- Email: [email protected]
Dopamine D1like receptors in the thymus of aged rats: A radioligand binding and autoradiographic study
Journal of Neuroimmunology ELSEVIER
Journal of Neuroimmunology 56 (1995) W-160
Dopamine D,-like receptors in the thymus of aged rats: A radioligand binding and autoradiographic study Albert0 Ricci a, Josh Antonio Vega b, Damiano Zaccheo ‘, Francesco Amenta a,* a Sezione di Anatomia Umana, Istituto di Farmacologia, Universitci di Camerino, L&aScalzino 5, 62032 Camerino, Italy b Departamento de Morfologia y Biologia Celular Universidad de Oviedo, Oviedo, Spain ’ Istituto di Anatomia Umana, Universitci di Genova, Geneva, Italy
Received 17 May 1994; revised 11 August 1994; accepted 13 September 1994
Abstract Age-dependent changes in the density and pattern of dopamine D,-like receptors were studied in the thymus of young (3 months), adult (12 months) and aged (24 months) male Wistar rats using combined radioligand binding and autoradiographic techniques. [3H]SCH 23390, which was used as a ligand, was specifically bound to sections of the thymus in a manner consistent with the labelling of dopamine D, receptor. The dissociation constant value was similar in the thymus of the three animal groups examined. The maximal density of binding sites, evaluated with conventional radioligand binding techniques, was significantly reduced in the thymus of adult in comparison with young rats and further reduced in aged animals. Silver grains which correspond to [3H]SCH 23390 binding sites were revealed by light microscope autoradiography primarily in the cortex of the thymus and in lesser amounts within thymic corpuscles. A progressive decrease in the density of silver grains more pronounced in the cortex than in thymic corpuscles was observed in the thymus of adult and old in comparison with young rats. The loss of silver grains revealed with autoradiography is more moderate than the decrease in the density of binding sites shown by radioligand binding. Silver grains developed per single cells (probably lymphocytes) of the thymic cortex were reduced between young and adult rats and further decreased in old rats. The above findings suggest that the age-related decline in the density of dopamine D, receptor assayed in the thymus is due in part to the reduced thymic mass with aging. The observation of a decreased expression of dopamine D, receptor in cells of the thymic cortex as a function of age suggests that this reduction cannot be attributed simply to loss of thymic lymphocytes. Keywords:
Thymus; Aging; Dopamine
receptor; Autoradiography
1. Introduction Aging is associated with changes in immune function, although the possible link between these changes and the most prevalent diseases of the elderly has not yet been documented (Beregi, 1986). Changes in the humoral and cellular immunity, in the function of lymphocytes as well as decreased production of antibodies accompanied by increased auto-antibodies have been documented in old age (Gilard et al., 1977; Callard, 1981; Kay and Makinodan, 1982). Moreover, morphological alterations of peripheral lymphocytes, and of splenic and lymph node lymphocytes occur in aged rodents and humans (Beregi, 1986).
* Corresponding
author. Fax (+ 39-737) 2538
0165-5728/95/$09.50
0 1995 Elsevier Science B.V. All rights reserved
SSDI 0165-5728(94)00141-3
; Rat
The thymus is a primary organ of the immune system and plays an important role in the interactions between the neuro-endocrine and immune systems. It is supplied with a rich autonomic innervation (Geppetti et al., 1987; Geppetti et al., 1988; Felten and Felten, 1989; Lorton et al., 1990; Bellinger et al., 19931, and is immunoreactive for several pituitary hormone epitopes (Battanero et al., 1992). Moreover, thymic hormones contributing to regulate immune functions, such as thymosins, have a role in communication between non-immune organs. They were also identified in the central nervous system and probably play a functional role in the hypothalamus (Rebov et al., 1981; Hall et al., 1985; Malaise et al., 1987; Ader et al., 1990; Khansari et al., 1990). A possible role of the catecholamine dopamine in the modulation of immune function is suggested by the
156
A. Ricci et al./Journal
of Neuroimmunology 56 (1995) 155-160
findings that dopamine receptor agonists can reduce or even prevent humoral or cell-mediated immune reactions in rodents and in man (Nagy et al., 1983; Chapdelaine et al., 1988). Moreover, the localization of dopamine D, receptors has been recently demonstrated in the rat thymus (Ricci et al., 1994). Since no information is available so far concerning the influence of aging on dopamine D,-like receptor sites in the rat thymus, we have investigated the topic using combined radioligand binding and autoradiographic techniques. A preliminary account of the present study has been presented at the II European Congress of Gerontology (Ricci et al., 1991).
2. Materials and methods 2.1. Animals and tissue treatment Male Wistar rats of 3 months (young, n = 10, 270 & 12 g body weight), of 12 months (adult, IZ= 10,350 _t 25 g body weight) and of 24 months (aged, n = 10,450 k 20 g body weight) were used. Rats were anaesthetised with ether and sacrificed by decapitation. The thorax was opened, and the thymuses were removed, weighed and washed in ice-cold 0.9% NaCl solution. Thymuses were embedded in a cryoprotectant medium (Tissueteck, Miles, USA) and frozen in a dry ice/acetone mixture. Serial 8-pm sections were obtained using a microtome cryostat and mounted on pre-weighed gelatine-coated microscope slides. A group of 10 consecutive slides 20 pm apart for each tissue block was stained with Toluidine blue to verify microanatomical details and to measure the area of the slide occupied by thymic tissue, which was evaluated by image analysis.
assessed by incubating some sections as above with 2.5 nM [3H]SCH 23390 in the presence of increasing concentrations of compounds active on different subtypes of dopamine receptors or on 5-hydroxytryptamine (5HT) receptors and on a-adrenoceptors. 2.3. Light microscope autoradiography For light microscope autoradiography, sections of the thymus were incubated with 2.5 nM [ 3H]SCH 23390, in the presence or absence of 1 PM (+)-butaclamol to define non-specific binding, and processed according to the protocol detailed elsewhere (Ricci et al., 1994). The density of silver grains developed in dark-field sections of the thymus of rats of different ages was assessed in a 500~pm2 area of the cortex and of the medulla by quantitative image analysis according to the procedure reported in an earlier study (Ricci et al., 1994). Measurements were made on five consecutive sections 20 Km apart of the thymus per rat (n = 10 per age group) incubated with 2.5 nM [3H]SCH 23390 alone or plus 1 PM (+ )-butaclamol to define nonspecific binding. In order to assess whether age-dependent changes in the expression of dopamine D, receptor (see Results) were attributable to loss of thymic cells expressing receptors or to a decrease of the receptor itself, three random areas of the thymic cortex per section were examined at a final magnification of X900 using a bright-field condenser. The number of silver grains developed around the border of 10 randomly selected cells (probably lymphocytes) was then counted by image analysis. Counts were made independently in sections incubated with [3H]SCH 23390 alone (total binding) or plus 1 PM (+)-butaclamol (non-specific binding). Specific binding values were then calculated by subtracting non-specific from total binding values.
