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J. U L T R A S T R U C T U R E RESEARCH 47, 26-40 (1974)

Ult rostructure of the Thymus in "Nude" Mice ~

ANDRl~ C. CORDIER 2

Department of Histology, University of Louvain, Minderbroedersstraat, 12, 3000 Louvain, Belgium

Received August 1, 1973

The thymus of the "Nude" mouse has been studied with electron microscopy. This thymus is formed of cysts whose walls are formed by ciliated cells, mucous cells, undifferentiated cells, and cells of glandular character. Between the cysts are found undifferentiated cells, mucous acini, and a few plasma cells and mast cells.

We have been unable to find some of'the structures that are specific for the normal thymus, viz. Hassall bodies, myoid cells, epithelial cells of the cortex, and medullary "cystic" cells of the medulla.

The comparison of the normal thymus with the Nude mouse thymus suggests that the deficiency in epithelial cells of the cortical type could be the cause of the absence of lymphocyte multiplication. The deficiency in medullary "cystic cells" might explain the absence of hormonal capacity of the Nude thymus. The primary site of thymic malformation in the nu/nu mouse probably resides in the ectoblastic component of the thymic anlage.

The thymus has a complex function, part of which is fulfilled by one or more substan-

ces secreted by epithelial cells (12, 23, 24, 42, 51, 52, 77). In mice homozygous for the Nude gene, most thymic functions are absent, resulting in an immunological disorder

sharing many of the features of certains immune deficiencies described in the human , particularly the Nezelof syndrome (50) and, to a lesser extent, ataxia-telangiectasia (59).

The " N u d e " mutat ion in the mouse was described by Flanagan in 1966 (19). The homozygote nu/nu, is hairless and retarded in growth. Its thymus is so small as

to escape detection upon dissection and macroscopic examination (53). The thymus- dependent areas of the lymph nodes and spleen are devoid of lymphocytes (14) and, more generally, of cells bearing the 0 antigen (65). Such mice lack cellular im-

muni ty (54, 58, 64, 70, 71, 84) and display considerable impairment of humora l im- muni ty (38, 48, 56, 57, 67, 72). The apparent absence of the thymus in nu/nu mice

1 This work was supported by Grant 10.013 from the Fonds de Recherche Fondamentale Collec- tive, Bruxelles, Belgium.

2 Research Fellow of the Fonds National de la Recherche Scientifique.

ULTRASTRUCTURE OF THYMUS IN "NUDE" MICE 27

represents a dysgenesis of the organ, ra ther than a genuine agenesis. Actua l ly , a

thymic rud imen t can be found in the 14-day fetus, but it does no t conta in any lympho-

cytes, and has a cystic appea rance in 1 to 2-day-o ld mice (55). Wort i s et al. (85) sug-

gest tha t the absence of lymphocytes in the thymus of " N u d e " mice results no t f rom

the absence of s tem cells, but f rom a deficiency in induct ive capac i ty of the thymic

epi thel ium.

The present s tudy of thymic rud iments at the u l t ras t ruc tura l level was unde r t aken

with the hope of clar ifying the morpho log ica l basis of this deficiency.

MATERIALS A N D METHODS

"Nude" mice were obtained by mating heterozygous Rex + / + nu males with N M R I female mice. The wavy-coated mice of the F1 generation that carried the Rex marker were discarded, and the smooth-coated carriers of the nu gene were paired inter se. This produced 20-25 % nu/nu mice in the F2 generation.

Fifteen mice of both sexes, 2-3 months old, were sacrificed by cervical dislocation. Cold (4°C) fixative was poured into the quickly opened thorax and the thymic region was removed in block. The thymic rudiments were dissected under immersion in the fixative, and then fixed for 2 hours in same solution. Two different fixatives were used, viz. paraformaldehyde- glutaraldehyde according to Karnovsky (35) and 2.5 % glutaraldehyde in 0.2 M cacodylate buffer at pH 7.2, containing 0.5 % Ca CI~ and 1 To sucrose. Two fragments were also fixed in the latter solution to which 1% of Alcian Blue 8GX had been added (2). After fixation, the fragments were rinsed in Ringer's saline, postfixed in 2 To osmium tetroxide, dehydrated in a graded ethanol series, stained in block with uranyl acetate, and embedded in Epon 812 (43).

