JOURNAL OF MORPHOLOGY 204305-312 (1990)

Thymus of Ram perezi; Presence of lnterdigitating Cells RAFAEL ALVAREZ Departamento de Biologia Celular y Anatomia, Universidad de Lebn, 24071 Lecin, Spain

ABSTRACT In the thymus of Rana perezi, as in other anuran amphibians, there exist two different portions, cortex and medulla. In both sections epithelio-reticular cells are observed as are lymphocytes, macrophages, and granulocytes. In addition, the medulla shows cysts and secretory, myoid, and hypertrophied epithelio-reticular cells. In the cortex, plasma cells and interdigitating cells were also observed. Interdig- itating cells make contact with lymphocytes. This finding provides morphological support for the hypothesized role of interdigitating cells in antigen presentation.

T-lymphocyte differentiation takes place in the thymus (Manning, '81). The thymus gland is observed in all vertebrate classes except cyclos- tomes. Nevertheless, lampreys show some im- mune reactions considered as T-dependent in higher vertebrates (Page and Rowley, '82; Ar- davin et al., '84). Thymic cytoarchitecture of all jawed vertebrates is similar. However, as the distinction between cortex and medulla is not clear in fish, caecilians, and urodeles, the anuran amphibians are the first group to show this divi- sion uniformly (Manning and Horton, '82). Both immune and non-immune components of the amphibian thymus have been researched (Curtis et al., '79; Bigaj and Plytycz, '84, '87).

Interdigitating cells, similar to those in the thymus and peripheral secondary lymphoid or- gans of higher vertebrates, have been confirmed in Rana temporaria (Bigaj and Plytycz, '84, '87), but not in Rana perezi (Garrido, '86) or in the Ranidae generally. The origin and functional significance of interdigitating cells are still mat- ters of controversy (Rausch et al., '77; Groscurth, '80; Plytycz and Bigaj, '83; Leceta et al., '84).

The present paper analyzes the structure and ultrastructure of the thymus of Rana perezi, a poorly studied anuran amphibian from the histo- logic and cytologic point of view.

MATERIALS AM) METHODS

Ninety-six adult frogs, Rana perezi, were col- lected in September 1983 in the vicinity of Le6n (Spain) and were kept for a year under regulated light and temperature conditions (12 L/12 D and 20/22"C). Animals were maintained in enclo- sures containing a receptacle with water, which was changed every second day. All frogs were fed live worms, flies, and grasshoppers every 3 days. Four males and four females were sacrificed af- ter anaesthesia with MS 222 (Sandoz) each

month through the course of a year. Thymus glands were immediately removed, fixed in 2.5 96 glutaraldehyde in Sorensen buffer at pH 7.4, postfixed in 1% OsO, in the same buffer, dehy- drated in acetone, and embedded in Araldite. Semithin sections stained with toluidine blue were examined by light microscopy. Ultrathin sections double-stained with uranil acetate and lead citrate were examined with a JEOL 100-C electron microscope.

RESULTS

Thymus of Rana perezi is divided into a cor- tex and medulla without a definite cortico- medullary boundary (Fig. 1). Thymus gland is enclosed within a connective tissue capsule from which trabeculae penetrate inwards carrying blood vessels (Fig. 2). These trabeculae pene- trate into the thymic stroma but do not shape thymic lobes (Fig. 1). Cellular migration be- tween trabeculae and the thymic stroma can sometimes be observed (Fig. 3).

Cortex In the cortex meshes of the network of epithe-

lio-reticular cells are filled mainly with lympho- cytes. The epithelio-reticular cells are polygonal and project cytoplasmic processes, some of which are united with other cells by desmosomes (Fig. 4). The nuclei of epithelio-reticular cells are also polygonal, with some deep invaginations. Some cellular contacts (but not cell junctions) can be observed between these cells and neighbouring lymphocytes (Fig. 4). Occasionally epithelio- reticular cells show a degenerated appearance (Fig. 5) manifest as electron-lucent cells in which euchromatin and heterochromatin is very scarce and the cytoplasm sparse. Plasma cells (Fig. 6) are located near the connective capsule or within a section of trabeculae showing no intercellular

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Fig, 1. Section of thymus of R a m perezi, showing the cortex (C) and medulla (M). Note a trabecula (t) start ing from the capsule (c). Scale line = 100 pm.

Fig. 4. Thymiccortex, showing lymphocytes (L), epithelio- reticular cells (epr) attached by desmosomes (arrowheads) and lymphocytes/epithelio-reticular cells contacts (arrows). Scale line = 1 pm.

Fig. 2. Trabecula (t) containing blood vessels (large ar- rows). Scale line = 2 pm. Fig. 5. Degenerate epithelio-reticular cell (arrows) and

lymphocytes (L). Scale line = 1 pm. Fig. 3. Cellular migration through the trahecula (t) to the

thymic stroma (asterisk). Scale line = 1 pm.