2.2. Radio&and binding experiments 2.4. Data analysis For the analysis of dopamine D,-like receptors, sections of the thymus of rats of the three age groups were incubated with [3H]SCH 23390 according to the procedure detailed elsewhere (Ricci et al., 1994). Sections were incubated with increasing concentrations (0.1-7.5 nM) of [3H]SCH 23390 alone or plus 1 PM (+ )butaclamol to define non-specific binding. The optimal incubation time (60 min), temperature (25°C) and radioligand concentration (2.5 nM) were assessed in a recent study of our group (see Ricci et al., 1994). At the end of incubation, sections were washed in ice-cold incubation buffer (2 X 5 min) to remove unbound radioligand and rinsed quickly in distilled water. Sections were then wiped onto Whatman GF-B glass fibre filters and counted by liquid scintillation spectrometry. The pharmacological specificity of [ 3H]SCH 23390 binding to sections of the thymus of different ages was
Data from the binding experiments were analysed by linear regression analysis of Scatchard plots of saturation isotherms. Competitor dissociation constant (Ki) values were determined according to the method of Cheng and Prusoff (1973). Further details on statistical analysis are reported in a recent paper of our group (Ricci et al., 1994). 2.5. Chemicals 13H]SCH 23390 ([RI-( + )-(-chloro-2,3,4,5_tetrahydro5-phenyl-lH-3benzazepin-al-hemimaleate) (specific activity 85 Ci/mmol) was obtained from the Amersham Radiochemical Centre (Buckinghamshire, UK). Other chemicals were obtained from the sources recently indicated (Ricci et al., 1994).
A. Ricci et al. /Journal
of Neuroimmunology 56 (1995) 155-160
BOUND/FREE
”
50
66
82
98 [ 3
130
114
HI-SCH
23390
146
162
178
194
bound
Fig. 1. Scatchard analysis of [3H]SCH 23390 binding to sections of the thymus of young ( n), adult ( A > and old (0) rats. The B,,, values calculated with this kind of analysis averaged 170&12 fmol/mg tissue in young rats, 120 f 9.3 fmol/mg tissue in adult rats (P < 0.001 vs. young) and 62k4.1 fmol/mg tissue in old rats (P < 0.001 vs. young and P < 0.001 vs. adult).
3. Results
Values of area occupied by thymic tissue proper in rats of the three age groups are shown in the Table 1. As can be seen, this area significantly decreases as a function of age (Table 1). [ 3H]SCH 23390 was specifically bound to sections of the thymus in the three age groups examined. Binding was time-, temperature- and concentration-dependent (data not shown). [3H]SCH 23390 binding to sections of the thymus of rats of the three age groups examined is concentration-dependent, belonging to a single class of high-affinity sites (Fig. 1). The dissociation constant (K,) values were similar in the thymus of young (K,: 1.6 + 0.04 nM), adult (K,: 1.5 &-0.05) and aged rats (K,: 1.6 + 0.07). The maximum binding density (B,,,) value was significantly reduced in adults in comparison with young rats and further decreased in aged animals (Fig. 1). The pharmacological profile of [3H]SCH 23390 binding to sections of the thymus of rats of different age is consistent with the labelling of dopamine D,-like receptors, SCH 23390 being the most powerful com-
157
petitor of [3H]SCH 23390 binding, followed in descending order by (+ )-butaclamol, fenoldopam, dopamine and haloperidol (data not shown). Isomers of sulpiride, bromocriptine, domperidone, ketanserin, phentolamine and serotonin were weak competitors of L3HlSCH 23390 binding or without effect (data not shown). Dopamine competed with [“HISCH 23390 binding in the submicromolar range. The K, value was 40.7 + 2.1 nM in young, 38.3 + 1.6 nM in adult and 39.3 + 2.8 in the thymus of old rats. Light microscope autoradiography revealed the accumulation of specific [3H]SCH 23390 binding sites represented by silver grains primarily in the cortex of the thymus (Fig. 2) and in lesser amounts within thymic corpuscles of the medulla (Fig. 2). The density of specific silver grains was decreased in adult in comparison with young rats and further reduced in aged animals (Fig. 2). The age-dependent loss of silver grains was more pronounced in the cortex than in the medulla (Fig. 3). Analysis of the number of silver grains developed within single cells of the thymic cortex (which probably represent lymphocytes) revealed a decrease of specific [3H]SCH 23390 binding sites in adult in comparison with young rats and a further decrease in old rats (by about 38% vs. young and by about 18% vs. adult) (Table 1). Comparative evaluation of the age-dependent changes in the density of [3H]SCH 23390 binding sites assessed by radioligand binding and autoradiographic techniques revealed a more pronounced loss of binding sites using radioligand binding techniques (Fig. 1) than autoradiography (Fig. 3).
4. Discussion Important changes occur in the organisation of lymphoid organs and the regulation of immune function in aging. Several experimental data suggest a possible role of the autonomic innervation of immune organs in the immunosenescence (Bellinger et al., 1993). The thymus undergoes a variety of anatomical and functional changes during life span. In fact, it has the maximum development during sexual maturation, begins to atrophy at puberty, and continues a progressive degenera-
Table 1 Influence of aging on the area of thymic tissue in sections of thymus and on the density of silver grains within single cells of the thymic cortex in rats of different ages Animal group
n
Area occupied by thymic tissue proper (mm’)
Number of specific silver grains per cell
Young Adult Old
10 10 10
2300 + 120 1soo+ 105 a 950 + 75 c
29.1 f 0.8 23.4 f 0.6 b 17.9 f 0.4 d
Values are means + SEM and were calculated as described in Materials and methods. a P < 0.05 vs. young; b P < 0.01 vs. young; ’ P < 0.01 vs. young or adult; d P < 0.01 vs. young and P < 0.05 vs. adult.
