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Ontogeny of cholinergic innervation of thymus in mouse
DEVELOPMENTAL AND COMPARATIVE IMMUNOLOGY, Vol. ii, pp. 627-635, 1987. 0145-305X87 $3.00 + .00 Printed in the USA. Copyright (c) Pergamon Journals Ltd. All rights reserved.
ONTOGENY OF CHOLINERGIC INNERVATION OF THYMUS IN MOUSE
Upendra Singh, Jamal A.Fatani.,Abdul Majeed Mohajir Department of Anatomy College of Medicine King Saud University Riyadh. Kingdom of Saudi Arabia.
Abstract In this study, identification of cholinergic nerves in the murine thymic rudiments during ontogenetic development, by immunonocytochemical localization of the enzyme cholin acetyltransferase (CHAT) was carried out. Fetal thymic rudiments from day 13 of gestation to the time of birth, and upto 8th post natal week were examined. Contrary to the earlier reports, the results indicate that the thymic rudiment is not innervated during early ontogenetic development and that the first ChAT positive nerve profiles were observed at around day 17/18 of gestattion. Significance of cholinergic innervation in thymocyte maturation is suggested.
INTRODUCTION In a previous study we have reported the presence of cholinergic nerves in the adult murine thymus by histochemical and immunocytochemical localization of enzymes acetylecholinestrase (AchE) and choline acytle transferase (CHAT) (i). Earlier these nerves had been reported in murine adult and embryonic thymus by labelling of horse radish peroxidase (HRP) and histochemical techniques (2). Further more it was also reported that those nerves are sparsely distributed in the embryonic thymic rudiments of the nude mice and that successful reconstitution of the immunodeficient mice, following implantation of thymic grafts, depended upon the ingrowth (sprouting) of these nerves from the host animal into the implant (3,4).These results suggest 627
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that perhaps the c h o l i n e r g i c i n n e r v a t i o n of the thymus plays an i m p o r t a n t role in the p r o c e s s of i n ~ h y m i c m a t u r a t i o n of T lymphocytes. In view of the fact that these e a r l i e r studies w e r e carried out by u s i n g h i s t o c h e m i c a l m e t h o d s w h i c h are less r e l i a b l e and s p e c i f i c for d e m o n s t r a t i o n of these nerves(5) the p r e s e n t study was u n d e r t a k e n to r e - e x a m i n e the p a t t e r n of c h o l i n e r g i c i n n e r v a t i o n of the thymus by i m m u n o c y t o c h e m i c a l l o c a l i z a t i o n of C h A T w h i c h is c o n s i d e r e d more specific and t h e r e f o r e r e l i a b l e m a r k e r for these nerves (6). The results of this study indicate that c o n t r a r y to e a r l i e r studies where c h o l i n e r g i c n e r v e s w e r e r e p o r t e d in the thymic r u d i m e n t of mice even b e f o r e they were p o p u l a t e d by the l y m p h o i d stem cells (7) c h o l i n e r g i c n e r v e s were d e t e c t e d in the later part of the o n t o g e n e t i c d e v e l o p m e n t of the thymus and in the post natal period. The s i g n i f i c a n c e of this i n n e r v a t i o n in r e l a t i o n to the f u n c t i o n of thymus as the p r i m a r y l y m p h o i d organ for the g e n e r a t i o n of T l y m p h o c y t e s is discussed.
Material
and M e t h o d s
Animals: Male and female adult BALB/c m i c e d were used. To o b t a i n embryos of various g e s t a t i o n periods, m a t i n g b e t w e e n the male and female ( two females and one m a l e ) a n i m a l s w e r e set up. The d a y of v a g i n a l plug was c o u n t e d as the d a y 0 s i m i l a r to the e a r l i e r study (8). The animals were m a i n t a i n e d in the animal house of King Saud U n i v e r s i t y , C o l l e g e of Medicine, Riyadh. Rea@ents: Most of the c h e m i c a l s and buffers were o b t a i n e d from S a r g e n t Welch.Co. Avidin, b i o t i n kits w e r e p u r c h a s e d from V e c t o r Labo r a t o r i e s and the a n t i b o d y against c h o l i n e acetyle t r a n s f e r a s e (IE6) was i n i t i a l l y p r o v i d e d by Dr. G.D. C r a w f o r d of the N e u r o Science division, City of Hope hospital, Duarte, C a l i f o r n i a and were