Sections (1-2 ~ thick) were cut on a Sorvall Porter Blum MT2B microtome fitted with glass knives, and stained with 1 To toluidine blue. Thymic cysts, as selected under the micro- scope, were processed to thin sections by means of a Reichert OMU3 microtome with dia- mond knife. The latter sections were mounted on uncoated copper grids, stained with uranyl acetate (82) and lead citrate (68), and examined with a Philips EM300 electron microscope under 60 kV tension.

RESULTS

In nu/nu mice 2-3 mon ths o ld the thymus was found to consist of 2 lobes approx i -

mate ly 0.5 m m in d iamete r (Fig. 1A), loca ted in the an te r ior med ia s t i num above the

cardiac auricles. The lobes were included in an adipose and connect ive tissue tha t

subdiv ided them into several lobules. Each lobule con ta ined cystic fo rmat ions of

var iable size. In the larger cysts the epithel ial l ining was cubic o r squamous , whereas

in the smal ler cysts it appea red pseudos t ra t i f i ed (Fig. 1B). Between these cysts were

small nodules of undi f fe rent ia ted cells, clusters of mucous cells, and some isola ted

cells. The lack of l y m p h o i d cells p reven ted any dis t inct ion between cor tex and me-

dulla. Blood vessels were numerous .

28 ANDR]~ C. CORDIER

The epithelium of the cysts rested on a basement membrane and consisted of several types of cells, all of them united by junction complexes (Fig. 2). The types most fre- quently encountered were: (a) the undifferentiated cell, (b) the ciliated cell, (c) dif- ferent types of glandular cells, and (d) the mucous cell.

a. Undifferentiated cells (Fig. 3) had a round or oval nucleus with finely granular chromatin. A nuclear body was often present. Mitochondria were numerous and fre- quently round. The Golgi and the rough endoplasmic reticulum were poorly devel- oped. Several small vesicles of smooth endoplasmic reticulum existed in the apical region. Near the basal end of the c611 the membrane formed many interdigitations with adjacent cells. At the apical pole, microvilli projected into the lumen of the cyst.

b. Ciliated cells (Fig. 4) were frequently cylindrical, with a basally located elongated nucleus having its chromatin condensed along the nuclear membrane. Elongated mitochondria were situated in the apical region between the vesicles of the rough endoplasmic reticulum. The Golgi apparatus was well developed, and polyribosomes were numerous. The cilia were of the type found in the trachea. Between the cilia were microvilli whose length approximated 2/3 that of the cilia.

c. Four types of glandular cells, could be distinguished by their secretory granules. i. Glandular cells of type I (Fig. 4) contained round granules of variable size (0.3

0.5 #m) and density. The content of the granules appeared homogeneous. Rough endoplasmic reticulum was situated above the basally located nucleus. A highly de- veloped smooth endoplasmic reticulum existed near the apical pole.

ii. Glandular cells of type i i (Figs. 5 and 6) contained at their apical pole dense granules, 0.15-0.22 #m in size and limited by a membrane. Their mitochondria were situated at the basal pole and their rough endoplasmic reticulum was arranged in elongated saccules, parallel to the axis of the cell. The apical pole displayed many microvilli.

iii. Glandular cells of type III (Fig. 7) contained granules of about 0.12 #m, with a dense core of 95 nm separated from the membrane of the granule by a clear space of 5 nm. These cells were quite rare and no sections showing the whole cell body were found, so that the present description remains very fragmentary.

iv. Glandular cells of type IV (Fig. 8) possessed large inhomogeneous granules of 1-2 #m at their apical pole. The Golgi and rough endoplasmic reticulum were well developed. The nucleus was basally situated and finely granular.

d. Mucous cells (Figs. 9 and 10) were similar to intestinal goblet cells and contained

FIG. 1. General appearance of the nu/nu thymus. (A) Low-power view showing the two thymic lobes (1, 2) with numerous cysts (C, arrows), x 18. (B) Electron micrograph of an area of one lobe showing three cysts whose lumens are lined by a squamous (L1), cubic (L2), and pseudostratified (L3) epithe- lium, respectively. × 14 400.

r • ~

L3

30 ANDRE C. CORDIER

clear homogeneous vacuoles that were closely packed in the apical pole of the cell. The rough endoplasmic reticulum was well developed.

