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 develop- ment, by immunonocytochemical localization of the enzy- me 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 exam- ined. 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 choliner- gic nerves in the adult murine thymus by histochemical and imm- unocytochemical 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 rudi- ments 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

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that perhaps the cholinergic innervation of the thymus plays an important role in the process of in~hymic maturation of T lymphocytes. In view of the fact that these earlier studies were carried out by using histochemical methods which are less reliable and specific for demonstration of these nerves(5) the present study was undertaken to re-examine the pattern of cholinergic innervation of the thymus by immunocytochemical localization of ChAT which is considered more specific and therefore reliable marker for these nerves (6). The results of this study indicate that contrary to earlier studies where cholinergic nerves were reported in the thymic rudiment of mice even before they were populated by the lymphoid stem cells (7) cholinergic nerves were detected in the later part of the ontogenetic development of the thymus and in the post natal period. The significance of this innervation in relation to the function of thymus as the primary lymphoid organ for the generation of T lymphocytes is discussed.

Material and Methods

Animals:

Male and female adult BALB/c miced were used. To obtain embryos of various gestation periods, mating between the male and female ( two females and one male ) animals were set up. The day of vaginal plug was counted as the day 0 similar to the earlier study (8). The animals were maintained in the animal house of King Saud University, College of Medicine, Riyadh.

Rea@ents:

Most of the chemicals and buffers were obtained from Sargent Welch.Co. Avidin, biotin kits were purchased from Vector Lab- oratories and the antibody against choline acetyle transferase (IE6) was initially provided by Dr. G.D. Crawford of the Neuro Science division, City of Hope hospital, Duarte, Calif- ornia and were later purchased from Boehringer Mannheim Biochemicals, Indianapolis.

Th[mic rudiments:

Thymic rudiments from day 13 to birth were removed asepti- cally and immediately fixed in 10% formaline. Post natal thymic lobes were obtained from one, four and eight week old animals, by surgically removing the thymic lobes from the mediastinal cavities of the animals, taking care to avoid any inclusion of mediastinal lymph nodes. All surgical proceedures were carried out under dissecting microscope as in previous study (8). The larger lobes were cut into small pieces and immediately fixed by immersion into 10% formaline.

Immunoc~tochemistry:

Embryonic and post natal thymic tissues were fixed in 10% buffered formaline ( Phosphate buffer Ph 7.4 ) for upto 24 hours after which they were washed amd immersed in 5% Surcose solution 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 histo- stick 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 anti- body (Boehringer) for 18 hours at 4°C in a staining chamber. The slides were washed and treated with rabit anti rat bioti- nated 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 obser- vations were consistent with the obeservations of our earlier studies (8), (Fig.l)

2. Fuorescence microscopy:

No fluorescent nerve profiles were observed within the emb- ryonic 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 cap- sular 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 prof- iles 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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630 CHOLINERGIC INNERVATION OF THYMUS Vol. ii, No. 3

FIG. 2

Fluorescent micrograph of a thirteen day fetal thymic rudiment following treatment with anti-ChAT antibody, showing lack of fluorescent profiles. X25

FIG. 3

Fluorescent micrograph of a sixteen day fetal thymic rudiment following treatment with anti-ChAT antibody showing lack of fluorescent profiles, similar to figure 2.X25

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FIG. 4

Fluorescent micrograph of a thymic rudiment from a newly born mouse following anti-ChAT antibody treatment, showing few fluorescent profiles in the deeper part of parenchyma in relation to the blood vessels. X25

FIG. 5

Fluorescent micrograph of one week old post natal thymus showing fluorescent profiles within the paren- chyma, following anti-ChAT antibody treatment. X25

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T

FIG. 6

Fluorescent micrograph of an~,adult thymus showing fluorescent profiles, following anti-ChAT antibody treatment. X25

Discussion

The results of this study indicate that the cholinergic nerve~ appear within the thymic rudiment around the time of birth. This conclusion is based on the results of immunocytochemical local- ization of the enzyme ChAT which is considered a specific mar- ker for the demonstration of cholinergic neurons and nerves (9). These results, however, are not in agreement with the earlier observations where these nerves were reported to be present within the embryonic thymic rudiments, even prior to the entry of lymphoid cells (7,4). In the mouse the thymic progenitor are known to enter the thymic rudiment around day 10 of gestation during ontogeny (i0). The earliest time at which the cholinergic nerves were observed in this study, how- ever, was just before birth (18/19 day of gestation). This discrepancy between these results and the results of Bulloch and Pomerantz (4) could be attributed to the differences in methods employed in the studies. In view of less reliability of the histochemical localization of acetylcholinesterase, as marker for cholinergic nerves, the results of previous studies need confirmation.