later p u r c h a s e d from B o e h r i n g e r M a n n h e i m Biochemicals, Indianapolis. T h [ m i c rudiments: T h y m i c r u d i m e n t s from day 13 to b i r t h w e r e r e m o v e d aseptically and i m m e d i a t e l y fixed in 10% formaline. Post natal thymic lobes were o b t a i n e d from one, four and eight week old animals, by s u r g i c a l l y r e m o v i n g the thymic lobes from the m e d i a s t i n a l cavities of the animals, t a k i n g care to avoid any i n c l u s i o n of m e d i a s t i n a l lymph nodes. All s u r g i c a l p r o c e e d u r e s w e r e c a r r i e d out under d i s s e c t i n g m i c r o s c o p e as in p r e v i o u s s t u d y (8). The larger lobes w e r e cut into small pieces and i m m e d i a t e l y fixed by i m m e r s i o n into 10% formaline. Immunoc~tochemistry: E m b r y o n i c and post natal thymic tissues were fixed in 10% b u f f e r e d f o r m a l i n e ( P h o s p h a t e b u f f e r Ph 7.4 ) for upto 24 hours after w h i c h they were w a s h e d amd immersed in 5% S u r c o s e s o l u t i o n for 12 hours. The thymic lobes were then frozen on
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metal stubs with OCT compound. 15 to 20 n thick sections were cut at -15°by a cryostat. The sections were mounted on histostick coated slides. The slides were stored at37 C for 48 to 72 hours before use for better sticking of the section. The slides were immersed in tris buffer (Ph 7.4) for fifteen minutes and then were coated with 1:50 of normal rabit serum followed by 4ng/ml solution of rat anti ChAT monoclonal antibody (Boehringer) for 18 hours at 4°C in a staining chamber. The slides were washed and treated with rabit anti rat biotinated antibody and ffluoriscinated avidin (Vector laboratories. The slides were examined under a fluorescent microscope and photographed. Results I. Light microscopy: Examination of thymic rudiments by toluidine blue showed few large basophilic cells in the thymic rudiments at 13/14 days of gestation. The number of such lymphoid cells increased with age. The distinction between the thymic cortex and medulla was evident only in the post natal thymic rudiments. These observations were consistent with the obeservations of our earlier studies (8), (Fig.l) 2. Fuorescence microscopy: No fluorescent nerve profiles were observed within the embryonic thymic rudiments of 13 to 18 days of gestation (Fig:2,~) In the thymic rudiment of 19 days of gestation and at birth few fuorescent nerve profiles were seen in relation to blood vessels in the deeper part of the thymic paranchyma. The capsular area and the superficial regions of the thymic tissue did not show such profiles (Fig:4). In one week old post natal thymic rudiments, numerous fluorescent profiles were seen mostly within the deep parachymal tissue which corresponds with the medullary region of the thymic tissue on examination by light microscope. The distribution of these fluorescent profiles increased with age and appeared most abundant in 6/8 week old thymic rudiments. (Fig:5,6).
FIG.
1
Section of a thirteen day fetal thymic rudiment showing rounded darkly stained (lymphoid) cells. H&E. X25.
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V
FIG.
2
F l u o r e s c e n t m i c r o g r a p h of a t h i r t e e n day fetal thymic r u d i m e n t f o l l o w i n g t r e a t m e n t w i t h a n t i - C h A T antibody, showing lack of f l u o r e s c e n t profiles. X25
FIG.
3
F l u o r e s c e n t m i c r o g r a p h of a sixteen day fetal thymic rudiment following treatment with anti-ChAT antibody showing lack of f l u o r e s c e n t profiles, similar to figure 2.X25
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4
F l u o r e s c e n t m i c r o g r a p h of a thymic r u d i m e n t from a newly born mouse f o l l o w i n g a n t i - C h A T antibody treatment, showing few f l u o r e s c e n t profiles in the d e e p e r part of p a r e n c h y m a in relation to the blood vessels. X25
FIG.
5
F l u o r e s c e n t m i c r o g r a p h of one week old post natal thymus s h o w i n g f l u o r e s c e n t profiles w i t h i n the parenchyma, f o l l o w i n g a n t i - C h A T a n t i b o d y treatment. X25
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T
FIG.