Between the cysts there were clusters composed of (e) undifferentiated cells and (f) mucous acini (Figs. 11 and 12).

e. Undifferentiated cells had a large nucleus, diffuse chromat in and several nucleoli. Their cytoplasmic organelles were poorly developed. These cells were united by desmo- somes and clusters of such cells were limited by a basement membrane (Fig. 11).

f. Mucous acini (Fig. 11) were formed of four or five cells. Such acini have been seen in cyst walls, but their cells never made contact with the lumen, contrary to the mucous cells described above (cf. d); in fact, mucous cells remained separated f rom the lumen by processes f rom undifferentiated cells (not shown). The rough endoplas-

mic reticulum of mucous acinar cells was well developed and formed parallel lamellae at the basal pole, as in salivary acini. Elongated epithelial cells were situated at the base of the mucous cells to which they were attached by desmosomes. Their cytoplasm was clear and contained numerous microfilaments (Fig. 12).

Cysts and glandular masses were embedded in connective tissue composed of fibro- blasts, adipocytes, and macrophages. Mas t cells, plasma cells, and occasional lympho- cytes were also found. Blood vessels were numerous. The double epithelial walls characteristic of thymic vessels (66, 83) and assumed to be the support of the blood/

thymus barrier (49) was not observed in the nu/nu thymic rudiments. Fenestrated capillaries, as generally found in glands, have been described in the normal thymus (40), and were also present in the nu/nu thymus.

DISCUSSION

The thymus of the " N u d e " mouse is essentially devoid of lymphocytes and rich in cystic formations.

The existence of cysts in the normal thymus has been noted in the mouse (1, 8, 18) and other animals (3, 5, 17, 21, 25, 39), as well as in man (41, 69). Their number increas- es with age (1) and after injection of various steroids (IO, 74). The origin of these cysts is a matter of controversy. Dear th (13) has suggested their format ion by degener-

FIG. 2. Junctional complexes in the epithelium. (A) Interdigitations at the basal pole of undifferen- tiated cells, x 8 000. (B) Desmosomes at the apical pole of ciliated cells. × 28 000. (C) Half-desmo- somes at the basal pole of an undifferentiated cell. x 106 000. (D) Unfrequent type of desmosome-like junctional complex, between two undifferentiated cells. This structure consists of up to 5 electron dense parallel bands, x 87 500. FIG. 3. Undifferentiated cells (UC) in the epithelium of a cyst. Note large nuclei (N) frequently con- taining a nuclear body (inset × 16 250). Numerous interdigitations (arrows) are present between adja- cent cells. A thick basement membrane (BM) separates the epithelium from the underlying cells among which three lymphocytes (Ly) can be seen x 7 550.

3 - 7 3 1 8 3 6 a r. Ultrastructure Research

32 ANDRE C. CORDIER

ation of the central port ion of epithelial masses. Spear (76) described them as being remnants of thymopharyngeal ducts whereas Shier (75) considered them to originate

f rom primitive thymic tubes or f rom epithelial inclusions. In the cyst walls of nu/nu thymuses we have described undifferentiated cells, ciliated

cells, glandular cells, and mucous cells. Several of these types also exist in the normal thymus. Arnesen (1) describes in normal A K R / O and W L O mice cystic structures also

limited by three types of cells, ciliated, mucous, and "pseudostratif ied." Isolated ciliated cells have been reported also f rom the thymus of the adult dog (81), the mouse (8, 28, 29), and hamster (7, 31), as well as f rom that of the human fetus (73).

Mucous cells have been reported in the thymus of the adult mouse (1, 8). The forma- t ion of large cysts, such as those of the " N u d e " mouse thymus and of mucous acini comparable to those of submaxillary gland, have been described in the thymus of an

8-month-old cat (16). Therefore it appears that the cysts and cells that occur in small number in the normal thymus make up the entire thymus of the " N u d e " mouse.

A secretory function has been at tr ibuted to the cysts of the mouse and rat thymus (39). The existence of similar strucutres in the thymus of the " N u d e " mouse, which is

immunologically incompetent, suggests that these structures do not have the funct ion of inducing immunological competence. Fur thermore it has been reported that several

endocrine glands of the " N u d e " mouse, e.g., the hypophysis, thyroid, and adrenal cortex, show the same changes that are described for these organs in the thymecto- mized mouse (63). Therefore, cystic format ions do not seem to intervene in the mul-

tiple interrelations of the thymus with endocrine glands, described by Pierpaoli et ai. (62).

