell mitogen concanavalin A (Con A, 0.5/~g/well, Sigma Chemical Co., St. Louis, MO) or lipoplysaccharide (LPS, 20/~g/well, Difco, Detroit, MI) as described previously [14]. Briefly, 2 x l05 spleen cells were cultured in flat-bottom microtiter plates with complete medium and mitogen. Cell cultures were performed in triplicate. The plates were incubated at 37 °C in a 95070 air/5070 CO 2 atmosphere for 56 h at which time 0.25/~Ci [3H]thymidine (spec. act. 6.7 Ci/mmol, New England Nuclear, Boston, MA) was added in 25/A. Cells were harvested 16--18 h later using an automated cell harvester and [3H]thymidine incorporation into DNA was assayed by liquid scintillation counting.
Thymus morphologicalanalysis The complete thymus was removed at the time of death and placed in 4°70 formaldehyde. Following fixation specimens were processed in the standard manner and embedded in paraffin. Sections (8/~m) were cut and stained with hematoxylin and eosin. Microscopic review revealed no significant alterations in the cortex or medulla of mice receiving pituitary grafts or in control animals. Thymus size was determined utilizing a Kontron Imager Processing System interfaced with an Olympus Vanox 5 microscope. Sections were through the widest portion of the embedded specimen. Thymus images were enhanced and discrimination depended on the difference in grey level value. The areas of each thymus were individually traced. Computer measurements were converted to mm 2 for presentation.
TABLE I T H E H U M O R A L A N T I B O D Y RESPONSE 1N Y O U N G MICE RECEIVING P I T U I T A R Y G R A F T S Treatment group
n
D P F C / I 0~ n u c l e a t e d spleen cells
Pituitary graft Sham graft Normal
5 5 5
550 ± 52 333 +- 46 192 ± 17
Each animal was grafted with a pituitary gland 10 days prior to immunization with 2 x l(Y SRBC. DPFC were assayed on day 5 after immunization. The antibody response of mice receiving a pituitary graft and s h a m grafted mice were significantly different ( P < 0.001).
15 In a separate series o f experiments, thymic reconstitution was evaluated in mice which had been transplanted 28 days previously with PG. Thymocytes were dispersed into a single cell suspension and nucleated cells were counted on a hemocytometer. Statistical analysis Data were analyzed using Student's two-tailed t-test for independent means. All experiments were repeated a minimum of three times and only the results from one representative experiment are shown. RESULTS Effect o f transplanting pituitary glands on the primary antibody response in young and aged mice Experiments were performed to determine the effects of transplanting pituitary glands into young and aged mice on the subsequent primary antibody response to SRBC. As shown in Table I placement o f pituitary glands under the kidney capsules of young adult mice for 10 days results in an enhanced primary antibody response to SRBC in these animals compared to sham grafted or normal control animals. The serum prolactin levels in these animals was 2--3-fold above normal basal levels [12]. The data presented in Table II show that immunization of aged mice in which pituitary glands were transplanted 10 days prior to immunization elevates the serum level of prolactin greater than two fold above that found in aged normal mice, but the splenic DPFC (IgM) responce is not elevated concomitantly. To more fully investigate the effects of elevated prolactin on the humoral immune responsiveness of aged animals the following experiments were performed. Aged
TABLE II THE EFFECTSOF A PITUITARYGRAFTON THE DPFC RESPONSE IN 24-MONTH-OLDMICE Treatment group
n
Serum prolactin (ng/mO
DPFC/I O~ nucleated spleen cells ±S.E.M.
Aged, pituitary graft Aged, shamgraft
8 5 5 5
169 - 24 45 ± 14 70 _+ 9 20 _+. 3
31 :l: 10 26 :l: 17 22 _+ 6 365 __. 86
Aged, normal
Young, normal
Aged mice were transplanted with a pituitary gland ten days prior to immunization. Direct plaque forming cells (DPFC) were determined 4 days after immunization. Statistical analysis indicates that none of the antibody responses of the three aged groups of mice were different. The antibody response elicited by younganimals differed from that observed with the aged group of mice(P < 0.001).
o.
5o'-
100.
150.
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$
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6
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# days after Immunization
4
pituitary graft
7
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0
500
1000~
1500
2000
Z500
3000
3500 young mice
# days after immunization
_y"
e~O
(.)
Fig. 1. The lgM primary antibody response of aged (22-month) or young (3 - 4 - m o n t h ) mice receiving pituitary grafts. Sham operated mice served as contrors. Twenty-eight days following transplantation the mice were immunized with 2 x lO~ SRBC and the direct plaque forming cells (DPFC) were determined on days 3--7. Statistical analysis indicates that the antibody response of animals with pituitary grafts is signi ficantly enhanced on days 6 and 7 (P < 0.05).
C..) L~. (1. r~
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300
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350
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50
90.
120.
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S
7
# days a f t e r i m m u n i z a t i o n
5
plhJItary graft ~ a m graft
CA)
0
SO0.
1000.
1500-
2000-
2500-
3000 0~0
I
# days after immunization
young mloe
(e)
Fig. 2. The lgG primary antibody response of aged (22-month) or young (3--4 month) mice receiving pituitary grafts. Twenty-eight days following transplantation the mice were immunized with 2 × 10s SRBC and indirect plaque forming cells (IPFC) were determined on days 5--7. The antibody response of animals with pituitary grafts is significantly enhanced on days 6 and 7 ( P < 0.05) as compared to sham control values.