158
A. Ricci et al. /Journal
of Neuroimmunology 56 (1995) 155-160
tion with aging (Boyd, 1932; Bellinger et al., 1988). The thymus of aged rats is supplied with a more dense autonomic innervation than young rats (Bellinger et al., 1990; Zirbes and Novotny, 1992). However, the func-
Silver
arain
densitv
x 500
urn2
350 300 ; 250 1 200
-
15010050 1 C o-l CORTEX
MEDULLA
Fig. 3. Quantitative analysis of the density of specific silver grains developed in the cortex (CORTEX) and in the medulla (MEDULLA) of the thymus of young (A), adult (B) and old (Cl rats after exposure of sections to a L3H]SCH 23390 concentration of 2.5 nM. Specific binding was obtained by subtracting non-specific from total binding as indicated in Materials and methods. Data are means +SEM. ‘P < 0.001 vs. young; * *P < 0.01 vs. young; lP < 0.001 vs. and P < 0.01 vs. adult.
tional significance of these observations has not yet been established (Bellinger et al., 1993). A possible role of dopamine in modulating immune function has been suggested (Nagy et al., 1983; Chapdelaine et al., 1988). The immunosuppressive effects of dopamine and its agonists are considered to be due primarily to inhibition of the release of anterior pituitary hormones stimulating immune function, rather than by a direct action on the immune system (for a review see Clark, 1990). Studies on the expression of dopamine receptors in circulating immune cells, completed before the introduction of molecular biology techniques to dopamine receptor research, have provided conflicting results (see Ricci et al., 1994). More recently the expression of dopamine D, receptor genes and their transcription in two pseudo genes in human lymphocytes have been demonstrated (Takahashi et al., 1992), as well as the presence of dopamine D, receptors in the thymus (Ricci et al., 1994). In the last lo-15 years two main subtypes of central and peripheral dopamine receptors, namely D, and D, receptors, have been identified (Gingrich and Caron,
Fig. 2. Autoradiographic localization of [3H]SCH 23390 binding in sections of the thymus of rats of 3 months (young, A), 12 months (adult, D) and 24 months (old, El. Sections were incubated with a 2.5 nM concentration of 13HlSCH 23390 alone (A, D and E) or plus 1 mM (+)-butaclamol (C) to generate non-specific binding. (A, C, D and E) are dark-field pictures; (B) is a bright-field picture of micrograph (A) stained with Toluidine blue to verify microanatomical details. C, cortex; M, medulla. The highest accumulation of silver grains occurred within the cortical tissue. Note the reduced density of silver grains in the adult in comparison with the young rat and the further decrease noticeable in the old rat. (X 160).
A. Ricci et al. /Journal
of Neuroimmunology 56 (1995) 155-160
1993). More recently, the application of molecular biology techniques to dopamine receptor research allowed the identification of five subtypes of dopamine receptors CD,-D, receptors) (Sibley and Monsma, 1992; Gingrich and Caron, 1993). D, and D, receptors belong to the D,-like receptor family, whereas D,, D, and D, sites belong to the D,-like receptor family (Sibley and Monsma, 1992; Gingrich and Caron, 1993). Consistent with data of a previous study of our group performed in young rats (Ricci et al., 1994) we have seen that the dopamine D,-like receptor radioligand [3HlSCH 23390 (Brodde, 1989) was specifically bound to section of the thymus of rats of different ages in a manner consistent with the labelling of D,-like receptors. In view of the capability of dopamine to compete with [3H]SCH 23390 binding in the submicromolar range, the site we have characterized probably belongs to the dopamine D, receptor subtype (Sibley and Monsma, 1992; Gingrich and Caron, 1993). This receptor is located primarily in the cortex, which is the thymic portion densely packed with thymocytes (T lymphocytes) (Amenta, 1991). This suggests that T lymphocytes express dopamine D, receptor. Aging is accompanied by a loss of dopamine D, receptor in the rat thymus. The findings that conventional radioligand binding techniques reveal more pronounced decrease of this receptor, suggest that this reduction is in part due to the decrease of functionally active thymic areas as a function of age. In fact, the loss of specific [3HlSCH 23390 binding sites averages the 30% and the 24% between young and adult rats, the 65% and the 46% between old and young rats and the 35% and the 22% between old and adult rats using radioligand binding and autoradiographic techniques, respectively. This suggests that quantitative autoradiographic techniques may be useful in assessing changes in the density of neurotransmitter receptors in organs undergoing regressive changes as a function of age such as the thymus (Boyd, 1932; Bellinger et al., 1988). The observation of a decreased expression of dopamine D, receptor in single cells (probably lymphocytes) of the thymic cortex as a function of age suggests that this reduction cannot be attributed simply to loss of thymic lymphocytes. Age-related changes of cerebral dopamine receptors consisting in a decrease of their density primarily between young and adult age have been documented (Amenta et al., 1991). The same occurs for the dopamine D,-like receptor of the thymus, the density of which is reduced with aging. However, unlike central D,-like dopamine receptors (Amenta et al., 19911, the most remarkable decrease of the thymic receptor occurs between middle-age and old subjects. The majority of investigations on aging of central dopamine receptors have been performed in the striatum, which is a tissue expressing dopamine D, rather than D, recep-
159
tors (Gingrich and Caron, 1993). It cannot be excluded that the two subtypes of dopamine D,-like receptors may have a different sensitivity to aging.
Acknowledgements
The present study was supported in part by a grant of the Italian National Research Council (CNR, Progetto Finalizzato Invecchiamento). The authors are greatly indebted to Ms. S. Beatty for the critical revision of the manuscript, and to Mr. G. Bonelli for photographic assistance.