6
F l u o r e s c e n t m i c r o g r a p h of an~,adult thymus s h o w i n g f l u o r e s c e n t profiles, f o l l o w i n g a n t i - C h A T a n t i b o d y treatment. X25 Discussion The results of this s t u d y i n d i c a t e that the c h o l i n e r g i c nerve~ appear w i t h i n the thymic r u d i m e n t around the time of birth. This c o n c l u s i o n is b a s e d on the results of i m m u n o c y t o c h e m i c a l locali z a t i o n of the enzyme ChAT w h i c h is c o n s i d e r e d a specific marker for the d e m o n s t r a t i o n of c h o l i n e r g i c n e u r o n s and nerves (9). T h e s e results, however, are not in a g r e e m e n t w i t h the e a r l i e r o b s e r v a t i o n s w h e r e these n e r v e s were r e p o r t e d to be p r e s e n t w i t h i n the e m b r y o n i c thymic rudiments, even p r i o r to the e n t r y of l y m p h o i d cells (7,4). In the mouse the thymic p r o g e n i t o r are known to enter the thymic r u d i m e n t around day 10 of g e s t a t i o n d u r i n g o n t o g e n y (i0). The e a r l i e s t time at w h i c h the c h o l i n e r g i c n e r v e s w e r e o b s e r v e d in this study, however, was just b e f o r e b i r t h (18/19 day of gestation). This d i s c r e p a n c y b e t w e e n these results and the results of B u l l o c h and P o m e r a n t z (4) could be a t t r i b u t e d to the d i f f e r e n c e s in m e t h o d s e m p l o y e d in the studies. In v i e w of less r e l i a b i l i t y of the h i s t o c h e m i c a l l o c a l i z a t i o n of a c e t y l c h o l i n e s t e r a s e , as m a r k e r for c h o l i n e r g i c nerves, the results of p r e v i o u s studies need confirmation. The f u n c t i o n a l s i g n i f i c a n c e of c h o l i n e r g i c i n n e r v a t i o n in r e l a t i o n to thymic l y m p h o p o i e s i s is not known. It is claimed, however, that the r e c o n s t i t u t i o n of the dy uhymic ( nude ) m i c e , f o l l o w i n g a thymic graft d e p e n d s on the e s t a b l i s h m e n t of c h o l i n e r g i c i n n e r v a t i o n w i t h i n the graft (3). Furthermore, the nude mice thymic r u d i m e n t s were also reported to be sparsely i n n e r v a t e d than the thymic rudiments of normal mice (4). These o b s e r v a t i o n do suggest a r e g u l a t o r y role for the i n n e r v a t i o n in the d e v e l o p m e n t of thymus. D i f f e r e n t i a t i o n of T lymphocytes
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requires an i n t e r a c t i o n b e t w e e n the l y m p h o i d p r o g i n a t o r cells w h i c h enter the thymus from the bone m a r r o w in the a d u l t and the yolk sac and liver in the fetus and the n o n l y m p h o i d cells of the thymic parenchyma, the so c a l l e d thymic m i c r o e n v i r o n ment (ii). These non l y m p h o i d cells are Ia positive and include the e p i t h e l i a l cells, m a c r o p h a g e s and d e n d r i t i c cells of the t h y m u s . S u p p o r t for the c o n c e p t of this i n t e r a c t i o n comes from the o b s e r v a t i o n of the i n h i b i t i o n of thymic l y m p h o p o i e s i s in organ c u l t u r e s on a d d i t i o n of a n t i - I a a n t i b o d y in the culture m e d i a (12) and the absence of Ia p o s i t i v e cells in the thymic r u d i m e n t s of the nude mice (13). It has been s u g g e s t e d that the r e c o g n i t i o n o f I a m o l i c u l e on the a c c e s s a r y cells, by the lymphoid "~lls, leads to the s e c r e t i o n of g r o w t h factor i n t e r l u k i n - I (IL-I) w h i c h in turns s t i m u l a t e s the p r o l i f e r a t i o n of t h y m o c y t e s w h i c h are i m m u n o r e a c t i v e and on antigen stimulation produce another factor i n t e r l u k i n - 2 (IL-2) (14). Tnis s u g g e s t i o n is based on the results of e x p e r i m e n t s on presumably immature t h y m o c y t e s isolated from the adult thymus and t h e r e f o r e may not truely r e p r e s e n t the events that occur d u r i n g the m a t u r a t i o n of fetal thymocytes. If mere r e c o g n i t i o n of I~ r m o l i c u l e on the a c c e s s a r y cells by the l y m p h o i d cells was e n o u g h for the for the g e n e r a t i o n of i m m u n o r e a c t i v e cells, such cells could have been p r e s e n t in the early thymic (13/14) rudiments, since these rudiments do possess both the lymphoid cells and Ia-: p o s i t i v e cells (13). On the other hand immuner e a c t i v e cells do not appear w i t h i n the thymus b e f o r e day 18/19 of g e s t a t i o n (15). It is likely t h e r e f o r e that in a d d i t i o n to the r e c o g n i t i o n of Ia~ molicule, some other r e g u l a t o r y mecha n i s m also plays a role in the p r o d u c t i o n of these T cell g r o w t h factors by the a c c e s s a r y cells of thymus and thereby r e g u l a t e thymic function. Previous studies have already i n d i c a t e d i n h i b i t i o n of th~ ~ic lymphope~ ,sis and the immuner e a c t i v i t y of T l y m p h o c y t e s (16, 17,18). In view of these studies it may be s u g g e s t e d that the c h o l i n e r g i c i n n e r v a t i o n has a s t i m u l a t o r y role, o p p o s i t e to the i n h i b i t o r y role of the a d r e n e r g i c n e r v e s in this process. Such a h y p o t h e s i s is r e a s o n a b l e to c o n s i d e r in view of the fact that the choln e r g i c i n n e r v a t i o n is c o n c e r n e d w i t h the s e c r e t o r y a c t i v i t y of most tissues of the body. It is possible, therefore, that c h o l i n e r g i c nerves could r e g u l a t e the s e c r e t o r y activity of n o n l y m p h o i d cells of the thymic parenchyma, w h i c h ia c o n s i d e r e d to be the source of various growth factors (19,20). The pattern of d i s t r i b u t i o n of c h o l i n e r g i c n e r v e s in the d e e p e r part of the thymic p a r e n c h y m a w h i c h is the site of most nonl y m p h o i d cells of the thymus, as o b s e r v e d in this study, further s t r e n g h t e n s this possibility. This study, p r o v i d e s a n a t o m i c a l e v i d e n c e for an i n t e r a c t i o n b e t w e e n the d e v e l o p m e n t and m a t u r a t i o n of the thymus and its a u t o n o m i c i n n e r v a t i o n on the basis of i m m u n o c y t o c h e m i c a l localization of ChAT n e r v e s d u r i n g o n t o g e n i t i c d e v e l o p m e n t of the thymus. Studies are in progress to further e l u c i d a t e the role of a u t o n o m i c nerves in the r e g u l a t i o n of thymic function.