Ciliated cells and mucous cells are numerous in certain cysts and absent in others.

It is difficult to assign to them a precise function in the thymus as can be done in other organs. This localization may perhaps merely express the capacity of the pha- ryngeal endoderm to originate these types of cell.

A m o n g the granulated types, those cells possessing some dense bodies surrounded by lighter aura (Fig. 7), are similar to secretory cells described in certain thymic tumors (44). The same granules have been found in an epithelial t h y m o m a producing A C T H

FI~. 4. Ciliated cells (CC) and glandular cells of type I. The glandular cells of type I (Gi) contain numerous vesicles of variable density. Smooth endoplasmic reticulum is abundant at the apical pole. x 5 700. FI~. 5. Glandular cells of type II (G~), containing many electron dense granules, numerous basalty located mitochondria, and slender parallel cisternae of rough endoplasmic reticulum. Numerous rnicrovilli are present at the cell surface. × 10 500. FIG. 6. Detail of granules with a diameter ranging from 15 to 22 #m in a glandular cell of type II. × 29 50{).

FtG. 7. Glandular cells of type lit (G3), containing numerous granules (average diameter 12/~m) with a dense core separated from the limiting membrane by a clear space, x 29 900.

34 ANDRI~ C. CORDIER

(36). Comparable granulations are found in cells of the pancreas or in A cells of the digestive tract (20).

Other cell types reported here have never before been described from the thymus. One of these types (Fig. 4) contains dense homogeneous granules and an abundance of smooth endoplasmic reticulum. These two characteristics are reminiscent of steroid hormone-secreting cells. Other cells (Figs. 5 and 6) have smaller granules and numer- ous villosities. ~[heir morphology does not permit comparisons with known cellular types.

Nuclear bodies are frequent in the undifferentiated cells of cyst walls. Clark (8) first noted their presence in the epithelial cells of the mouse. Their distribution in the human thymus has been studied by Henry and Petts (26), who found them in epithelial cells and also in lymphocytes and macrophages. These authors associated the presence of nuclear bodies with a state of "cellular hyperactivity." We have found similar structures in thymic plasma cells.

In thymic rudiments of the "Nude" mouse, we have never observed four of the elements that are specific for the thymus, viz. Hassall bodies, myoid cells, cortical epithelial cells, and the medullary "cystic cell" of Mandel (45).

The Hassall bodies are relatively poorly developed even in normal mice (8, 29). Electron microscope studies on Hassall bodies in the mouse and guinea pig were published by Khonen and Weiss (37), and during the estrogen-induced involu- tion of the thymus in the guinea pig by Izard (32). Hassall bodies are composed of squamous cells characterized by the presence of numerous tonofilaments and desmo- somes (45). The cells in the core of the Hassall body are comparable to keratinized cells of the epidermis, and their peripheral cells contain grains of keratohyalin (32).

I t is interesting to note that there are no Hassall bodies in the thymus of infants with lymphopenic agammaglobulinemia of the "Swiss type" (4, 60) or the Nezelof syndrome (50), which is the human genetic immunological deficiency most similar to the Nude anomaly in the mouse. The Hassall bodies are also not present in the Louis- Barr syndrome (ataxia-telangiectasia) (60), nor in that described by Buckely et al. (6), in which the immunoglobulins show a diminution comparable to that described in the "Nude" mouse (72).

Myoid cells exist in normal thymuses. Their relation with myasthenia gravis has been discussed (78, 79). We cannot interpret their absence in the "Nude" mouse.

FIG. 8. Glandular cell of type IV (G~), with large granules (diameter 1 eml) of variable density. Golgi complex and endoplasmic reticulurn are well developed, x 8 700. F~G. 9. Mucous cells from epithelium of a cyst, containing mucous droplets of variable density. x 18 250.

F~o. 10. Mucous cell with the typical aspect of a goblet cell. Note tight packing of mucous droplets. x 14 800.