References Ader, R., Felten, D. and Cohen, N. (1990) Interactions between the brain and the immune system. Annu. Rev. Pharmacol. Toxicol. 30, 561-602. Amenta, P.S. (1991) Histology and Human Microanatomy. Piccin, Padova, pp. 249-255. Amenta, F., Zaccheo, D. and Collier, W.L. (1991) Neurotransmitters, neuroreceptors and aging. Mech. Aging Devel. 61, 249-273. Battanero, E., De Leeuw, F.-J., Jansen, G.H., Van Wichen, D.F., Huber, .I., Schuurman, H.-J. (19921 The neural and neuroendocrine component of the human thymus. II. Hormone immunoreactivity. Brain Behav. Immun. 6, 249-264. Bellinger, D.L., Felten, S.Y. and Felten, D.L. (1988) Maintenance of noradrenergic sympathetic innervation in the involuted thymus of aged Fischer 344 rat. Brain Behav. Immunol. 2, 133-150. Bellinger, D.L., Lorton, D., Romano, T., Olschowka, J.A., Felten, S.Y. and Felten, D.L. (1990) Neuropeptide innervation of lymphoid organs. Ann. N.Y. Acad. Sci. 594, 17-33. Bellinger, D.L., Felten, S.Y., Ackerman, K.D., Lorton, D., Madden, K.S. and Felten, D.L. (19931 Noradrenergic sympathetic innervation of lymphoid organs during development, aging, and in autoimmune disease. In: F. Amenta (Ed.), Aging of the Autonomic Nervous System. CRC Press, Boca Raton, FL, pp. 243-284. Beregi, E. (1986) Relationships between aging of the immune system and aging of the whole organism. In: M. Bergener, M. Ermini and H.B. Staehelin (Eds.1, Dimensions in Aging. Academic Press, London, pp. 35-49. Boyd, E. (1932) The weight of the thymus gland in health and disease. Am. J. Dis. Child. 43, 1162-1168. Brodde, O.E. (1989) Affinity of dopamine agonists and antagonists for peripheral dopamine receptors: comparison of functional studies with radioligand binding studies. In: J.P. Hieble (Ed.), Cardiovascular Function of Peripheral Dopamine Receptors. Marcel Dekker, New York, Base& pp. 27-72. Callard, R.E. (1981) Aging of the immune system. In: M.M.B. Kay and T. Makinodan (Eds.1, Handbook of Immunology in Aging. CRC Press, Boca Raton, FL, p. 103. Chapdelaine, J.M., Warren, Th.C., Whalen, J.D., Grob, P.M. and Wooley, P.H. (1988) Effect of bromocriptine on collagen- induced arthritis. Arthr. Rheum. 31 (SuppI.) S89-S93. Cheng, Y.C. and Prusoff, W.H. (1973) Relationship between inhibition constant (K,) and the concentration of inhibitor which causes 50 per cent inhibition (IC,,,) of an enzymatic reaction. Biochem. Pharmacol. 22, 3099-3103. Clark, B.J. (1990) Postjunctional dopamine receptor stimulants. In: F. Amenta (Ed.), Peripheral Dopamine Pathophysiology. CRC Press, Boca Raton, FL, pp. 253-255.
160
A. Ricci et al. /Journal
of Neuroimmunology
Felten, D.L. and Felten, S.Y. (1989) Innervation of the thymus. In: M.D. Kendall and M.A. Ritter (Eds.), Thymus Update. Harwood, London, pp. 73-88. Geppetti, P., Maggi, C.A., Zecchi-Orlandini, S., Santicioli, P., Meli, A., Frilli, S., Spillantini, M.G. and Amenta, F. (1987) Substance P-like immunoreactivity in capsaicin-sensitive structures of the rat thymus. Regul. Pept. 18, 321-329. Geppetti, P., Frilli, S., Renzi, D., Santicioli, I., Maggi, C.A., Theodorsson, E. and Fanciullacci, M. (1988) Distribution of calcitonin gene-related peptide-like immunoreactivity in various rat tissues: Correlation with substance P and other tachykinins and sensitivity to capsaicin. Regul. Pept. 23, 289-298. Gilard, J.P., Paychere, M., Cuevas, M. and Fernandez, B. (1977) Cell-mediated immunity in ageing population. Clin. Exp. Immunol. 25, 85-91. Gingrich, J.A. and Caron, M.G. (1993) Recent advances in the molecular biology of dopamine receptors. Annu. Rev. Neurosci. 16, 299-321. Hall, N.R., McGillis, J.P., Spagnelo, B.L. and Goldstein, A.L. (1985) Evidence that thymosins and other biologic response modifiers can function as neuroactive immunotransmitters. J. Immunol. 135, 806S-811s. Kay, M.M.B. and Makinodan, T. (1982) The ageing immune system. In: A. Viidik (Ed.), Lectures on Gerontology. Academic Press, London, New York, p. 143. Khansari, D.N., Murgo, A.J. and Faith, R.E. (1990) Effects of stress on the immune system. Immunol. Today 11, 170-175. Lorton, D., Bellinger, D.L., Felten, S.Y. and Felten, D.L. (1990)
56 (1995) 155-160
Substance P innervation of the rat thymus. Peptides 11, 12691275. Malaise, M.G., Hazee-Hagelstein, M.T., Reuter, A.M., VrindsGevaert, Y., Goldstein, G. and Franchimont, P. (1987) Thymopoietin and thymopentin enhanced the labels of ACTH, betaendorphin and beta-lipotropin from rat pituitary cells in vitro. Acta Endocrinol. 115, 455-460. Nagy, E., Berczi, I., Wren, G.E., Asa, S.L. and Kowacs, K. (1983) Immunomodulation by bromocriptine. Immunopharmacology 6, 231-235. Rebov, R.W., Miyake, A., Low, T.L.K. and Goldstein, A.L. (1981) Thymosin stimulates secretion of luteinizing hormone-releasing factor. Science 214, 669-671. Ricci, A., Vega, J.A., Zaccheo, D. and Amenta, F. (1991) Dopamine receptors in the rat thymus decrease with age. Rev. Esp. Geriatr. Gerontol. 26, 173. Ricci, A., Vega, J.A. and Amenta, F. (1994) Pharmacological characterization and autoradiographic localization of dopamine D,-like receptors in the thymus. J. Neuroimmunol. 50, 133-138. Sibley, D.R. and Monsma, F.J. Jr. (1992) Molecular biology of dopamine receptors. Trends Pharmacol. Sci. 13, 61-69. Takahashi, N., Nagai, Y., Ueno, S., Seaki, Y. and Yanagihara, T. (1992) Human peripheral blood lymphocytes express D, dopamine receptor gene and transcribe the two pseudogenes. FEBS Lett. 314, 23-25. Zirbes, T. and Novotny, G.E.K. (1992) Quantification of thymic innervation in juvenile and aged rats. Acta Anat. 145, 283-288.