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Acknowledgement The authors are thankful to Mr. Abdur Rehman for his technical help and for the microphotography, Mr Imdad Elahi Quraishi for typing the manuscript a n d King Saud University, College of Medicine, Research Center, for the financial support.
References i. Fatani,J.A., M.A.Qayyum.,L.Mehta.,and U.Singh. (1986) Parasympathetic innervation of the thymus: A histochemical and immunocytochemical study. J.Anat. 147:115-119. 2. Bulloch,K.and R.Y.Moore (1981). Innervation of the thymus gland by the brain stem and spinal cord in mouse and rat. Am.J.Anat.162:157-166. 3. Bulloch,K., M.R.Cullen., M.L.Davis and R.H.Schwartz. (1983) Neuroimmunology of the thymus gland. Neurology. 33.184-187. 4. Bulloch,K., and W.Pomerentz. (1984) Autonomic ~rvo~1~ system innervation of the thymic related lymphoid tissue in wild type and nude mice. J.Comp.Neurol. 228:57-68. 5. Levey,A.I., B.H. Wainer.,E.J.Mufson., and M.M.Mesulam. (1983) Co-localization of acetylcholinesterase and choline a c e t y l transferase in the rat cerebellum.Neuroscience. 9:9-22. 6. Eckenstein,F.and M.V.Sofroniew. (1983) Identification of central cholinergic neurons containing both choline acetyltransferase and acetylcholinesterase and of central neuron containing only acetylcholinesterase. J.Neuroscience 3:2286-2291. 7. Bulloch,K. (1982) A light and ultrastructural analysis of the innervation of the thymus gland during prenatal period. Soc. Neursci.Abst. 8:72. 8. Singh,U. (1979) Effect of catecholamine on imphopoiesis in fetal mouse thymic explants. J. Anat. 129:279-292. 9. Houser,C.R.,G.D.Crawford,R.P.Barber.,P.M.Salvaterra., and J.E.Vaughn. (1983) Organization and morphological characteristics of cholinergic neurons: an immunocytochemical study with monoclonal antibody to choline acetyltransferase. Brain.Res. 266:79-119. 10. Moore,M.A.S.and J.J.T.Owen. (1967) Experimental studies on the development of the thymus. J.Exp.Med.126:715-725. ii. Stutman,O. (1978) Intrathymic and extrathymic T cell maturation,Transplant. Rev.42:138-184. 12. Deluca,D. (1986) Ia positive nonlymphoid cells and cell development in murine fetal thymus organ cultures: Monoclonalant-Ia antibodies inhibit the development of T cells. J. Immunol.136:430-439. 13. Vliet,E.V.,E.J.Jenkinson,R.Kingston.,J.J.T. Owen and W. VanEwijk. (1985) Stroma cell types in the developing thymus of the normal and nude mouse embryo. Eur.J.Immunol.15:675-681. 14. Rock,K.L. and B. Benacerraf. (1984) The role of Ia molicule in the activation of T lymphocytes.J.Immunol.132:1654-1662.
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15. Robinson, J.H. and J.J.T.Owen.(1976) Generation of T cell function in organ cultures of fetal mouse thymus. I. Mitogen responsiveness. Clin. Exp. Immunol. 23:347-354. 16. Besedovsky,H.O., A.Ray.,E.Sorkin.,M.D.Prada and H.H.Keller. (1979) Immunoregulation mediated by the sympathetic nervous system. Cell. Immunol. 48:346-355. 17. Williams, J.M.,R.G.Peterson.,P.A.Shea.,J.F.Schedtje.,D.C. Bauer and D.L.Felton. (1981) Sympathetic innervation of murine thymus and spleen. Evidence for a functional link between the nervous system and immune system. Brain Res. Bull. 6:83-94. 18. Singh,U. (1985)Lymphopoiesis in the nude fetal thymus following sympathec£omy.Cell. Immunol. 93:222-228. 19. Palacious,R., C.Fernandez, and P.Sideras. (1980) Development and continuous growth in culture of Interlukin-2 producing lymphocytes from athymic nu/nu mice. Eur. J.Immunol. 12:777-782. 20. Smith,K.A., L.B.Sachman.,J.J.Oppenheim, and M.F.Favata.(1930) Functional relationship of interlukins. J.Exp. Med. 151: 1551-1556. Received: November, 1986 Accepted: February, 1987
ONTOGENY OF CHOLINERGIC INNERVATION OF THYMUS IN MOUSE
Upendra Singh, Jamal A.Fatani.,Abdul Majeed Mohajir Department of Anatomy College of Medicine King Saud University Riyadh. Kingdom of Saudi Arabia.
