CELLULAR IMMUNOLOGY 93,222-228 (1985)

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Lymphopoiesis in the Nude Fetal Thymus following Sympathectomy

UPENDRA SINGH’

Depurtment ofAnatomy, School of Medicine, University of Utah, Salt Lake City, Utah 841 I2

Received October 2, 1984: accepted November 6. 1984

This study was carried out to examine the innervation of the nude fetal thymus during ontogeny and to see if lymphopoietic activity would occur within these thymic lobes in the absence of sympathetic neuronal input. Fetal thymic rudiments from nu/nu mice were removed and examined for galoxylic acid-induced histotluorescence to detect the catecholaminergic nerves. Some of these lobes were organ cultured for 5 to 7 days in the presence of deoxyguanosine to eliminate any existing lymphoid cells within the rudiments. Such “nonlym- phoid” thymic rudiments were implanted into the anterior eye chambers of syngenic BALB/c mice (heterozygous) from which cervical sympathetic ganglia and part of the sympathetic chain had been surgically removed (right side) one week earlier. The left side was only sham operated. The thymic implants were allowed to grow for up to 21 days on both sides; they were then removed and examined by histofluorescence, immunofluorescence, and light microscopy. The results indicate for the first time that the nude fetal thymus is innervated by sympathetic nerves and that following sympathectomy the nude thymus is able to sustain lymphopoietic activity and generate lymphoid cells which have characteristics present on thymocytes during in vivo development in normal mice, such as binding to peanut agglutinin and expression of Thy-l antigen. The relationship between the presence of sympathetic inhibitory influence and the thymic atrophy seen in the nude mice during ontogeny, is being investigated. o 1985 Academic

Press. Inc.

INTRODUCTION Recent studies indicate that the central nervous system modulates the immuno-

reactivity of lymphocytes (1, 2) and that the thymus, which is a primary lymphoid organ for the generation of peripheral T lymphocytes, may depend upon a contribution from the neural crest for its normal development (3). Furthermore, chemical and surgical sympathectomy have been shown to have a stimulatory effect on the immunoreactivity of peripheral T lymphocytes (4, 5). However, the role of central nervous system in the maturation of thymocytes, particularly during ontogeny, has not been studied. In our preliminary studies it has been observed that the developing thymic rudiment receives sympathetic nerve fibers at around Day 17 or 18 of gestation and that these nerves increase in concentration in the adult (6). In addition, it was also observed that the nonlymphoid fetal thymic rudiments, if implanted into the anterior eye chambers of mice following surgical cervical

’ Present address: Department of Anatomy, College of Medicine, King Saud University, P.O. Box 2925, Riyadh, Saudi Arabia.

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sympathectomy on one side, had greater immunoreactivity in mitogen and mixed lymphocyte reactions as compared to their counterparts from the sham-operated side (7, 8). In view of these inhibitory influences of the sympathetic nerves on developing thymus, this study was initiated to examine the presence of sympathetic innervation in the nude fetal thymus during ontogeny and to explore the possibility of having lymphopoiesis in these thymic rudiments in an environment free from inhibitory influences of sympathetic nerves during ontogenetic development, by grafting these rudiments into the anterior eye chambers following cervical surgical sympathectomy. The results of the study indicate for the first time that it is possible to have continued lymphopoiesis in a nude fetal thymic rudiment in the absence of sympathetic nerves, and that the cell yield of such rudiments is greater than the rudiments innervated by the sympathetic nerves. The lymphoid cells generated within the implanted grafts have characteristics of thymocytes, such as the binding to peanut agglutinin (PNA) and the expression of Thy-l antigen.

MATERIAL AND METHODS

Thymic rudiments. Mating was set between male and female nu/nu mice from an inbred colony of BALB/c heterozygous and nude mice in the Department of Dermatology, University of Utah. Thirteen days before birth, fetal thymic rudiments were carefully removed from the embryos. These rudiments were then treated with 1.3 mA4 deoxyguanosine to eliminate any lymphoid cells which may have been present within the rudiments (9) before grafting into the eye. Some of them were then grown as organ cultures for another 2 weeks at 37’C, as previously described (lo), to ascertain the presence or absence of any residual lymphoid cells within the rudiments, by histological examination. Other rudiments were frozen at -70°C for histofluorescence microsocopy.