Journal of Neuroimmunology 56 (1995) W-160
Dopamine D,-like receptors in the thymus of aged rats: A radioligand binding and autoradiographic study Albert0 Ricci a, Josh Antonio Vega b, Damiano Zaccheo ‘, Francesco Amenta a,* a Sezione di Anatomia Umana, Istituto di Farmacologia, Universitci di Camerino, L&aScalzino 5, 62032 Camerino, Italy b Departamento de Morfologia y Biologia Celular Universidad de Oviedo, Oviedo, Spain ’ Istituto di Anatomia Umana, Universitci di Genova, Geneva, Italy
Received 17 May 1994; revised 11 August 1994; accepted 13 September 1994
Abstract Age-dependent changes in the density and pattern of dopamine D,-like receptors were studied in the thymus of young (3 months), adult (12 months) and aged (24 months) male Wistar rats using combined radioligand binding and autoradiographic techniques. [3H]SCH 23390, which was used as a ligand, was specifically bound to sections of the thymus in a manner consistent with the labelling of dopamine D, receptor. The dissociation constant value was similar in the thymus of the three animal groups examined. The maximal density of binding sites, evaluated with conventional radioligand binding techniques, was significantly reduced in the thymus of adult in comparison with young rats and further reduced in aged animals. Silver grains which correspond to [3H]SCH 23390 binding sites were revealed by light microscope autoradiography primarily in the cortex of the thymus and in lesser amounts within thymic corpuscles. A progressive decrease in the density of silver grains more pronounced in the cortex than in thymic corpuscles was observed in the thymus of adult and old in comparison with young rats. The loss of silver grains revealed with autoradiography is more moderate than the decrease in the density of binding sites shown by radioligand binding. Silver grains developed per single cells (probably lymphocytes) of the thymic cortex were reduced between young and adult rats and further decreased in old rats. The above findings suggest that the age-related decline in the density of dopamine D, receptor assayed in the thymus is due in part to the reduced thymic mass with aging. The observation of a decreased expression of dopamine D, receptor in cells of the thymic cortex as a function of age suggests that this reduction cannot be attributed simply to loss of thymic lymphocytes. Keywords:
Thymus; Aging; Dopamine
receptor; Autoradiography
1. Introduction Aging is associated with changes in immune function, although the possible link between these changes and the most prevalent diseases of the elderly has not yet been documented (Beregi, 1986). Changes in the humoral and cellular immunity, in the function of lymphocytes as well as decreased production of antibodies accompanied by increased auto-antibodies have been documented in old age (Gilard et al., 1977; Callard, 1981; Kay and Makinodan, 1982). Moreover, morphological alterations of peripheral lymphocytes, and of splenic and lymph node lymphocytes occur in aged rodents and humans (Beregi, 1986).
* Corresponding
author. Fax (+ 39-737) 2538
0165-5728/95/$09.50
0 1995 Elsevier Science B.V. All rights reserved
SSDI 0165-5728(94)00141-3
; Rat
The thymus is a primary organ of the immune system and plays an important role in the interactions between the neuro-endocrine and immune systems. It is supplied with a rich autonomic innervation (Geppetti et al., 1987; Geppetti et al., 1988; Felten and Felten, 1989; Lorton et al., 1990; Bellinger et al., 19931, and is immunoreactive for several pituitary hormone epitopes (Battanero et al., 1992). Moreover, thymic hormones contributing to regulate immune functions, such as thymosins, have a role in communication between non-immune organs. They were also identified in the central nervous system and probably play a functional role in the hypothalamus (Rebov et al., 1981; Hall et al., 1985; Malaise et al., 1987; Ader et al., 1990; Khansari et al., 1990). A possible role of the catecholamine dopamine in the modulation of immune function is suggested by the
156
A. Ricci et al./Journal
of Neuroimmunology 56 (1995) 155-160
findings that dopamine receptor agonists can reduce or even prevent humoral or cell-mediated immune reactions in rodents and in man (Nagy et al., 1983; Chapdelaine et al., 1988). Moreover, the localization of dopamine D, receptors has been recently demonstrated in the rat thymus (Ricci et al., 1994). Since no information is available so far concerning the influence of aging on dopamine D,-like receptor sites in the rat thymus, we have investigated the topic using combined radioligand binding and autoradiographic techniques. A preliminary account of the present study has been presented at the II European Congress of Gerontology (Ricci et al., 1991).
2. Materials and methods 2.1. Animals and tissue treatment Male Wistar rats of 3 months (young, n = 10, 270 & 12 g body weight), of 12 months (adult, IZ= 10,350 _t 25 g body weight) and of 24 months (aged, n = 10,450 k 20 g body weight) were used. Rats were anaesthetised with ether and sacrificed by decapitation. The thorax was opened, and the thymuses were removed, weighed and washed in ice-cold 0.9% NaCl solution. Thymuses were embedded in a cryoprotectant medium (Tissueteck, Miles, USA) and frozen in a dry ice/acetone mixture. Serial 8-pm sections were obtained using a microtome cryostat and mounted on pre-weighed gelatine-coated microscope slides. A group of 10 consecutive slides 20 pm apart for each tissue block was stained with Toluidine blue to verify microanatomical details and to measure the area of the slide occupied by thymic tissue, which was evaluated by image analysis.
assessed by incubating some sections as above with 2.5 nM [3H]SCH 23390 in the presence of increasing concentrations of compounds active on different subtypes of dopamine receptors or on 5-hydroxytryptamine (5HT) receptors and on a-adrenoceptors. 2.3. Light microscope autoradiography For light microscope autoradiography, sections of the thymus were incubated with 2.5 nM [ 3H]SCH 23390, in the presence or absence of 1 PM (+)-butaclamol to define non-specific binding, and processed according to the protocol detailed elsewhere (Ricci et al., 1994). The density of silver grains developed in dark-field sections of the thymus of rats of different ages was assessed in a 500~pm2 area of the cortex and of the medulla by quantitative image analysis according to the procedure reported in an earlier study (Ricci et al., 1994). Measurements were made on five consecutive sections 20 Km apart of the thymus per rat (n = 10 per age group) incubated with 2.5 nM [3H]SCH 23390 alone or plus 1 PM (+ )-butaclamol to define nonspecific binding. In order to assess whether age-dependent changes in the expression of dopamine D, receptor (see Results) were attributable to loss of thymic cells expressing receptors or to a decrease of the receptor itself, three random areas of the thymic cortex per section were examined at a final magnification of X900 using a bright-field condenser. The number of silver grains developed around the border of 10 randomly selected cells (probably lymphocytes) was then counted by image analysis. Counts were made independently in sections incubated with [3H]SCH 23390 alone (total binding) or plus 1 PM (+)-butaclamol (non-specific binding). Specific binding values were then calculated by subtracting non-specific from total binding values.