Abstract In this study, identification of cholinergic nerves in the murine thymic rudiments during ontogenetic development, by immunonocytochemical localization of the enzyme cholin acetyltransferase (CHAT) was carried out. Fetal thymic rudiments from day 13 of gestation to the time of birth, and upto 8th post natal week were examined. Contrary to the earlier reports, the results indicate that the thymic rudiment is not innervated during early ontogenetic development and that the first ChAT positive nerve profiles were observed at around day 17/18 of gestattion. Significance of cholinergic innervation in thymocyte maturation is suggested.
INTRODUCTION In a previous study we have reported the presence of cholinergic nerves in the adult murine thymus by histochemical and immunocytochemical localization of enzymes acetylecholinestrase (AchE) and choline acytle transferase (CHAT) (i). Earlier these nerves had been reported in murine adult and embryonic thymus by labelling of horse radish peroxidase (HRP) and histochemical techniques (2). Further more it was also reported that those nerves are sparsely distributed in the embryonic thymic rudiments of the nude mice and that successful reconstitution of the immunodeficient mice, following implantation of thymic grafts, depended upon the ingrowth (sprouting) of these nerves from the host animal into the implant (3,4).These results suggest 627
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that perhaps the c h o l i n e r g i c i n n e r v a t i o n of the thymus plays an i m p o r t a n t role in the p r o c e s s of i n ~ h y m i c m a t u r a t i o n of T lymphocytes. In view of the fact that these e a r l i e r studies w e r e carried out by u s i n g h i s t o c h e m i c a l m e t h o d s w h i c h are less r e l i a b l e and s p e c i f i c for d e m o n s t r a t i o n of these nerves(5) the p r e s e n t study was u n d e r t a k e n to r e - e x a m i n e the p a t t e r n of c h o l i n e r g i c i n n e r v a t i o n of the thymus by i m m u n o c y t o c h e m i c a l l o c a l i z a t i o n of C h A T w h i c h is c o n s i d e r e d more specific and t h e r e f o r e r e l i a b l e m a r k e r for these nerves (6). The results of this study indicate that c o n t r a r y to e a r l i e r studies where c h o l i n e r g i c n e r v e s w e r e r e p o r t e d in the thymic r u d i m e n t of mice even b e f o r e they were p o p u l a t e d by the l y m p h o i d stem cells (7) c h o l i n e r g i c n e r v e s were d e t e c t e d in the later part of the o n t o g e n e t i c d e v e l o p m e n t of the thymus and in the post natal period. The s i g n i f i c a n c e of this i n n e r v a t i o n in r e l a t i o n to the f u n c t i o n of thymus as the p r i m a r y l y m p h o i d organ for the g e n e r a t i o n of T l y m p h o c y t e s is discussed.
Material
and M e t h o d s
Animals: Male and female adult BALB/c m i c e d were used. To o b t a i n embryos of various g e s t a t i o n periods, m a t i n g b e t w e e n the male and female ( two females and one m a l e ) a n i m a l s w e r e set up. The d a y of v a g i n a l plug was c o u n t e d as the d a y 0 s i m i l a r to the e a r l i e r study (8). The animals were m a i n t a i n e d in the animal house of King Saud U n i v e r s i t y , C o l l e g e of Medicine, Riyadh. Rea@ents: Most of the c h e m i c a l s and buffers were o b t a i n e d from S a r g e n t Welch.Co. Avidin, b i o t i n kits w e r e p u r c h a s e d from V e c t o r Labo r a t o r i e s and the a n t i b o d y against c h o l i n e acetyle t r a n s f e r a s e (IE6) was i n i t i a l l y p r o v i d e d by Dr. G.D. C r a w f o r d of the N e u r o Science division, City of Hope hospital, Duarte, C a l i f o r n i a and were later p u r c h a s e d from B o e h r i n g e r M a n n h e i m Biochemicals, Indianapolis. T h [ m i c rudiments: T h y m i c r u d i m e n t s from day 13 to b i r t h w e r e r e m o v e d aseptically and i m m e d i a t e l y fixed in 10% formaline. Post natal thymic lobes were o b t a i n e d from one, four and eight week old animals, by s u r g i c a l l y r e m o v i n g the thymic lobes from the m e d i a s t i n a l cavities of the animals, t a k i n g care to avoid any i n c l u s i o n of m e d i a s t i n a l lymph nodes. All s u r g i c a l p r o c e e d u r e s w e r e c a r r i e d out under d i s s e c t i n g m i c r o s c o p e as in p r e v i o u s s t u d y (8). The larger lobes w e r e cut into small pieces and i m m e d i a t e l y fixed by i m m e r s i o n into 10% formaline. Immunoc~tochemistry: E m b r y o n i c and post natal thymic tissues were fixed in 10% b u f f e r e d f o r m a l i n e ( P h o s p h a t e b u f f e r Ph 7.4 ) for upto 24 hours after w h i c h they were w a s h e d amd immersed in 5% S u r c o s e s o l u t i o n for 12 hours. The thymic lobes were then frozen on