Q"
0
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J --4
18 160
(A) i 14-0 120
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(B)
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100
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young
oged
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Fig. 3. The mitogen responsivenessof spleen cells obtained from aged (22-month) or young (55--70-day) mice receivingpituitary grafts. Either l0 days or 28 days after transplantation spleen cells were obtained from these animals or sham controls and cultured with the T-cell mitogen Con A (0.5 tag/well, panel A) or the B-cell mitogen LPS (20 tag/well, panel B). The Con A response of spleen cells from young, pituitary grafted mice is significantly enhanced compared to sham control values (P < 0.01). The Con A responsiveness of spleen cells obtained from aged animal receiving pituitary grafts l0 days previously did not differ from sham control values while the Con A responses of spleen cells obtained from aged animals bearing these grafts for 28 days did (P < 0.05). Transplantation of pituitary glands into either young or aged animals does not enhance the LPS responsivenessof their spleen cells studied aftcr l0 days.
mice were t r a n s p l a n t e d with P G 28 days prior to i m m u n i z a t i o n a n d the kinetics of the p r i m a r y a n t i b o d y response were evaluated. The data in Fig. l a show that transp l a n t a t i o n o f pituitary g l a n d does e n h a n c e the D P F C response of aged mice compared to sham controls a n d that, the kinetics o f the response is shifted to the right. However, this e n h a n c e m e n t is still less t h a n 10°70 o f the response observed with y o u n g a n i m a l s (Fig. lb). Similar results are o b t a i n e d with the I P F C (IgG) response except that the kinetics o f I P F C a p p e a r a n c e is not altered (Fig. 2a). The I P F C response o f aged a n i m a l s receiving P G does not a p p r o a c h that o f the y o u n g a n i m a l s (Fig. 2b).
Effect o f transplanting pituitary grafts on T-cell and B-cell mitogen responsiveness o f aged animals T o d e t e r m i n e the effects o f t r a n s p l a n t i n g pituitary grafts on T cell a n d B-cell f u n c t i o n in both y o u n g a n d old mice, spleen cells from pituitary gland t r a n s p l a n t recipients a n d sham controls were cultured in vitro with the T-cell mitogen, C o n A or the B-cell m i t o g e n , LPS. The data in Fig. 3a show that t r a n s p l a n t a t i o n of pitui-
19 TABLE 111 THYMUS SIZE IN AGED MICE RECEIVING PITUITARY GLANDS Treatment
n
Thymus size
Thymocyte no.
group Old, pituitary graft Old, S h a m Old, Normal
8 4 6
(ram2)
( × 109
4.83 + 0.93 6.63 ± 0.43 5.56 :I: 0.97
102 4- 18 145 4- 22 ND
Thymus size was measured on formalin-fixed hematoxylin and eosin stained sections from mice which received a pituitary graft 2 weeks prior to sacrifice. Thymus size (mean :1: S.E.M.) using image analysis on each individual thymus were calculated. Thymus nucleated cell counts were performed on mice which had received a pituitary graft 28 days prior to sacrifice. There was no significant difference among the values obtained for thymus size or thymocyte number in the three different treatment groups. ND = not done.
tary gland can enhance splenic T-cell mitogen responsiveness in young animals, but has no effect on B-cell responsiveness (Fig. 3b). The mitogen responses of spleen cells obtained f r o m aged mice which had received pituitary grafts or sham operations were determined (Fig. 3 a,b). The results demonstrate that the Con A (Fig. 3a) or LPS (Fig. 3b) responsiveness of spleen cells from aged animal regardless of whether they had received P G is markedly decreased compared to values obtained with young animals. Furthermore, the results show that the mitogen responsiveness o f spleen cells obtained from either animals with pituitary grafts or sham control animals are virtually identical. The Con A responsiveness of spleen cells obtained from aged animals receiving pituitary grafts is enhanced approximately 2-fold at 28 days but not 10 days after the transplant when compared to sham control values (Fig. 3a). There is no difference between the LPS responsiveness of spleen cells obtained from aged or young animals receiving pituitary grafts and sham control animals. Effect o f transplantation o f a pituitary gland on reconstitution o f the thymus Thymus size was evaluated on hematoxylin and eosin stained thymus tissue from pituitary gland transplanted, sham and normal old mice. The data in Table III indicate that thymus glands from mice with elevated serum prolactin are no larger than thymus glands from sham control or normal old mice. Similar results are seen in mice which have been transplanted for 28 days with pituitary grafts. In these experiments the number of nucleated cells in the thymus from aged mice with pituitary grafts and sham controls also was quantified. The data show that animals receiving pituitary grafts do not exhibit thymic reconstitution, because the number of thymocytes obtained from thymus glands of pituitary transplanted or sham controls do not differ (Table III).