PRESENCE OF INTERDIGITATING CELLS 307

Fig. 6. Plasma cell (arrow) and lymphocytes. x9ooo. Fig. 9. Thymic medulla, showing lymphcqk, epithelio- reticular cells (epr), and hypertrophied epithelio-reticular

Fig. 7. Degenerate plasma cell (mow). Scale line = 1 pm. cells (arrows) associated with cysts. Scale line = 2 pm.

Fig. 8. Interdigitating cell (i) and lymphocytes in contact Fig. 10. Cyst with cilia. Scale line = 0.5 pm.

Fig. 11. Cyst with microvilli. Scale line = 1 pm. (arrows). Scale line = 1 pm.

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junctions. The nuclei of plasma cells are eccen- tric and the nucleolus pronounced. Cytoplasm is mainly filled with rough endoplasmic reticulum whose more-or-less massive cisternae contain a moderately electron-dense material. Occasion- ally plasma cells shows degenerated appearance (Fig. 7).

The interdigitating cells are located near the capsule. The nuclei of interdigitating cells have peripheral condensed chromatin (Fig. 8). The cytoplasm is electron-lucent and contains scant organelles, mainly small and long shaped pro- files of smooth endoplasmic reticulum. The cell processes make numerous contacts, especially with neighbouring lymphocytes (Fig. 8).

Medulla As in the cortex, lymphocytes and epithelio-

reticular cells are observed (Fig. 9); some epithe- lio-reticular cells are hypertrophic and consti- tute uni- or multicellular cysts. Ultrastructurally, lymphocytes and epithelio-reticular cells are sim- ilar to those described in the cortex, the surfaces of the intra- or intercellular cysts usually being ciliated (Fig. 10) or less frequently covered with microvilli (Fig. 11). Distributed among the med- ullary parenchyma are three secretory cell types. One type is a round cell with an eccentric and slightly smoothed nuclear envelope, and shows a variable number of small electron-dense gran- ules and some electron-lucent vesicles in the cytoplasm (Fig. 12). Other secretory cells (Fig. 13) are polarized globated cells showing an obvi- ous nucleolus. Their cytoplasmic granules are medium-sized and moderately electron-dense, sometimes showing a slightly eccentric and very electron-dense body within. The last secretory cell type (Fig. 14) varies in shape and has big secretory granules of variable electron-density.

Myoid cells (Fig. 15) are the largest cells in the medulla (and in the thymus). They are round or oval cells with round or oval nuclei located at a slightly eccentric position in the cell and typi- cally display a prominent nucleolus. The cyto- plasm is almost completely filled with myofila- ments (Fig. 16). There also exist some degen- erating myoid cells (Fig. 17).

Throughout cortex and medulla of the thy- mus, we also observe macrophages and granulo- cytes. Macrophages (Fig. 18) usually show large phagolysosomes and they always have electron- dense bodies, probably primary lysosomes. The heterophils (Fig. 19), with a lobulated nucleus, exhibit two characteristic types of cytoplasmic granules, both of them round or oval in shape. The former are small and electron-dense, and the latter are larger and more electron-lucent. The eosinophils (Fig. 20), also with a lobulated

nucleus, show two types of dense bodies. The most widespread ones are large, round, and elec- tron-dense (sometimes there are pale vesicles sharing granule and hyaloplasm); the other ones, which are less common, are small, oval, and less electron-dense. Basophils (Fig. 21) are scarcer yet and exhibit a characteristic non-lobulated nucleus and a cytoplasm completely filled with large, round, and electron-dense granules. These granules contain a homogeneous matrix some- times showing filamentous, more electron-dense structures.

DISCUSSION

The clear demarcation between cortex and medulla in the Rana perezi (and other anuran amphibian) thymus was previously described by Manning and Horton ('82). In Rana perezi, as in urodele (Henry and Charlemagne, '81) and in other anurans (Bigaj and Plytycz, '84) epithelio- reticular cells constitute the basic network of the thymic cortex. An irregular nucleus with some deep invaginations is characteristic of these cells in amphibians (Henry and Charlemagne, '81; Bigaj and Plytycz, '84). The contacts between epithelio-reticular cells and lymphocytes resem- ble those of the "nurse" cells in the mammalian thymus (Wekerle et al., '80; Wijngaert et al., '83) and other similar cells in peripheral lymphoid organs (Manconi et al., '84). The role of these cells could be related to the maturation process of some subtypes of T-lymphocytes.

The presence of macrophages, as observed in Rana perezi, is another common feature of the vertebrate thymus (Cooper and Zapata, '86) and is probably related to removal of degenerate or abortive lymphocytes (Scollay et al., '80; Duijves- tijn et al., '82).

Plasma cells are similar to those described in anuran amphibians (Cowden and Dyer, '71) and the majority of vertebrates (Cooper and Zapata, '86). According to Du Pasquier and Horton ('82) they pass through the thymus, and Plytycz and Bigaj ('83) relate them and granulocytes to degen- erative processes. Nevertheless, plasma cells have been observed in Rana perezi thymus both in an apparently depressed thymic state and in the apparently active organ. Medullary hypertrophic epithelio-reticular cells are similar to those de- scribed in Rana temporaria (Bigaj and Plytycz, '84), constituting the thymic cysts. Their func- tional significance is still a matter of controversy since the hypothesis that they produce and se- crete thymic hormones (Bigaj and Plytycz, '84) has been rejected (Goldstein et al., '81).