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metal stubs with OCT compound. 15 to 20 n thick sections were cut at -15°by a cryostat. The sections were mounted on histostick coated slides. The slides were stored at37 C for 48 to 72 hours before use for better sticking of the section. The slides were immersed in tris buffer (Ph 7.4) for fifteen minutes and then were coated with 1:50 of normal rabit serum followed by 4ng/ml solution of rat anti ChAT monoclonal antibody (Boehringer) for 18 hours at 4°C in a staining chamber. The slides were washed and treated with rabit anti rat biotinated antibody and ffluoriscinated avidin (Vector laboratories. The slides were examined under a fluorescent microscope and photographed. Results I. Light microscopy: Examination of thymic rudiments by toluidine blue showed few large basophilic cells in the thymic rudiments at 13/14 days of gestation. The number of such lymphoid cells increased with age. The distinction between the thymic cortex and medulla was evident only in the post natal thymic rudiments. These observations were consistent with the obeservations of our earlier studies (8), (Fig.l) 2. Fuorescence microscopy: No fluorescent nerve profiles were observed within the embryonic thymic rudiments of 13 to 18 days of gestation (Fig:2,~) In the thymic rudiment of 19 days of gestation and at birth few fuorescent nerve profiles were seen in relation to blood vessels in the deeper part of the thymic paranchyma. The capsular area and the superficial regions of the thymic tissue did not show such profiles (Fig:4). In one week old post natal thymic rudiments, numerous fluorescent profiles were seen mostly within the deep parachymal tissue which corresponds with the medullary region of the thymic tissue on examination by light microscope. The distribution of these fluorescent profiles increased with age and appeared most abundant in 6/8 week old thymic rudiments. (Fig:5,6).
FIG.
1
Section of a thirteen day fetal thymic rudiment showing rounded darkly stained (lymphoid) cells. H&E. X25.
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FIG.
2
F l u o r e s c e n t m i c r o g r a p h of a t h i r t e e n day fetal thymic r u d i m e n t f o l l o w i n g t r e a t m e n t w i t h a n t i - C h A T antibody, showing lack of f l u o r e s c e n t profiles. X25
FIG.
3
F l u o r e s c e n t m i c r o g r a p h of a sixteen day fetal thymic rudiment following treatment with anti-ChAT antibody showing lack of f l u o r e s c e n t profiles, similar to figure 2.X25
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4
F l u o r e s c e n t m i c r o g r a p h of a thymic r u d i m e n t from a newly born mouse f o l l o w i n g a n t i - C h A T antibody treatment, showing few f l u o r e s c e n t profiles in the d e e p e r part of p a r e n c h y m a in relation to the blood vessels. X25
FIG.
5
F l u o r e s c e n t m i c r o g r a p h of one week old post natal thymus s h o w i n g f l u o r e s c e n t profiles w i t h i n the parenchyma, f o l l o w i n g a n t i - C h A T a n t i b o d y treatment. X25
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FIG.
6
F l u o r e s c e n t m i c r o g r a p h of an~,adult thymus s h o w i n g f l u o r e s c e n t profiles, f o l l o w i n g a n t i - C h A T a n t i b o d y treatment. X25 Discussion The results of this s t u d y i n d i c a t e that the c h o l i n e r g i c nerve~ appear w i t h i n the thymic r u d i m e n t around the time of birth. This c o n c l u s i o n is b a s e d on the results of i m m u n o c y t o c h e m i c a l locali z a t i o n of the enzyme ChAT w h i c h is c o n s i d e r e d a specific marker for the d e m o n s t r a t i o n of c h o l i n e r g i c n e u r o n s and nerves (9). T h e s e results, however, are not in a g r e e m e n t w i t h the e a r l i e r o b s e r v a t i o n s w h e r e these n e r v e s were r e p o r t e d to be p r e s e n t w i t h i n the e m b r y o n i c thymic rudiments, even p r i o r to the e n t r y of l y m p h o i d cells (7,4). In the mouse the thymic p r o g e n i t o r are known to enter the thymic r u d i m e n t around day 10 of g e s t a t i o n d u r i n g o n t o g e n y (i0). The e a r l i e s t time at w h i c h the c h o l i n e r g i c n e r v e s w e r e o b s e r v e d in this study, however, was just b e f o r e b i r t h (18/19 day of gestation). This d i s c r e p a n c y b e t w e e n these results and the results of B u l l o c h and P o m e r a n t z (4) could be a t t r i b u t e d to the d i f f e r e n c e s in m e t h o d s e m p l o y e d in the studies. In v i e w of less r e l i a b i l i t y of the h i s t o c h e m i c a l l o c a l i z a t i o n of a c e t y l c h o l i n e s t e r a s e , as m a r k e r for c h o l i n e r g i c nerves, the results of p r e v i o u s studies need confirmation. The f