20 DISCUSSION The results of this study demonstrate that transplantation of pituitary glands under the kidney capsules of old animals can modulate the primary antibody response. While aged mice bearing transplanted pituitarys for l0 days did not evidence an enhanced antibody response, those which carried the transplanted pituitarys for 28 days were elevated when compared to values obtained from sham controls. Both the IgM and IgG antibody responses were increased in these transplanted aged animals. Not only was the IgM antibody response enhanced to more (approximately 27-fold) than the lgG antibody response (7-fold) but the peak day of antibody production was 1 day later. Although aged animals bearing pituitary grafts exhibited enhanced IgM and IgG primary antibody responses, these responses were still approximately 10-fold lower than that observed in young animals. The explanation for the enhanced antibody response observed in aged animals with pituitary transplants will require a more detailed study to delineate the mechanisms responsible. The results also revealed no change in the thymic architecture or thymocyte numbers in transplanted aged animals as compared to sham control animals. This suggests that the thymus has not been reconstituted and is not exporting lymphocytes. It is possible that the thymic stromal cells are producing hormones which influence the function of T-cells [l 5]. The data do provide some support for this notion in that the T-cell responsiveness of spleen cells from aged animals which carried pituitary grafts for 28 days were enhanced relative to those values obtained from sham control animals. However, this T-cell responsiveness observed was approximately 3-fold less than the response observed with spleen ceils from young animals. Previous reports by others [10] have shown that the implantation of a GH and prolactin secreting G H 3 cell line reconstitutes the thymus and restores T-cell function in old rats. The discrepancies between these observations and those in the present study may relate to a number of potentially important differences. First, in our studies we employed a mouse model while in the other study aged rats were used. Secondly, the source of the transplanted tissue employed is different in that Kelley and co-workers [10] transplanted allogeneic GH~ tumor cells into aged rats while we employed pituitary grafts. Finally, it is known that the GH~ tumor cells produce both GH and prolactin as well as probably other undefined factors [16] while the pituitary glands secrete only prolactin after approximately one week following transplantation into animals [17]. It is possible that GH is a more potent immunoenhancer than prolactin. Indeed, it has been demonstrated that administration of ovine GH to aged rats does enhance mitogen induced T-cell proliferation and natural killer cell activity [l 1], but these animals failed to exhibit a restoration of thymic architecture. Finally, the immune potentiation observed in aged rats the GH 3 tumor cells could result from some other immunoenhancer produced by these tumor cells or the result of the synthesis of cytokines by lymphoid cells mounting an immune attack against these allogeneic cells.
21
thesis o f cytokines by lymphoid cells mounting an immune attack against these allogeneic cells. It is clear that young adult mice receiving pituitary grafts have markedly enhanced antibody responses [12] while pituitary grafted aged animals have for the most part lost this ability. The explanation for this age-related difference is presently unknown. One possibility is that lymphocytes obtained from aged animals may not possess prolactin receptors as do lymphocytes from young adults [18] or that other intrinsic age related changes have taken place which prevent the elevation of prolactin from enhancing immune function [5]. Conversely, extrinsic changes such as the alteration of other hormone levels may have occurred which prevent prolactin from enhancing immune function [6]. For example, prolactin may be affecting a secondary sex hormones or other hormones which can influence lymphocyte function [l 9]. Alteration of the activity of these secondary hormones may override the immunopotentiating effects of prolactin on immune function. In summary, the data indicate that young animals receiving pituitary grafts exhibit a marked increase in their humoral antibody response to SRBC. When similar experiments were performed on aged animals, the pituitary grafted animals had an increase in their antibody responses as compared to sham control animals. However, this manuever did not restore the antibody response to levels observed in young animals. Collectively these data indicate that increasing prolactin levels in aged mice does not restore the immune function or thymic architecture to that of young mice.
ACKNOWLEDGEMENTS
Supported in part by USPHS grant numbers NS 17423, NS 22512. REFERENCES 1 2
3 4 5 6 7
8
B.A. Helfrich, M. Segre and D. Segre, Age-related changes in the degeneracy of the mouse T-cell repertoire. Cell. Immunol., 118 (1989) I--9. M.L. Thoman and W.O. Weigle, Partial restoration of Con A-induced proliferation, IL-2 receptor expression and IL-2 synthesis in aged murine lymphocytes by phorbol myristate acetate and ionomycin. Cell. Immunology, 114 (1988) l - I I. J.F. Albright and T. Makinodan, Growth and senescence of antibody forming cells. J. Cell. Physiol., (Suppl. 1)(1966) 185--206. W.M. Adler, T. Takiguchi and R.T. Smith, Effect of age upon primary alloantigen recognition by mouse spleen cells. J. lmmunol., 107(1971) 1357--1362. G.B. Price and T. Makinodan, Immunologic deficiencies in senescence. I. Characterization of intrinsic deficiencies. J. lmrnunol., 108 (1972) 403--412. G.B. Price and T. Makinodan, Immunologic deficiencies in senescence. II. Characterization of extrinsic deficiencies. J. lmmunol., 108 (1972) 41 3--417. N. Fabris, W. Pierpaoli and E. Sorkin, Hormones and the immunologic capacity. Ill. The immunodeficiency disease of the hypopituitary Snell-Bagg dwarf mouse. Clin. Exp. Immunol., 9 0971) 209--225. N. Fabris, W. Pierpaoli and E. Sorkin, Hormones and the immunologic capacity. IV. Restorative effects of developmental hormones or of lymphocytes on the immunodeficiency syndrome of the dwarf mouse. Clin. Exp. lmmunol. 9(1971)227--240.