Gland cells described in this study are also similar to those previously described in other amphibians (Bigaj and Plytycz, '84). Sundler et

PRESENCE OF INTERDIGITATING CELLS 309

Fig. 12. Secretory-like cell. Scale line = 1 pm.

Fig. 13. Secretmylike cell (arrows). Scale line = 1 pm.

Fig. 14. Secretory-like cell (arrows). Scale l i e = 1 pm.

Fig. 15. Thymic medulla, showing secretory-like cells (ar- rows) and myoid cells (cm). Scale line = 10 pm.

Fig. 16. Distribution of myoid cell myofilaments. n, nu- cleus. Scale lme = 0.25 pm.

Fig. 17. Degenerate myoid cell. Scale line = 1 pm.

310 R. ALVAREZ

Fig. 18. Macrophage (arrows). Scale line = 1 pm.

Fig. 19. Heterophil. Scale line = 1 pm.

Fig. 20. Eosinophil. Scale line = 1 pm.

Fig. 21. Basophil. Scale l i e = 1 pm.

al. ('78) suggest that certain medullary gland cells in the chicken thymus are equivalent to other chicken cells containing neurotensin and somatostatin. This fact and the close resem- blance between gland cells described here and the Rana perezi integumentary mucous cells observed by Lopez ('a), could support the hy- pothesis of Pearse ('68) on a possible common origin for certain integumentary, intestinal, and thymic cells. In any case the functional meaning of gland cells in the thymic medulla remains unclear. The different morphologic patterns we observe in these gland cells and their granules support the idea that there exist several cellular types. The possible Hassal's bodies described by some authors (Fabrizzio and Charipper, '41; Bigaj and Plytycz, '84) are absent in Rana perezi.

Thymic myoid cells of Rana perezi are similar to those described by other authors. The dif- ferent morphological conditions observed may indicate a cellular turnover which takes place in

the adult thymus, as other authors have already indicated in various vertebrates (Toro et al., '69; Bigaj and Plytycz, '84). Their functional signifi- cance is still not clear, despite the fact that Hanzlikova ('79) using histochemical methods demonstrated homology with the skeletal mus- cle fibres. Mandel ('68) suggests they are resid- ual in the thymus from embryogenesis, whereas Henry and Charlemagne ('80) state that they derive from epithelio-reticular cells. According to Toro et al. ('69)' Henry and Charlemagne ('81), and Plytycz and Bigaj ('83)' myoid cells are involved in promoting circulation of tissue fluids within the thymus. Finally, Rimmer ('80) consid- ers that they are a self-antigen source contribut- ing to the natural self-tolerance of development, and may contribute muscle antigens.

In the present study a distinct cortico-medul- lary region has not been observed. In higher vertebrates it is in this cortico-medullary region that thymocyte migration takes place, and which

PRESENCE OF INTERDIGITATING CELLS 311

shows an important vascularization and is en- riched in interdigitating cells (Olah et d., '75). However, we have observed in Rana perezi im- ages of cellular migration similar to those de- scribed by Bigaj and Plytycz ('84) between con- nective trabeculae and the thymic stroma. In these highly vascularized places some cellular fragments, probably from interdigitating cells, have been observed. Interdigitating cells in the thymic cortex resemble those described in the peripheral lymphoid organs (Veldman, '70; Vil- lena et al., '83) and thymus (Kendall and Frazier, '79; Fonfria et al., '82) of higher vertebrates and also in reptilian thymus and spleen (Leceta and Zapata, '85), and in the thymus (Bigaj and Ply- tycz, '84, '87) and spleen (Barrutia et al., '85) of amphibians. The role of these interdigitating cells is unclear. According to Groscurth ('80) they are involved in the presentation of antigens to lymphocytes, and Bigaj and Plytycz ('84) sug- gested they create a special microenvironment. In any case they are closely related to specific T-microenvironments (Villena et al., '83). Their origin has also been discussed; Rausch et al. ('77) upholds a reticular origin, but Leceta et al. ('84) suggest it is monocytic. Cellular contacts ob- served in this study between interdigitating cells and lymphocytes can be a morphological sup- port for the Groscurth ('80) hypothesis. With regard to their origin, the absence of circulating or stable monocytes in the Rana perezi thymus can support the hypothesis of Rausch et al. ('77). But the hypothesis of Leceta e t al. ('84) is also supported if the monocytic transformation does not take place in the thymus.

ACKNOWLEDGMENTS

Thanks to Dr. Paulino de Paz for a critical review of the manuscript; to Dr. Agustin Zapata, who initiated my interest in the immune system; and to the anonymous reviewers who assisted me in improving the manuscript.

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