The functional significance of cholinergic innervation in relation to thymic lymphopoiesis is not known. It is claimed, however, that the reconstitution of the dy uhymic ( nude ) mice,following a thymic graft depends on the establishment of cholinergic innervation within the graft (3). Furthermore, the nude mice thymic rudiments were also reported to be sparsely innervated than the thymic rudiments of normal mice (4). These observation do suggest a regulatory role for the innervation in the development of thymus. Differentiation of T lymphocytes

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requires an interaction between the lymphoid proginator cells which enter the thymus from the bone marrow in the adult and the yolk sac and liver in the fetus and the nonlymphoid cells of the thymic parenchyma, the so called thymic microenviron- ment (ii). These non lymphoid cells are Ia positive and include the epithelial cells, macrophages and dendritic cells of the thymus.Support for the concept of this interaction comes from the observation of the inhibition of thymic lymphopoiesis in organ cultures on addition of anti-Ia antibody in the culture media (12) and the absence of Ia positive cells in the thymic rudiments of the nude mice (13). It has been suggested that the recognition ofIa molicule on the accessary cells, by the lymphoid "~lls, leads to the secretion of growth factor interlukin-I (IL-I) which in turns stimulates the proliferation of thymocytes which are immunoreactive and on antigen stimul- ation produce another factor interlukin-2 (IL-2) (14). Tnis suggestion is based on the results of experiments on presum- ably immature thymocytes isolated from the adult thymus and therefore may not truely represent the events that occur during the maturation of fetal thymocytes. If mere recognition of I~ r molicule on the accessary cells by the lymphoid cells was enough for the for the generation of immunoreactive cells, such cells could have been present in the early thymic (13/14) rudiments, since these rudiments do possess both the lymphoid cells and Ia-: positive cells (13). On the other hand immune- reactive cells do not appear within the thymus before day 18/19 of gestation (15). It is likely therefore that in addition to the recognition of Ia~ molicule, some other regulatory mech- anism also plays a role in the production of these T cell growth factors by the accessary cells of thymus and thereby regulate thymic function. Previous studies have already indicated inhibition of th~ ~ic lymphope~ ,sis and the immune- reactivity of T lymphocytes (16, 17,18). In view of these studies it may be suggested that the cholinergic innervation has a stimulatory role, opposite to the inhibitory role of the adrenergic nerves in this process. Such a hypothesis is reasonable to consider in view of the fact that the chol- nergic innervation is concerned with the secretory activity of most tissues of the body. It is possible, therefore, that cholinergic nerves could regulate the secretory activity of nonlymphoid cells of the thymic parenchyma, which ia considered to be the source of various growth factors (19,20). The

pattern of distribution of cholinergic nerves in the deeper part of the thymic parenchyma which is the site of most non- lymphoid cells of the thymus, as observed in this study, further strenghtens this possibility.

This study, provides anatomical evidence for an interaction between the development and maturation of the thymus and its autonomic innervation on the basis of immunocytochemical local- ization of ChAT nerves during ontogenitic development of the thymus. Studies are in progress to further elucidate the role of autonomic nerves in the regulation of thymic function.

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Acknowledgement

The authors are thankful to Mr. Abdur Rehman for his tech- nical help and for the microphotography, Mr Imdad Elahi Quraishi for typing the manuscript and 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 acetyl- 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 acetyl- transferase 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 character- istics 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 matura- tion,Transplant. Rev.42:138-184.

12. Deluca,D. (1986) Ia positive nonlymphoid cells and cell development in murine fetal thymus organ cultures: Mono- clonalant-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. Van- Ewijk. (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