22 9 10
I1
12 13 14 15 16
17 18
19
M. Utsuyama and K. Hirokawa, Hypertrophy of the thymus and restoration of immune functions in mice and rats by gonadectomy. Mech. Ageing Dev., 47 (1989) 175-- 185. K.W. Kelley, S. Brief, H.J. Westly, J. Novakofski, P.J. Bechtel, J. Simon and E.B. Walker, GH~ pituitary adenoma cells can reverse thymic aging in rats. Proc. NatL Acad. Sci., 83 (1986) 5663-5667. D.R. Davila, S. Brief, J. Simon, R.E. Hammer, R.L. Brinster and K.W.Kelley. Role of growth hormone in regulating T-dependent immune events in aged, nude and transgenic rodents. J. Neurosci. Res., 18 (1987) 108-- 1! 16. R.J. Cross, J.L. Campbell and T.L. Roszman, Potentiation of antibody responsiveness after the transplantation of a syngeneic pituitary gland. J. Neuroimmunology, 25 (1989) 29-- 35. A.J. Cunningham and A. Szenberg, Further improvements in the plaque technique for detecting single antibody forming cells. Immunology, 14 (1968) 599--600. R.J. Cross and T.L. Roszman, Central catecholamine depletion impairs in vivo immunity but not in vitro lymphocyte activation. J. NeuroimmunoL, 19 (1988) 33--45. N.R. Hall and A.L. Goldstein, Thymosin modulation of the immune system. In The NeuroImmune-Endocrine Connection, CW. Cotman (ed.), Raven Press, New York, 1987. F.R. Boockfor, J.P. Hoeffler and L.S Frawley Cultures of GH 3 cells are functionally heterogeneous: thyrotropin-releasing hormone, estradiol and cortisol cause reciprocal shifts in the proportions of growth hormone and prolactin secretors. Endocrinology, 117 (1985) 418--420. R.A. Adler, The anterior pituitary-grafted rat: a valid model of chronic hyperprolactinemia. EndocrineRev., 7(1986) 302-- 313. D.H. Russell, R. Kibler, L. Matrisian, D.F. Larson, B. Poulos and B.E. Magun, Prolactin receptors on human T and B lymphocytes: antagonisn of prolactin binding by cyclosporin. J. lmmunoL, 134 (1985) 3027--3031. A.E. Boyd and S. Reichlin, Galactorrhea-amenorrhea, bromocryptine and the dopamine receptor. N. Engl. J. Med., 295 (1975)954--956.
11
TRANSPLANTATION OF PITUITARY GRAFTS FAIL TO RESTORE IMMUNE FUNCTION AND TO RECONSTITUTE THE THYMUS GLANDS OF AGED MICE
RICHARD J. CROSS j'*, JENNIFER L. CAMPBELL', WILLIAM R. MARKESBERYb and THOMAS L. ROSZMANj •Department of Microbiology and Immunology and bSanders-Brown Center on Aging and the Departments of Pathology and Neurology, University of Kentucky Medical Center, Lexington K Y 40536
(U.S.A.) (Received August 12th, 1989) (Revision received April 30th, 1990)
SUMMARY
There is evidence to indicate that the neuroendocrine and immune systems can interact. Thus, neuroendocrine hormones can modulate a variety of immune functions and there have been attempts to manipulate the neuroendocrine system of aged animals to enhance immune function. We have previously shown that the transplantation of a syngeneic pituitary gland under the kidney capsule of young adult mice elevates serum prolactin and enhances immune responsiveness. In the present study pituitary glands were transplanted under the kidney capsule of 22-month-old mice to determine if this maneuver can enhance a number of immunologic parameters. The results demonstrate that aged animals bearing transplanted pituitary grafts for 10 days did not exhibit any enhancement in their primary antibody response to sheep red blood cells, splenic T or B-cell mitogen responsiveness or restoration of thymic architecture. When these immunologic assessments are performed on animals bearing pituitary grafts for 28 days, the IgM and IgG primary antibody responses and splenic T-cell responsiveness are enhanced but repopulation of the thymus still does not occur. Importantly, this enhancement does not restore immunocompetence to levels observed in young mice.
Key words: Aging; Immune response; Prolactin; Pituitary graft; Thymus
*To whom aft correspondence should be addressed at: Department of Microbiology and Immunology, MS 401, University of Kentucky Medical Center, 800 Rose St., Lexington, KY 40536-0084, U.S.A. 0047-6374/90/$03.50 Printed and Published in Ireland
© 1990 Elsevier Scientific Publishers Ireland Ltd.
12
INTRODUCTION
It is well-documented that immune function declines with advancing age. This decline is characterized by thymic involution, impaired T-cell proliferation [1], diminished interleukin-2 (IL-2) synthesis [2], impaired humoral antibody production [31 and cell-mediated immune responsiveness [4]. These deficits may result from intrinsic defects within the specific cellular compartments of the immune response [5] or may be due to extrinsic changes which impinge on immunocompetent cells and thus impair function [6]. One such group of extrinsic factors are those derived from the neuroendocrine system. Pituitary hormones appear to be important not only in the modulation but also the development of the immune system [7,8], particularly thymic function. It is possible to successfully restore immunocompetence in hormonally deficient mice. Using hypopituitary dwarf mice which have abnormal thymic development and subsequent decreased immune function, Fabris et al. [8] have restored immune responsiveness by administering growth hormone (GH) and thyroxine or GH alone. Similarly, age-related impairment of immune responsiveness can be restored by two different manipulations of the neuroendocrine system. Utsuyoma and Hirokawa [91 gonadectomized old male and female mice and observed a regeneration of the thymus as well as an improvement in the humoral antibody response to sheep red blood cells (SRBC). Thymic regeneration was more pronounced in male mice while females showed the greatest enhancement of the humoral antibody response, lmmunofluorescence analysis showed an increase in the percentage of splenic T-cells and B-cells, although the response to SRBC in gonadectomized old mice never approached the response found in young mice. This enhancement of immune function may result from the observed decrease in secondary sex hormones (testosterone, estradiol) or a concomitant increase in luteinizing hormone as a result of the loss of negative feedback, although these levels were not measured. There also is evidence to suggest that neuroendocrine manipulation