u n c t i o n a l s i g n i f i c a n c e of c h o l i n e r g i c i n n e r v a t i o n in r e l a t i o n to thymic l y m p h o p o i e s i s is not known. It is claimed, however, that the r e c o n s t i t u t i o n of the dy uhymic ( nude ) m i c e , f o l l o w i n g a thymic graft d e p e n d s on the e s t a b l i s h m e n t of c h o l i n e r g i c i n n e r v a t i o n w i t h i n the graft (3). Furthermore, the nude mice thymic r u d i m e n t s were also reported to be sparsely i n n e r v a t e d than the thymic rudiments of normal mice (4). These o b s e r v a t i o n do suggest a r e g u l a t o r y role for the i n n e r v a t i o n in the d e v e l o p m e n t of thymus. D i f f e r e n t i a t i o n of T lymphocytes
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requires an i n t e r a c t i o n b e t w e e n the l y m p h o i d p r o g i n a t o r cells w h i c h enter the thymus from the bone m a r r o w in the a d u l t and the yolk sac and liver in the fetus and the n o n l y m p h o i d cells of the thymic parenchyma, the so c a l l e d thymic m i c r o e n v i r o n ment (ii). These non l y m p h o i d cells are Ia positive and include the e p i t h e l i a l cells, m a c r o p h a g e s and d e n d r i t i c cells of the t h y m u s . S u p p o r t for the c o n c e p t of this i n t e r a c t i o n comes from the o b s e r v a t i o n of the i n h i b i t i o n of thymic l y m p h o p o i e s i s in organ c u l t u r e s on a d d i t i o n of a n t i - I a a n t i b o d y in the culture m e d i a (12) and the absence of Ia p o s i t i v e cells in the thymic r u d i m e n t s of the nude mice (13). It has been s u g g e s t e d that the r e c o g n i t i o n o f I a m o l i c u l e on the a c c e s s a r y cells, by the lymphoid "~lls, leads to the s e c r e t i o n of g r o w t h factor i n t e r l u k i n - I (IL-I) w h i c h in turns s t i m u l a t e s the p r o l i f e r a t i o n of t h y m o c y t e s w h i c h are i m m u n o r e a c t i v e and on antigen stimulation produce another factor i n t e r l u k i n - 2 (IL-2) (14). Tnis s u g g e s t i o n is based on the results of e x p e r i m e n t s on presumably immature t h y m o c y t e s isolated from the adult thymus and t h e r e f o r e may not truely r e p r e s e n t the events that occur d u r i n g the m a t u r a t i o n of fetal thymocytes. If mere r e c o g n i t i o n of I~ r m o l i c u l e on the a c c e s s a r y cells by the l y m p h o i d cells was e n o u g h for the for the g e n e r a t i o n of i m m u n o r e a c t i v e cells, such cells could have been p r e s e n t in the early thymic (13/14) rudiments, since these rudiments do possess both the lymphoid cells and Ia-: p o s i t i v e cells (13). On the other hand immuner e a c t i v e cells do not appear w i t h i n the thymus b e f o r e day 18/19 of g e s t a t i o n (15). It is likely t h e r e f o r e that in a d d i t i o n to the r e c o g n i t i o n of Ia~ molicule, some other r e g u l a t o r y mecha n i s m also plays a role in the p r o d u c t i o n of these T cell g r o w t h factors by the a c c e s s a r y cells of thymus and thereby r e g u l a t e thymic function. Previous studies have already i n d i c a t e d i n h i b i t i o n of th~ ~ic lymphope~ ,sis and the immuner e a c t i v i t y of T l y m p h o c y t e s (16, 17,18). In view of these studies it may be s u g g e s t e d that the c h o l i n e r g i c i n n e r v a t i o n has a s t i m u l a t o r y role, o p p o s i t e to the i n h i b i t o r y role of the a d r e n e r g i c n e r v e s in this process. Such a h y p o t h e s i s is r e a s o n a b l e to c o n s i d e r in view of the fact that the choln e r g i c i n n e r v a t i o n is c o n c e r n e d w i t h the s e c r e t o r y a c t i v i t y of most tissues of the body. It is possible, therefore, that c h o l i n e r g i c nerves could r e g u l a t e the s e c r e t o r y activity of n o n l y m p h o i d cells of the thymic parenchyma, w h i c h ia c o n s i d e r e d to be the source of various growth factors (19,20). The pattern of d i s t r i b u t i o n of c h o l i n e r g i c n e r v e s in the d e e p e r part of the thymic p a r e n c h y m a w h i c h is the site of most nonl y m p h o i d cells of the thymus, as o b s e r v e d in this study, further s t r e n g h t e n s this possibility. This study, p r o v i d e s a n a t o m i c a l e v i d e n c e for an i n t e r a c t i o n b e t w e e n the d e v e l o p m e n t and m a t u r a t i o n of the thymus and its a u t o n o m i c i n n e r v a t i o n on the basis of i m m u n o c y t o c h e m i c a l localization of ChAT n e r v e s d u r i n g o n t o g e n i t i c d e v e l o p m e n t of the thymus. Studies are in progress to further e l u c i d a t e the role of a u t o n o m i c nerves in the r e g u l a t i o n of thymic function.