36 ANDRI~ C. CORDIER

The epithelial cell, characteristic of the thymic cortex, is a stellate cell having long cytoplasmic extensions which contain tonofilaments and dense vesicular formations in addition to numerous small vesicles; the nucleus possesses a voluminous nucleolus (46). Lymphocytes are always in intimate contact with these cells. The frequency of mitotic lymphocytes is significantly greater in the proximity of epithelial cells than in the cortex (47). This epithelial cell, therefore, may be responsible for the multiplica- tion of the lymphocytes in the normal thymus. The absence of this type of epithelial cells in the "Nude" mouse thymus could explain the lymphopenia described by Pante- louris (53) and by Wortis (84), as well as the absence of lymphocytes in the thymus.

The "cystic cell" of Mandel (not to be confused with cells from the cyst walls) occurs in the medulla of the thymus. No cell answering its description was observed in the course of our study in "Nude" thymuses, although we had no difficulty in identi- fying it in normal controls. It has been described in the mouse (8, 29) the golden ham- ster (31), the guinea pig (33, 45), the rat (61, 80), and the human fetus and newborn (27). Its cytoplasm shows numerous vacuoles lined by a membrane with microvilli. These vacuoles may be empty or filled with a granular or homogeneous PAS-positive material (45). This cell has been shown to incorporate labeled glucosamine (9), and its n umbers increase during regeneration following involution after injection of hydro- cortisone (30) or bacterial endotoxin (22). The "cystic cell" shows cytoplasmic charac- teristics indicative of a secretory function and has been proposed as the cell which produces the thymic factor (8, 45, 61, 80) that is commonly held responsible for distant blood-mediated, inductive effect of the thymus. For instance, mice thymectomized at birth regained their immunity if grafted with thymus enclosed in a diffusion cham- ber (42, 52); 8 days after implantation, these chambers contained only "epithelial" cells. The immunological deficiency in the "Nude" mouse might in fact be due to the absence of a well defined cell type, the "cystic cell." It would be of particular interest to study in the electron microscope the thymus of infants afflicted with immunological deficiency. Bockman et al. (4) have described the ultrastructure of the thymus of an infant suffering from agammaglobulinemia o f the Swiss type, but their report con- tains no mention of the presence or the absence of the medullary "cystic cell" type.

Several cell types from mouse thymuses have been connected with different kinds of pathology, e.g., the "dark" cells of Izard and de Harven (34), which are said to be increased in leukemic AKR mice, and a particular type of epithelial cell reported to exhibit specific changes in NZB mice (15). Studies in progress at our laboratory will attempt to correlate these different cell types with those described in the present work.

Fro. 11. Clusters of mucous glandular cells (MC) and undifferentiated cells (UC) localized between the thymic cysts, x 2 600.

!i ~ ~ , • i~,~i!i~ili~

~ ~ ~,,!'~i~i ~ii'iiii~, ~

38 ANDRI~ C. CORDIER

FIG. 12. Detail of intercystic structures. The basal pole of a mucous cell (MC) demonstrates numerous parallel cisternae of rough endoplasmic reticulum. Outside the glandular cluster, some clear epithelial cells (CE) can be seen. They contain numerous bundles of microfilaments (arrows) and are bound together and to the mucous cells by typical desmosomes (D). The cellular clusters are surrounded by a basement membrane (BM). × 19 400.

The thymus of the mouse is der ived f rom the e n d o d e r m of the 3rd pharyngea l

p o u c h and ec toderm of the 3rd b ranch ia l slit (11). The absence of hair and Hassal l

bodies in the " N u d e " mouse suggests tha t the pr imi t ive defect lays in the ec todermal

par t of the thymus. The concomi t an t absence of epi thel ial cells of the thymic cortex

and of cystic cells of the medu l l a may be indicat ive of the ectoblast ic origin of these

two cell types. The endodermic po r t i on would then develop into cystic format ions .

The presence of ci l iated and mucous cells indicates the mult ipl ic i ty of the evolu t ionary

potent ia l s of b ranch ia l a rch derivatives.

I wish to thank Professor J. F. Heremans, whose suggestions and criticism were most help- ful. The skillful technical assistance of Miss Alberte Lefevre and of Mrs Jacqueline Vander Perre for secretorial work and Mr Jef Goossens for photographic work is also gratefully acknowledged.

ULTRASTRUCTURE OF THYMUS IN "NUDE" MICE 39

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