can improve the immune response status of aged animals. There are reports that restoration of Tcell function can be achieved by implanting G H 3 tumor cells which secrete both GH and prolactin into aged rats [10]. Two months after implantation, these 18-monthold rats had repopulated their thymus glands to near normal histology and the thymocytes from these animals had normal mitogen-induced proliferative responses and increased IL-2 synthesis compared to non-implanted 18-month-old rats. Similarly treated 24-month-old rats also exhibited restoration of thymus cellularity and thymocyte proliferative responses although the reconstitution of immune function in these animals was not as complete as that found in 18-month-old animals. These neuroendocrine effects on the immune response of aged mice were more directly examined by treating old rats with 1.5 mg of G H / d a y for 5 weeks [11]. This treatment enhanced both the proliferative response of spleen cells to mitogens as well as natural killer cell activity, but, reconstitution of thymus gland, or I1-2 pro-
13 duction did not occur following G H treatment. Because control groups using spleen cells from young rats were not included, it is difficult to determine if G H treatment induced a full recovery of immunocompetence. We have previously shown that elevation of serum prolactin by implanting syngeneic pituitary grafts under the kidney capsule enhances the humoral antibody response of young mice [12]. The present studies were performed to determine whether elevated serum prolactin resulting from grafting aged mice with pituitary glands could restore immune responsiveness. The data presented indicate that elevation of prolactin does not restore antibody responsiveness, splenic mitogen responsiveness nor thymus cellularity in 22-month-old mice ten days following pituitary gland transplantation. However, when pituitary grafts remain in place for 28 days antibody responsiveness is enhanced compared to sham grafted animals, but this enhancement is considerably less than response observed In young animals. Moreover, thymic regeneration does not occur. MATERIALSAND METHODS
Animals Female C57BL/6 22-month-old female mice were obtained from the National Institute on Aging. Young, 55--70-day-old C57BL/6 female controls were purchased from Charles River Laboratories, Wilmington, MA. The animals were maintained on sterile lab chow and water ad libitum and housed in Microisolator boxes. Reagents SRBC were obtained from one animal maintained by the Division of Laboratory Animal Resources, University of Kentucky. The mouse prolactin radioimmunoassay kit was provided by Dr. A.F. Parlow through the Pituitary Hormone and Antisera Center, Torrance, CA. Calculations of serum prolactin were made using the mouse prolactin standard provided with the kit as previously described [ 11 ]. Pituitary gland transplantation Pituitary gland transplantation was performed as described previously [12]. Briefly, mice were anesthetized with sodium pentobarbital, the skin and body wall excised and kidney exposed. A syngeneic pituitary gland was placed under the kidney capsule and the body wall and skin were closed with silk sutures. Surgical control groups (sham) were treated identically except for the implantation of the graft. Immunization and PFC assay Mice were immunized with 2 x 10s SRBC (i.p.) 14 or 28 days after pituitary gland transplantation. Mice were killed by cervical dislocation under metofane anesthesia. The spleens were teased into a single cell suspension, counted on a hemocytometer in 3°7o acetic acid to lyse erythrocytes and adjusted to the proper concentration
14
for the assay. Direct plaque forming cells (DPFC) which secrete lgM were enumerated using the method of Cunningham and Szenberg [13]. Indirect lgG secreting PFC were developed with rabbit anti-mouse IgG antibody at a final dilution of 1/50.
Mitogen-inducedlymphocyte blastogenesis Dispersed spleen cells were cultured in complete medium (Dulbecco's modified MEM supplemented with penicillin-streptomycin, glutamine, non-essential amino acids, vitamins, 10070 heat inactivated fetal calf serum and 50 mM 2-mercaptoethanol) with the T-cell mitogen concanavalin A (Con A, 0.5/~g/well, Sigma Chemical Co., St. Louis, MO) or lipoplysaccharide (LPS, 20/~g/well, Difco, Detroit, MI) as described previously [14]. Briefly, 2 x l05 spleen cells were cultured in flat-bottom microtiter plates with complete medium and mitogen. Cell cultures were performed in triplicate. The plates were incubated at 37 °C in a 95070 air/5070 CO 2 atmosphere for 56 h at which time 0.25/~Ci [3H]thymidine (spec. act. 6.7 Ci/mmol, New England Nuclear, Boston, MA) was added in 25/A. Cells were harvested 16--18 h later using an automated cell harvester and [3H]thymidine incorporation into DNA was assayed by liquid scintillation counting.
Thymus morphologicalanalysis The complete thymus was removed at the time of death and placed in 4°70 formaldehyde. Following fixation specimens were processed in the standard manner and embedded in paraffin. Sections (8/~m) were cut and stained with hematoxylin and eosin. Microscopic review revealed no significant alterations in the cortex or medulla of mice receiving pituitary grafts or in control animals. Thymus size was determined utilizing a Kontron Imager Processing System interfaced with an Olympus Vanox 5 microscope. Sections were through the widest portion of the embedded specimen. Thymus images were enhanced and discrimination depended on the difference in grey level value. The areas of each thymus were individually traced. Computer measurements were converted to mm 2 for presentation.
TABLE I T H E H U M O R A L A N T I B O D Y RESPONSE 1N Y O U N G MICE RECEIVING P I T U I T A R Y G R A F T S Treatment group
n
D P F C / I 0~ n u c l e a t e d spleen cells
Pituitary graft Sham graft Normal
5 5 5
550 ± 52 333 +- 46 192 ± 17
Each animal was grafted with a pituitary gland 10 days prior to immunization with 2 x l(Y SRBC. DPFC were assayed on day 5 after immunization. The antibody response of mice receiving a pituitary graft and s h a m grafted mice were significantly different ( P < 0.001).