Sympathectomy and anterior eye chamber grafting. Deoxyguanosine-treated organ- cultured thymic rudiments were then surgically implanted into the anterior eye chambers of syngenic heterozygous adult mice which had been either sham operated (left) or sympathectomized (right) surgically under general anesthesia, by removing the cervical sympathetic ganglion along with a part of the sympathetic chain, 1 week earlier. Confirmation of sympathectomy was made by the appearance of drooping of the eye lid (ptosis) on the operated side following the operation and by histological examination of the tissue removed. Four experiments were carried out using three to five animals in each experiment. The implants were allowed to grow for up to 21 days; they were then removed for histological examination, histoflu- orescence, and immunofluorescence microscopy.

Light microscopy. For histological examination the tissues were fixed in 40% buffered formaldehyde and embedded in wax. Sections (5 pm) were cut and stained by hematoxylin-eosin (H&E) and toluidine blue stains.

Histojluorescence microscopy. Fetal thymic rudiments or grafts removed from the eyes were immediately frozen at -70°C and 16-pm-thick frozen sections were cut for examination by galoxylic acid-induced fluorescence by the method of de la Terre (11). Every alternate five sections were used for histofluorescence or histological examination.

Fluorescence microscopy. Frozen sections were treated with fluorescinated peanut agglutinin (Calbiochem-Behring, La Jolla, Calif.) at 1:200 dilution and examined

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under an epi-illuminating fluorescent microscope, for the presence of PNA+ cells. Isolated cells removed from the grafts were counted on hemocytometer and also examined by indirect immunofluorescence microscopy using monoclonal anti-Thy- 1.2 antibody and fluorescinated rat anti-mouse antibody (kindly provided by Dr. Lee Roberts, Department of Dermatology, University of Utah).

RESULTS

Macroscopic and Light Microscopic Examination

Examination of animals after the sympathectomy operation showed presence of drooping of the eye lid on the operated side (Fig. 1A) and the examination of the

FIG. 1. (A) A BALB/c heterozygous mouse I day after sympathectomy on the right side, showing drooping of the eyelid (ptosis) on that side. (B) A nu/nu 16day fetal thymic rudiment implanted within the anterior eye chamber of a syngenic heterozygous host. (C) A I-pm-thick section of a 16-day nu/nu thymic rudiment showing a cluster of epithelial cells without any basophilic cells (X40). (D) A l-pm- thick section of a 16-day nu/nu fetal thymic rudiment organ cultured for 5 days with 1.3 mM deoxyguanosine followed by further organ culture for 15 days, showing absence of any lymphoid (basophilic) cells on staining with toluidine blue (X40).

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implant under a dissecting microscope showed that the implant had grown within the anterior eye chamber and was vascularized by the blood vessels from the eye (Fig. 1B).

The implants on the operated side had grown two to three times larger in size compared to the implants on the sham-operated side. Histological examination of the tissue removed after sympathectomy by H&E staining showed large nerve cells characteristic of a sympathetic ganglia. Staining of the thymic rudiments by toluidine blue showed occasional basophilic cells (lymphoid) on removal (Fig. 1C) but not on subsequent organ cultures (Fig. 1 D). Histological examination of the frozen sections from the grafts by toluidine blue staining showed some round basophilic cells within the implants on the sham-operated side but the density of such cells was much higher on the operated side (Figs. 2A and C).

FIG. 2. (A) A section from the sham-operated side showing a few basophilic cells stained with toluidine blue (X25). (B) Fifth section from the one shown in A, showing histofluorescent fibers (arrow) on treatment with galoxylic acid (X25). (C) A section from the operated side showing an abundance of lymphoid cells (basophilic) on staining with toluidine blue (X25). (D) Fifth section from the one in C, showing absence of fluorescent fibers on treatment with galoxylic acid (X25).

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Histofluorescence and Immunofuorescence Microscopy

Examination of the implanted grafts for histofluorescence fibers indicated that there were no fluorescent fibers on the sympathectomized side either into the iris or within the implants whereas such fibers were present in these tissues on the sham- operated side (Figs. 2B and D). The results of histofluorescence microscopy revealed that the fetal nu/nu thymus receives sympathetic nerve fibers around Day 17 of gestation and that these fibers increase in density in later periods of ontogeny up to birth. The remains of the atrophic thymus in postnatal life receives sympathetic nerve fibers along with the blood vessels which supply that tissue (Figs. 3A and B). Staining of frozen sections with fluorescinated PNA indicated that most cells present in the grafts were PNA+ (Figs. 3C and D). Examination of the isolated cells from the grafts by indirect immunofluorescence microscopy using monoclonal anti-Thy-

FIG. 3. (A) Histofluorescence micrograph of Il-day nude fetal thymus showing fluorescent nerve fibers (X25). (B) Histofluorescence micrograph of the blood vessels and remains of the thymic gland of an adult nude mouse, showing fluorescent nerve fibers. (C) A frozen section from the graft implanted on the operated side of the anterior eye chamber, showing lymphoid cells (toluidine blue: X25). (D) Micrograph of the fifth section from the one in C, showing fluorescent cells following staining with fluorescinated peanut agglutinin (X25).