2.2. Radio&and binding experiments 2.4. Data analysis For the analysis of dopamine D,-like receptors, sections of the thymus of rats of the three age groups were incubated with [3H]SCH 23390 according to the procedure detailed elsewhere (Ricci et al., 1994). Sections were incubated with increasing concentrations (0.1-7.5 nM) of [3H]SCH 23390 alone or plus 1 PM (+ )butaclamol to define non-specific binding. The optimal incubation time (60 min), temperature (25°C) and radioligand concentration (2.5 nM) were assessed in a recent study of our group (see Ricci et al., 1994). At the end of incubation, sections were washed in ice-cold incubation buffer (2 X 5 min) to remove unbound radioligand and rinsed quickly in distilled water. Sections were then wiped onto Whatman GF-B glass fibre filters and counted by liquid scintillation spectrometry. The pharmacological specificity of [ 3H]SCH 23390 binding to sections of the thymus of different ages was
Data from the binding experiments were analysed by linear regression analysis of Scatchard plots of saturation isotherms. Competitor dissociation constant (Ki) values were determined according to the method of Cheng and Prusoff (1973). Further details on statistical analysis are reported in a recent paper of our group (Ricci et al., 1994). 2.5. Chemicals 13H]SCH 23390 ([RI-( + )-(-chloro-2,3,4,5_tetrahydro5-phenyl-lH-3benzazepin-al-hemimaleate) (specific activity 85 Ci/mmol) was obtained from the Amersham Radiochemical Centre (Buckinghamshire, UK). Other chemicals were obtained from the sources recently indicated (Ricci et al., 1994).
A. Ricci et al. /Journal
of Neuroimmunology 56 (1995) 155-160
BOUND/FREE
”
50
66
82
98 [ 3
130
114
HI-SCH
23390
146
162
178
194
bound
Fig. 1. Scatchard analysis of [3H]SCH 23390 binding to sections of the thymus of young ( n), adult ( A > and old (0) rats. The B,,, values calculated with this kind of analysis averaged 170&12 fmol/mg tissue in young rats, 120 f 9.3 fmol/mg tissue in adult rats (P < 0.001 vs. young) and 62k4.1 fmol/mg tissue in old rats (P < 0.001 vs. young and P < 0.001 vs. adult).
3. Results
Values of area occupied by thymic tissue proper in rats of the three age groups are shown in the Table 1. As can be seen, this area significantly decreases as a function of age (Table 1). [ 3H]SCH 23390 was specifically bound to sections of the thymus in the three age groups examined. Binding was time-, temperature- and concentration-dependent (data not shown). [3H]SCH 23390 binding to sections of the thymus of rats of the three age groups examined is concentration-dependent, belonging to a single class of high-affinity sites (Fig. 1). The dissociation constant (K,) values were similar in the thymus of young (K,: 1.6 + 0.04 nM), adult (K,: 1.5 &-0.05) and aged rats (K,: 1.6 + 0.07). The maximum binding density (B,,,) value was significantly reduced in adults in comparison with young rats and further decreased in aged animals (Fig. 1). The pharmacological profile of [3H]SCH 23390 binding to sections of the thymus of rats of different age is consistent with the labelling of dopamine D,-like receptors, SCH 23390 being the most powerful com-
157
petitor of [3H]SCH 23390 binding, followed in descending order by (+ )-butaclamol, fenoldopam, dopamine and haloperidol (data not shown). Isomers of sulpiride, bromocriptine, domperidone, ketanserin, phentolamine and serotonin were weak competitors of L3HlSCH 23390 binding or without effect (data not shown). Dopamine competed with [“HISCH 23390 binding in the submicromolar range. The K, value was 40.7 + 2.1 nM in young, 38.3 + 1.6 nM in adult and 39.3 + 2.8 in the thymus of old rats. Light microscope autoradiography revealed the accumulation of specific [3H]SCH 23390 binding sites represented by silver grains primarily in the cortex of the thymus (Fig. 2) and in lesser amounts within thymic corpuscles of the medulla (Fig. 2). The density of specific silver grains was decreased in adult in comparison with young rats and further reduced in aged animals (Fig. 2). The age-dependent loss of silver grains was more pronounced in the cortex than in the medulla (Fig. 3). Analysis of the number of silver grains developed within single cells of the thymic cortex (which probably represent lymphocytes) revealed a decrease of specific [3H]SCH 23390 binding sites in adult in comparison with young rats and a further decrease in old rats (by about 38% vs. young and by about 18% vs. adult) (Table 1). Comparative evaluation of the age-dependent changes in the density of [3H]SCH 23390 binding sites assessed by radioligand binding and autoradiographic techniques revealed a more pronounced loss of binding sites using radioligand binding techniques (Fig. 1) than autoradiography (Fig. 3).
4. Discussion Important changes occur in the organisation of lymphoid organs and the regulation of immune function in aging. Several experimental data suggest a possible role of the autonomic innervation of immune organs in the immunosenescence (Bellinger et al., 1993). The thymus undergoes a variety of anatomical and functional changes during life span. In fact, it has the maximum development during sexual maturation, begins to atrophy at puberty, and continues a progressive degenera-
Table 1 Influence of aging on the area of thymic tissue in sections of thymus and on the density of silver grains within single cells of the thymic cortex in rats of different ages Animal group
n
Area occupied by thymic tissue proper (mm’)
Number of specific silver grains per cell
Young Adult Old
10 10 10
2300 + 120 1soo+ 105 a 950 + 75 c
29.1 f 0.8 23.4 f 0.6 b 17.9 f 0.4 d
Values are means + SEM and were calculated as described in Materials and methods. a P < 0.05 vs. young; b P < 0.01 vs. young; ’ P < 0.01 vs. young or adult; d P < 0.01 vs. young and P < 0.05 vs. adult.