15 In a separate series o f experiments, thymic reconstitution was evaluated in mice which had been transplanted 28 days previously with PG. Thymocytes were dispersed into a single cell suspension and nucleated cells were counted on a hemocytometer. Statistical analysis Data were analyzed using Student's two-tailed t-test for independent means. All experiments were repeated a minimum of three times and only the results from one representative experiment are shown. RESULTS Effect o f transplanting pituitary glands on the primary antibody response in young and aged mice Experiments were performed to determine the effects of transplanting pituitary glands into young and aged mice on the subsequent primary antibody response to SRBC. As shown in Table I placement o f pituitary glands under the kidney capsules of young adult mice for 10 days results in an enhanced primary antibody response to SRBC in these animals compared to sham grafted or normal control animals. The serum prolactin levels in these animals was 2--3-fold above normal basal levels [12]. The data presented in Table II show that immunization of aged mice in which pituitary glands were transplanted 10 days prior to immunization elevates the serum level of prolactin greater than two fold above that found in aged normal mice, but the splenic DPFC (IgM) responce is not elevated concomitantly. To more fully investigate the effects of elevated prolactin on the humoral immune responsiveness of aged animals the following experiments were performed. Aged
TABLE II THE EFFECTSOF A PITUITARYGRAFTON THE DPFC RESPONSE IN 24-MONTH-OLDMICE Treatment group
n
Serum prolactin (ng/mO
DPFC/I O~ nucleated spleen cells ±S.E.M.
Aged, pituitary graft Aged, shamgraft
8 5 5 5
169 - 24 45 ± 14 70 _+ 9 20 _+. 3
31 :l: 10 26 :l: 17 22 _+ 6 365 __. 86
Aged, normal
Young, normal
Aged mice were transplanted with a pituitary gland ten days prior to immunization. Direct plaque forming cells (DPFC) were determined 4 days after immunization. Statistical analysis indicates that none of the antibody responses of the three aged groups of mice were different. The antibody response elicited by younganimals differed from that observed with the aged group of mice(P < 0.001).
o.
5o'-
100.
150.
2O0
$
5
6
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# days after Immunization
4
pituitary graft
7
CA)
0
500
1000~
1500
2000
Z500
3000
3500 young mice
# days after immunization
_y"
e~O
(.)
Fig. 1. The lgM primary antibody response of aged (22-month) or young (3 - 4 - m o n t h ) mice receiving pituitary grafts. Sham operated mice served as contrors. Twenty-eight days following transplantation the mice were immunized with 2 x lO~ SRBC and the direct plaque forming cells (DPFC) were determined on days 3--7. Statistical analysis indicates that the antibody response of animals with pituitary grafts is signi ficantly enhanced on days 6 and 7 (P < 0.05).
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300
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350
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50
90.
120.
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7
# days a f t e r i m m u n i z a t i o n
5
plhJItary graft ~ a m graft
CA)
0
SO0.
1000.
1500-
2000-
2500-
3000 0~0
I
# days after immunization
young mloe
(e)
Fig. 2. The lgG primary antibody response of aged (22-month) or young (3--4 month) mice receiving pituitary grafts. Twenty-eight days following transplantation the mice were immunized with 2 × 10s SRBC and indirect plaque forming cells (IPFC) were determined on days 5--7. The antibody response of animals with pituitary grafts is significantly enhanced on days 6 and 7 ( P < 0.05) as compared to sham control values.
Q"
0
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Ul q) (3
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J --4
18 160
(A) i 14-0 120
r,3 t:D
k\',4 PG-10d ahom-10d PG-28d 1~] ahom-28d
(B)
PG-IOd ~aham-10d
100
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80
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60 4-0 20 0
young
oged
oged
i ni
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oged
Fig. 3. The mitogen responsivenessof spleen cells obtained from aged (22-month) or young (55--70-day) mice receivingpituitary grafts. Either l0 days or 28 days after transplantation spleen cells were obtained from these animals or sham controls and cultured with the T-cell mitogen Con A (0.5 tag/well, panel A) or the B-cell mitogen LPS (20 tag/well, panel B). The Con A response of spleen cells from young, pituitary grafted mice is significantly enhanced compared to sham control values (P < 0.01). The Con A responsiveness of spleen cells obtained from aged animal receiving pituitary grafts l0 days previously did not differ from sham control values while the Con A responses of spleen cells obtained from aged animals bearing these grafts for 28 days did (P < 0.05). Transplantation of pituitary glands into either young or aged animals does not enhance the LPS responsivenessof their spleen cells studied aftcr l0 days.
mice were t r a n s p l a n t e d with P G 28 days prior to i m m u n i z a t i o n a n d the kinetics of the p r i m a r y a n t i b o d y response were evaluated. The data in Fig. l a show that transp l a n t a t i o n o f pituitary g l a n d does e n h a n c e the D P F C response of aged mice compared to sham controls a n d that, the kinetics o f the response is shifted to the right. However, this e n h a n c e m e n t is still less t h a n 10°70 o f the response observed with y o u n g a n i m a l s (Fig. lb). Similar results are o b t a i n e d with the I P F C (IgG) response except that the kinetics o f I P F C a p p e a r a n c e is not altered (Fig. 2a). The I P F C response o f aged a n i m a l s receiving P G does not a p p r o a c h that o f the y o u n g a n i m a l s (Fig. 2b).