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Acknowledgement The authors are thankful to Mr. Abdur Rehman for his technical help and for the microphotography, Mr Imdad Elahi Quraishi for typing the manuscript a n d King Saud University, College of Medicine, Research Center, for the financial support.
References i. Fatani,J.A., M.A.Qayyum.,L.Mehta.,and U.Singh. (1986) Parasympathetic innervation of the thymus: A histochemical and immunocytochemical study. J.Anat. 147:115-119. 2. Bulloch,K.and R.Y.Moore (1981). Innervation of the thymus gland by the brain stem and spinal cord in mouse and rat. Am.J.Anat.162:157-166. 3. Bulloch,K., M.R.Cullen., M.L.Davis and R.H.Schwartz. (1983) Neuroimmunology of the thymus gland. Neurology. 33.184-187. 4. Bulloch,K., and W.Pomerentz. (1984) Autonomic ~rvo~1~ system innervation of the thymic related lymphoid tissue in wild type and nude mice. J.Comp.Neurol. 228:57-68. 5. Levey,A.I., B.H. Wainer.,E.J.Mufson., and M.M.Mesulam. (1983) Co-localization of acetylcholinesterase and choline a c e t y l transferase in the rat cerebellum.Neuroscience. 9:9-22. 6. Eckenstein,F.and M.V.Sofroniew. (1983) Identification of central cholinergic neurons containing both choline acetyltransferase and acetylcholinesterase and of central neuron containing only acetylcholinesterase. J.Neuroscience 3:2286-2291. 7. Bulloch,K. (1982) A light and ultrastructural analysis of the innervation of the thymus gland during prenatal period. Soc. Neursci.Abst. 8:72. 8. Singh,U. (1979) Effect of catecholamine on imphopoiesis in fetal mouse thymic explants. J. Anat. 129:279-292. 9. Houser,C.R.,G.D.Crawford,R.P.Barber.,P.M.Salvaterra., and J.E.Vaughn. (1983) Organization and morphological characteristics of cholinergic neurons: an immunocytochemical study with monoclonal antibody to choline acetyltransferase. Brain.Res. 266:79-119. 10. Moore,M.A.S.and J.J.T.Owen. (1967) Experimental studies on the development of the thymus. J.Exp.Med.126:715-725. ii. Stutman,O. (1978) Intrathymic and extrathymic T cell maturation,Transplant. Rev.42:138-184. 12. Deluca,D. (1986) Ia positive nonlymphoid cells and cell development in murine fetal thymus organ cultures: Monoclonalant-Ia antibodies inhibit the development of T cells. J. Immunol.136:430-439. 13. Vliet,E.V.,E.J.Jenkinson,R.Kingston.,J.J.T. Owen and W. VanEwijk. (1985) Stroma cell types in the developing thymus of the normal and nude mouse embryo. Eur.J.Immunol.15:675-681. 14. Rock,K.L. and B. Benacerraf. (1984) The role of Ia molicule in the activation of T lymphocytes.J.Immunol.132:1654-1662.
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15. Robinson, J.H. and J.J.T.Owen.(1976) Generation of T cell function in organ cultures of fetal mouse thymus. I. Mitogen responsiveness. Clin. Exp. Immunol. 23:347-354. 16. Besedovsky,H.O., A.Ray.,E.Sorkin.,M.D.Prada and H.H.Keller. (1979) Immunoregulation mediated by the sympathetic nervous system. Cell. Immunol. 48:346-355. 17. Williams, J.M.,R.G.Peterson.,P.A.Shea.,J.F.Schedtje.,D.C. Bauer and D.L.Felton. (1981) Sympathetic innervation of murine thymus and spleen. Evidence for a functional link between the nervous system and immune system. Brain Res. Bull. 6:83-94. 18. Singh,U. (1985)Lymphopoiesis in the nude fetal thymus following sympathec£omy.Cell. Immunol. 93:222-228. 19. Palacious,R., C.Fernandez, and P.Sideras. (1980) Development and continuous growth in culture of Interlukin-2 producing lymphocytes from athymic nu/nu mice. Eur. J.Immunol. 12:777-782. 20. Smith,K.A., L.B.Sachman.,J.J.Oppenheim, and M.F.Favata.(1930) Functional relationship of interlukins. J.Exp. Med. 151: 1551-1556. Received: November, 1986 Accepted: February, 1987