158
A. Ricci et al. /Journal
of Neuroimmunology 56 (1995) 155-160
tion with aging (Boyd, 1932; Bellinger et al., 1988). The thymus of aged rats is supplied with a more dense autonomic innervation than young rats (Bellinger et al., 1990; Zirbes and Novotny, 1992). However, the func-
Silver
arain
densitv
x 500
urn2
350 300 ; 250 1 200
-
15010050 1 C o-l CORTEX
MEDULLA
Fig. 3. Quantitative analysis of the density of specific silver grains developed in the cortex (CORTEX) and in the medulla (MEDULLA) of the thymus of young (A), adult (B) and old (Cl rats after exposure of sections to a L3H]SCH 23390 concentration of 2.5 nM. Specific binding was obtained by subtracting non-specific from total binding as indicated in Materials and methods. Data are means +SEM. ‘P < 0.001 vs. young; * *P < 0.01 vs. young; lP < 0.001 vs. and P < 0.01 vs. adult.
tional significance of these observations has not yet been established (Bellinger et al., 1993). A possible role of dopamine in modulating immune function has been suggested (Nagy et al., 1983; Chapdelaine et al., 1988). The immunosuppressive effects of dopamine and its agonists are considered to be due primarily to inhibition of the release of anterior pituitary hormones stimulating immune function, rather than by a direct action on the immune system (for a review see Clark, 1990). Studies on the expression of dopamine receptors in circulating immune cells, completed before the introduction of molecular biology techniques to dopamine receptor research, have provided conflicting results (see Ricci et al., 1994). More recently the expression of dopamine D, receptor genes and their transcription in two pseudo genes in human lymphocytes have been demonstrated (Takahashi et al., 1992), as well as the presence of dopamine D, receptors in the thymus (Ricci et al., 1994). In the last lo-15 years two main subtypes of central and peripheral dopamine receptors, namely D, and D, receptors, have been identified (Gingrich and Caron,
Fig. 2. Autoradiographic localization of [3H]SCH 23390 binding in sections of the thymus of rats of 3 months (young, A), 12 months (adult, D) and 24 months (old, El. Sections were incubated with a 2.5 nM concentration of 13HlSCH 23390 alone (A, D and E) or plus 1 mM (+)-butaclamol (C) to generate non-specific binding. (A, C, D and E) are dark-field pictures; (B) is a bright-field picture of micrograph (A) stained with Toluidine blue to verify microanatomical details. C, cortex; M, medulla. The highest accumulation of silver grains occurred within the cortical tissue. Note the reduced density of silver grains in the adult in comparison with the young rat and the further decrease noticeable in the old rat. (X 160).
A. Ricci et al. /Journal
of Neuroimmunology 56 (1995) 155-160
1993). More recently, the application of molecular biology techniques to dopamine receptor research allowed the identification of five subtypes of dopamine receptors CD,-D, receptors) (Sibley and Monsma, 1992; Gingrich and Caron, 1993). D, and D, receptors belong to the D,-like receptor family, whereas D,, D, and D, sites belong to the D,-like receptor family (Sibley and Monsma, 1992; Gingrich and Caron, 1993). Consistent with data of a previous study of our group performed in young rats (Ricci et al., 1994) we have seen that the dopamine D,-like receptor radioligand [3HlSCH 23390 (Brodde, 1989) was specifically bound to section of the thymus of rats of different ages in a manner consistent with the labelling of D,-like receptors. In view of the capability of dopamine to compete with [3H]SCH 23390 binding in the submicromolar range, the site we have characterized probably belongs to the dopamine D, receptor subtype (Sibley and Monsma, 1992; Gingrich and Caron, 1993). This receptor is located primarily in the cortex, which is the thymic portion densely packed with thymocytes (T lymphocytes) (Amenta, 1991). This suggests that T lymphocytes express dopamine D, receptor. Aging is accompanied by a loss of dopamine D, receptor in the rat thymus. The findings that conventional radioligand binding techniques reveal more pronounced decrease of this receptor, suggest that this reduction is in part due to the decrease of functionally active thymic areas as a function of age. In fact, the loss of specific [3HlSCH 23390 binding sites averages the 30% and the 24% between young and adult rats, the 65% and the 46% between old and young rats and the 35% and the 22% between old and adult rats using radioligand binding and autoradiographic techniques, respectively. This suggests that quantitative autoradiographic techniques may be useful in assessing changes in the density of neurotransmitter receptors in organs undergoing regressive changes as a function of age such as the thymus (Boyd, 1932; Bellinger et al., 1988). The observation of a decreased expression of dopamine D, receptor in single cells (probably lymphocytes) of the thymic cortex as a function of age suggests that this reduction cannot be attributed simply to loss of thymic lymphocytes. Age-related changes of cerebral dopamine receptors consisting in a decrease of their density primarily between young and adult age have been documented (Amenta et al., 1991). The same occurs for the dopamine D,-like receptor of the thymus, the density of which is reduced with aging. However, unlike central D,-like dopamine receptors (Amenta et al., 19911, the most remarkable decrease of the thymic receptor occurs between middle-age and old subjects. The majority of investigations on aging of central dopamine receptors have been performed in the striatum, which is a tissue expressing dopamine D, rather than D, recep-
159
tors (Gingrich and Caron, 1993). It cannot be excluded that the two subtypes of dopamine D,-like receptors may have a different sensitivity to aging.
Acknowledgements
The present study was supported in part by a grant of the Italian National Research Council (CNR, Progetto Finalizzato Invecchiamento). The authors are greatly indebted to Ms. S. Beatty for the critical revision of the manuscript, and to Mr. G. Bonelli for photographic assistance.