Effect o f transplanting pituitary grafts on T-cell and B-cell mitogen responsiveness o f aged animals T o d e t e r m i n e the effects o f t r a n s p l a n t i n g pituitary grafts on T cell a n d B-cell f u n c t i o n in both y o u n g a n d old mice, spleen cells from pituitary gland t r a n s p l a n t recipients a n d sham controls were cultured in vitro with the T-cell mitogen, C o n A or the B-cell m i t o g e n , LPS. The data in Fig. 3a show that t r a n s p l a n t a t i o n of pitui-
19 TABLE 111 THYMUS SIZE IN AGED MICE RECEIVING PITUITARY GLANDS Treatment
n
Thymus size
Thymocyte no.
group Old, pituitary graft Old, S h a m Old, Normal
8 4 6
(ram2)
( × 109
4.83 + 0.93 6.63 ± 0.43 5.56 :I: 0.97
102 4- 18 145 4- 22 ND
Thymus size was measured on formalin-fixed hematoxylin and eosin stained sections from mice which received a pituitary graft 2 weeks prior to sacrifice. Thymus size (mean :1: S.E.M.) using image analysis on each individual thymus were calculated. Thymus nucleated cell counts were performed on mice which had received a pituitary graft 28 days prior to sacrifice. There was no significant difference among the values obtained for thymus size or thymocyte number in the three different treatment groups. ND = not done.
tary gland can enhance splenic T-cell mitogen responsiveness in young animals, but has no effect on B-cell responsiveness (Fig. 3b). The mitogen responses of spleen cells obtained f r o m aged mice which had received pituitary grafts or sham operations were determined (Fig. 3 a,b). The results demonstrate that the Con A (Fig. 3a) or LPS (Fig. 3b) responsiveness of spleen cells from aged animal regardless of whether they had received P G is markedly decreased compared to values obtained with young animals. Furthermore, the results show that the mitogen responsiveness o f spleen cells obtained from either animals with pituitary grafts or sham control animals are virtually identical. The Con A responsiveness of spleen cells obtained from aged animals receiving pituitary grafts is enhanced approximately 2-fold at 28 days but not 10 days after the transplant when compared to sham control values (Fig. 3a). There is no difference between the LPS responsiveness of spleen cells obtained from aged or young animals receiving pituitary grafts and sham control animals. Effect o f transplantation o f a pituitary gland on reconstitution o f the thymus Thymus size was evaluated on hematoxylin and eosin stained thymus tissue from pituitary gland transplanted, sham and normal old mice. The data in Table III indicate that thymus glands from mice with elevated serum prolactin are no larger than thymus glands from sham control or normal old mice. Similar results are seen in mice which have been transplanted for 28 days with pituitary grafts. In these experiments the number of nucleated cells in the thymus from aged mice with pituitary grafts and sham controls also was quantified. The data show that animals receiving pituitary grafts do not exhibit thymic reconstitution, because the number of thymocytes obtained from thymus glands of pituitary transplanted or sham controls do not differ (Table III).
20 DISCUSSION The results of this study demonstrate that transplantation of pituitary glands under the kidney capsules of old animals can modulate the primary antibody response. While aged mice bearing transplanted pituitarys for l0 days did not evidence an enhanced antibody response, those which carried the transplanted pituitarys for 28 days were elevated when compared to values obtained from sham controls. Both the IgM and IgG antibody responses were increased in these transplanted aged animals. Not only was the IgM antibody response enhanced to more (approximately 27-fold) than the lgG antibody response (7-fold) but the peak day of antibody production was 1 day later. Although aged animals bearing pituitary grafts exhibited enhanced IgM and IgG primary antibody responses, these responses were still approximately 10-fold lower than that observed in young animals. The explanation for the enhanced antibody response observed in aged animals with pituitary transplants will require a more detailed study to delineate the mechanisms responsible. The results also revealed no change in the thymic architecture or thymocyte numbers in transplanted aged animals as compared to sham control animals. This suggests that the thymus has not been reconstituted and is not exporting lymphocytes. It is possible that the thymic stromal cells are producing hormones which influence the function of T-cells [l 5]. The data do provide some support for this notion in that the T-cell responsiveness of spleen cells from aged animals which carried pituitary grafts for 28 days were enhanced relative to those values obtained from sham control animals. However, this T-cell responsiveness observed was approximately 3-fold less than the response observed with spleen ceils from young animals. Previous reports by others [10] have shown that the implantation of a GH and prolactin secreting G H 3 cell line reconstitutes the thymus and restores T-cell function in old rats. The discrepancies between these observations and those in the present study may relate to a number of potentially important differences. First, in our studies we employed a mouse model while in the other study aged rats were used. Secondly, the source of the transplanted tissue employed is different in that Kelley and co-workers [10] transplanted allogeneic GH~ tumor cells into aged rats while we employed pituitary grafts. Finally, it is known that the GH~ tumor cells produce both GH and prolactin as well as probably other undefined factors [16] while the pituitary glands secrete only prolactin after approximately one week following transplantation into animals [17]. It is possible that GH is a more potent immunoenhancer than prolactin. Indeed, it has been demonstrated that administration of ovine GH to aged rats does enhance mitogen induced T-cell proliferation and natural killer cell activity [l 1], but these animals failed to exhibit a restoration of thymic architecture. Finally, the immune potentiation observed in aged rats the GH 3 tumor cells could result from some other immunoenhancer produced by these tumor cells or the result of the synthesis of cytokines by lymphoid cells mounting an immune attack against these allogeneic cells.