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1.2 antibody and fluorescinated rat anti-mouse antibody showed that 73 + 4% cells were Thy-l positive. Not enough cells could be isolated, however, to carry out any functional assays on these cells, due to low cell yield from the implants.

DISCUSSION

The results of this study indicate for the first time that a nonlymphoid nu/nu fetal mouse thymus implanted into the anterior eye chamber for 3 weeks is repopulated by thymic stem cells which differentiate along the T-cell pathway to some extent, as seen in the normal mice. Previous studies using an organ culture system had shown that 13- to 17-day fetal nude thymic rudiments do not become lymphoid in organ culture ( 12, 13). In this study there were also no lymphoid cells visible in the organ cultures at the time of implantation nor in the subsequent organ cultures in vitro at 37°C for up to 15 days. It is reasonable to speculate, therefore, that the lymphoid cells were most likely derived from the host following implantation into the anterior eye chambers following their vascularization in 3 to 5 days and that they were thymocytes (as indicated by binding to PNA and by the presence of Thy-l antigens) rather than peripheral T or B lymphocytes which may have found their way into the grafts following implantation. The lymphopoietic activity within the thymus appears to be inhibited by the sympathetic nervous system as seen by histological examination of the grafts and their cell yields from the sham-operated side. It should be pointed out, however, that the cell yield from the operated side was also lower in this study compared to the cell yield obtained from implants of normal mice thymic rudiments grown for similar times (about 3 X 106/lobe).

The diminished lymphopoietic activity on the sham-operated side seen in this study may have been due to some inhibitory effect of the sympathetic nerves on the proliferation of thymocytes following their entry into the grafted rudiment by binding on beta adrenoceptors, which are present on these cells (14, 15) or by modulating the activities of other cells within the thymic stroma, which secrete factors such as the thymic hormones or interlukin, which are known to promote thymocyte proliferation and at least some of them mediate their actions through beta adrenoceptors (16, 17). On the other hand, it is equally possible that on the operated side, in addition to the removal of these inhibitory influences, there may have been an unopposed stimulatory effect of the parasympathetic nerves which may also have innervated the implants from the iris and at least in vitro such cholinergic neurotransmitters have been found to stimulate lymphopoiesis in thymic organ cultures (10). The results of this study further support our earlier in vitro observations on the effect of catecholamines on lymphopoiesis in the embryonic thymus (10) and provide further support to the notion of a neuroendocrine modulation of the immune system, a concept which is gathering strength on the basis of anatomical and physiological evidence demonstrating direct links between these two systems (18, 19).

It may be argued, however, that the differences seen between the two sets of grafts may be due to some differences in the blood supply or the permeability of the blood vessels due to the differences in the sympathetic input on the two sides, which in turn may have affected the lymphopoiesis or the repopulation of the implants. It may well be, indeed, that autonomic neuronal input plays an important role in these processes in vivo too, and may be essential for a continued and long-term

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integrity of the thymic microenvironment, and that may have been one of the reasons why monolayers of epithelial cells have not been very successful in supporting continued lymphopoiesis (20-22). The role of the autonomic nervous system in the development of thymus, therefore, needs further investigation. The work is being carried further to study lymphopoiesis, and the mode of repopulation of fetal “empty nonlymphoid” thymic implants using this experimental protocol. This will provide an excellent model for the study of the role of the neuronal input in the process of maturation and repopulation of thymus, both in normal and nude mice.

ACKNOWLEDGMENTS

The author thanks Ms. Karen Evans and Ms. June Stephenson for typing the manuscript, Ms. Jeannette Taylor for the technical help, and Dr. Lee Roberts, Department of Dermatology, for kindly providing the animals and the antibodies. The project was supported by Grant A l-20 169-O 1 from the National Institutes of Health.

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