References Ader, R., Felten, D. and Cohen, N. (1990) Interactions between the brain and the immune system. Annu. Rev. Pharmacol. Toxicol. 30, 561-602. Amenta, P.S. (1991) Histology and Human Microanatomy. Piccin, Padova, pp. 249-255. Amenta, F., Zaccheo, D. and Collier, W.L. (1991) Neurotransmitters, neuroreceptors and aging. Mech. Aging Devel. 61, 249-273. Battanero, E., De Leeuw, F.-J., Jansen, G.H., Van Wichen, D.F., Huber, .I., Schuurman, H.-J. (19921 The neural and neuroendocrine component of the human thymus. II. Hormone immunoreactivity. Brain Behav. Immun. 6, 249-264. Bellinger, D.L., Felten, S.Y. and Felten, D.L. (1988) Maintenance of noradrenergic sympathetic innervation in the involuted thymus of aged Fischer 344 rat. Brain Behav. Immunol. 2, 133-150. Bellinger, D.L., Lorton, D., Romano, T., Olschowka, J.A., Felten, S.Y. and Felten, D.L. (1990) Neuropeptide innervation of lymphoid organs. Ann. N.Y. Acad. Sci. 594, 17-33. Bellinger, D.L., Felten, S.Y., Ackerman, K.D., Lorton, D., Madden, K.S. and Felten, D.L. (19931 Noradrenergic sympathetic innervation of lymphoid organs during development, aging, and in autoimmune disease. In: F. Amenta (Ed.), Aging of the Autonomic Nervous System. CRC Press, Boca Raton, FL, pp. 243-284. Beregi, E. (1986) Relationships between aging of the immune system and aging of the whole organism. In: M. Bergener, M. Ermini and H.B. Staehelin (Eds.1, Dimensions in Aging. Academic Press, London, pp. 35-49. Boyd, E. (1932) The weight of the thymus gland in health and disease. Am. J. Dis. Child. 43, 1162-1168. Brodde, O.E. (1989) Affinity of dopamine agonists and antagonists for peripheral dopamine receptors: comparison of functional studies with radioligand binding studies. In: J.P. Hieble (Ed.), Cardiovascular Function of Peripheral Dopamine Receptors. Marcel Dekker, New York, Base& pp. 27-72. Callard, R.E. (1981) Aging of the immune system. In: M.M.B. Kay and T. Makinodan (Eds.1, Handbook of Immunology in Aging. CRC Press, Boca Raton, FL, p. 103. Chapdelaine, J.M., Warren, Th.C., Whalen, J.D., Grob, P.M. and Wooley, P.H. (1988) Effect of bromocriptine on collagen- induced arthritis. Arthr. Rheum. 31 (SuppI.) S89-S93. Cheng, Y.C. and Prusoff, W.H. (1973) Relationship between inhibition constant (K,) and the concentration of inhibitor which causes 50 per cent inhibition (IC,,,) of an enzymatic reaction. Biochem. Pharmacol. 22, 3099-3103. Clark, B.J. (1990) Postjunctional dopamine receptor stimulants. In: F. Amenta (Ed.), Peripheral Dopamine Pathophysiology. CRC Press, Boca Raton, FL, pp. 253-255.
160
A. Ricci et al. /Journal
of Neuroimmunology
Felten, D.L. and Felten, S.Y. (1989) Innervation of the thymus. In: M.D. Kendall and M.A. Ritter (Eds.), Thymus Update. Harwood, London, pp. 73-88. Geppetti, P., Maggi, C.A., Zecchi-Orlandini, S., Santicioli, P., Meli, A., Frilli, S., Spillantini, M.G. and Amenta, F. (1987) Substance P-like immunoreactivity in capsaicin-sensitive structures of the rat thymus. Regul. Pept. 18, 321-329. Geppetti, P., Frilli, S., Renzi, D., Santicioli, I., Maggi, C.A., Theodorsson, E. and Fanciullacci, M. (1988) Distribution of calcitonin gene-related peptide-like immunoreactivity in various rat tissues: Correlation with substance P and other tachykinins and sensitivity to capsaicin. Regul. Pept. 23, 289-298. Gilard, J.P., Paychere, M., Cuevas, M. and Fernandez, B. (1977) Cell-mediated immunity in ageing population. Clin. Exp. Immunol. 25, 85-91. Gingrich, J.A. and Caron, M.G. (1993) Recent advances in the molecular biology of dopamine receptors. Annu. Rev. Neurosci. 16, 299-321. Hall, N.R., McGillis, J.P., Spagnelo, B.L. and Goldstein, A.L. (1985) Evidence that thymosins and other biologic response modifiers can function as neuroactive immunotransmitters. J. Immunol. 135, 806S-811s. Kay, M.M.B. and Makinodan, T. (1982) The ageing immune system. In: A. Viidik (Ed.), Lectures on Gerontology. Academic Press, London, New York, p. 143. Khansari, D.N., Murgo, A.J. and Faith, R.E. (1990) Effects of stress on the immune system. Immunol. Today 11, 170-175. Lorton, D., Bellinger, D.L., Felten, S.Y. and Felten, D.L. (1990)
56 (1995) 155-160
Substance P innervation of the rat thymus. Peptides 11, 12691275. Malaise, M.G., Hazee-Hagelstein, M.T., Reuter, A.M., VrindsGevaert, Y., Goldstein, G. and Franchimont, P. (1987) Thymopoietin and thymopentin enhanced the labels of ACTH, betaendorphin and beta-lipotropin from rat pituitary cells in vitro. Acta Endocrinol. 115, 455-460. Nagy, E., Berczi, I., Wren, G.E., Asa, S.L. and Kowacs, K. (1983) Immunomodulation by bromocriptine. Immunopharmacology 6, 231-235. Rebov, R.W., Miyake, A., Low, T.L.K. and Goldstein, A.L. (1981) Thymosin stimulates secretion of luteinizing hormone-releasing factor. Science 214, 669-671. Ricci, A., Vega, J.A., Zaccheo, D. and Amenta, F. (1991) Dopamine receptors in the rat thymus decrease with age. Rev. Esp. Geriatr. Gerontol. 26, 173. Ricci, A., Vega, J.A. and Amenta, F. (1994) Pharmacological characterization and autoradiographic localization of dopamine D,-like receptors in the thymus. J. Neuroimmunol. 50, 133-138. Sibley, D.R. and Monsma, F.J. Jr. (1992) Molecular biology of dopamine receptors. Trends Pharmacol. Sci. 13, 61-69. Takahashi, N., Nagai, Y., Ueno, S., Seaki, Y. and Yanagihara, T. (1992) Human peripheral blood lymphocytes express D, dopamine receptor gene and transcribe the two pseudogenes. FEBS Lett. 314, 23-25. Zirbes, T. and Novotny, G.E.K. (1992) Quantification of thymic innervation in juvenile and aged rats. Acta Anat. 145, 283-288.