21
thesis o f cytokines by lymphoid cells mounting an immune attack against these allogeneic cells. It is clear that young adult mice receiving pituitary grafts have markedly enhanced antibody responses [12] while pituitary grafted aged animals have for the most part lost this ability. The explanation for this age-related difference is presently unknown. One possibility is that lymphocytes obtained from aged animals may not possess prolactin receptors as do lymphocytes from young adults [18] or that other intrinsic age related changes have taken place which prevent the elevation of prolactin from enhancing immune function [5]. Conversely, extrinsic changes such as the alteration of other hormone levels may have occurred which prevent prolactin from enhancing immune function [6]. For example, prolactin may be affecting a secondary sex hormones or other hormones which can influence lymphocyte function [l 9]. Alteration of the activity of these secondary hormones may override the immunopotentiating effects of prolactin on immune function. In summary, the data indicate that young animals receiving pituitary grafts exhibit a marked increase in their humoral antibody response to SRBC. When similar experiments were performed on aged animals, the pituitary grafted animals had an increase in their antibody responses as compared to sham control animals. However, this manuever did not restore the antibody response to levels observed in young animals. Collectively these data indicate that increasing prolactin levels in aged mice does not restore the immune function or thymic architecture to that of young mice.
ACKNOWLEDGEMENTS
Supported in part by USPHS grant numbers NS 17423, NS 22512. REFERENCES 1 2
3 4 5 6 7
8
B.A. Helfrich, M. Segre and D. Segre, Age-related changes in the degeneracy of the mouse T-cell repertoire. Cell. Immunol., 118 (1989) I--9. M.L. Thoman and W.O. Weigle, Partial restoration of Con A-induced proliferation, IL-2 receptor expression and IL-2 synthesis in aged murine lymphocytes by phorbol myristate acetate and ionomycin. Cell. Immunology, 114 (1988) l - I I. J.F. Albright and T. Makinodan, Growth and senescence of antibody forming cells. J. Cell. Physiol., (Suppl. 1)(1966) 185--206. W.M. Adler, T. Takiguchi and R.T. Smith, Effect of age upon primary alloantigen recognition by mouse spleen cells. J. lmmunol., 107(1971) 1357--1362. G.B. Price and T. Makinodan, Immunologic deficiencies in senescence. I. Characterization of intrinsic deficiencies. J. lmrnunol., 108 (1972) 403--412. G.B. Price and T. Makinodan, Immunologic deficiencies in senescence. II. Characterization of extrinsic deficiencies. J. lmmunol., 108 (1972) 41 3--417. N. Fabris, W. Pierpaoli and E. Sorkin, Hormones and the immunologic capacity. Ill. The immunodeficiency disease of the hypopituitary Snell-Bagg dwarf mouse. Clin. Exp. Immunol., 9 0971) 209--225. N. Fabris, W. Pierpaoli and E. Sorkin, Hormones and the immunologic capacity. IV. Restorative effects of developmental hormones or of lymphocytes on the immunodeficiency syndrome of the dwarf mouse. Clin. Exp. lmmunol. 9(1971)227--240.
22 9 10
I1
12 13 14 15 16
17 18
19
M. Utsuyama and K. Hirokawa, Hypertrophy of the thymus and restoration of immune functions in mice and rats by gonadectomy. Mech. Ageing Dev., 47 (1989) 175-- 185. K.W. Kelley, S. Brief, H.J. Westly, J. Novakofski, P.J. Bechtel, J. Simon and E.B. Walker, GH~ pituitary adenoma cells can reverse thymic aging in rats. Proc. NatL Acad. Sci., 83 (1986) 5663-5667. D.R. Davila, S. Brief, J. Simon, R.E. Hammer, R.L. Brinster and K.W.Kelley. Role of growth hormone in regulating T-dependent immune events in aged, nude and transgenic rodents. J. Neurosci. Res., 18 (1987) 108-- 1! 16. R.J. Cross, J.L. Campbell and T.L. Roszman, Potentiation of antibody responsiveness after the transplantation of a syngeneic pituitary gland. J. Neuroimmunology, 25 (1989) 29-- 35. A.J. Cunningham and A. Szenberg, Further improvements in the plaque technique for detecting single antibody forming cells. Immunology, 14 (1968) 599--600. R.J. Cross and T.L. Roszman, Central catecholamine depletion impairs in vivo immunity but not in vitro lymphocyte activation. J. NeuroimmunoL, 19 (1988) 33--45. N.R. Hall and A.L. Goldstein, Thymosin modulation of the immune system. In The NeuroImmune-Endocrine Connection, CW. Cotman (ed.), Raven Press, New York, 1987. F.R. Boockfor, J.P. Hoeffler and L.S Frawley Cultures of GH 3 cells are functionally heterogeneous: thyrotropin-releasing hormone, estradiol and cortisol cause reciprocal shifts in the proportions of growth hormone and prolactin secretors. Endocrinology, 117 (1985) 418--420. R.A. Adler, The anterior pituitary-grafted rat: a valid model of chronic hyperprolactinemia. EndocrineRev., 7(1986) 302-- 313. D.H. Russell, R. Kibler, L. Matrisian, D.F. Larson, B. Poulos and B.E. Magun, Prolactin receptors on human T and B lymphocytes: antagonisn of prolactin binding by cyclosporin. J. lmmunoL, 134 (1985) 3027--3031. A.E. Boyd and S. Reichlin, Galactorrhea-amenorrhea, bromocryptine and the dopamine receptor. N. Engl. J. Med., 295 (1975)954--956.