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Thymic epithelial cysts in the pigeon
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Thymic epithelial cysts in the pigeon Yasuro Atoji, Yoshio Yamamoto and Yoshitaka Suzuki Laboratory of Veterinary Anatomy, Faculty of Agriculture, Gifu University, Yanagido, Gifu 501-1193, Japan
Summary. Medullary epithelial cells forming cysts in the thymus of pigeon chicks were examined in order to clarify these morphological characteristics by immunohistochemical and electron microscopic techniques. Light microscopically, cysts were divided into intracellular and intercellular types. Intracellular cysts were positive for the periodic-acid Schiff reaction, while the intercellular type was not. With immunohistochemical staining for keratin, the edges of the intercellular cysts were moderately stained, whereas the cytoplasm of epithelial cells was weakly positive. Heterophils, eosinophils and lymphocytes were included in most of the intercellular cysts. Ultrastructurally, there were two types of epithelial cells which contained intracellular cysts densely packed with microvilli. They differed with regard to the shape of the rough endoplasmic reticulum and bundles of keratin filaments, and in the presence of large vacuoles. The walls of the intercellular cysts were composed of a single type of epithelial cell with a pale nucleus and bundles of keratin filaments. Small vesicles measuring 150 to 200 nm in diameter were numerous in the apical cytoplasm beneath the microvilli. In intercellular cysts, there were abundant heterophils, eosinophils, lymphocytes and macrophages which mostly appeared to be living cells. This evidence suggests that the intercellular cysts might provide a specific microenvironment for leukocytes in the pigeon thymus. Key words: Epithelial cell - Cyst - Leukocytes - Thymus - Ultrastructure
increasing evidence that epithelial cells also serve an endocrine function (Hadden 1992; Kendall 1994). In birds, epithelial cells play an important role, as they do in mammals. In the chicken, this is particularly so for the production of thymic hormones and the expression of peptide receptors (March and Scanes 1994; Atoji et al. 1996, 1997; Hipak et al. 1996). One type of epithelial cell in the thymic medulla is characterized by the formation of cysts lined with microvilli. Two types of cysts have been observed in the thymic medulla of birds (Frazier 1973; Isler 1976; Kendall and Frazier 1979; Hashimoto et al. 1982). One is an intracellular cyst formed in a single cell, a phenomenon also observed in mammals. Another type is an intercellular cyst formed by two or more epithelial cells, which is unique to birds. In cysts of the latter type, in addition to homogeneous material, there are sometimes lymphocytes and granulocytes which appear to have degenerated (Kendall and Frazier 1979; Gulati et al. 1995). The function of both types of cyst remains unknown. Recently, a virus infection in the pigeon has attracted attention in veterinary medicine (Minamoto et al., 1988; Estes and Cohen, 1989). To clarify the immune response against the virus, it is very important to understand the mechanism of the central lymphoid tissues of the thymus and bursa of Fabricius in the pigeon. However, there is, unfortunately, no histological description of them to our knowledge. Therefore we investigated the structure of the lymphoid tissues of the pigeon. In the present study, we examined mainly the ultrastructure of epithelial cysts in the normal pigeon thymus with special reference to the aggregation of leukocytes within them.
Introduction Thymic epithelial cells are associated with a thymic microenvironment, in particular the differentiation and maturation of T lymphocytes. Recently there has been
Materials and methods
Correspondence to: Y. Atoji
Seven chicks of the pigeon, Columba livia, (2 to 4 weeks old, 3 males and 4 females) were used in the present study. Food and
Ann Anat (1999) 181: 365-370 © Urban & Fischer Verlag http:llwww.urbanfischer.deljournalslannanat
0940-9602/99/181/4-365 $12.00/0
water were given ad libitum. They were anesthetized with sodium pentobarbital (25 mg/kg, i.v.) and then perfused transcardially with Ringer's solution followed by Bouin's fixative for light microscopy or by a mixture of 2% paraformaldehyde and 2% glutaraldehyde in 0.1 M phosphate buffer (pH 7.3) for electron microscopy. After perfusion, the thymuses were removed and fixed in the respective solutions. For light microscopy, thymuses from five chicks were embedded in paraffin, and thin sections (4 gm thickness) were stained with hematoxylin and eosiu, and for the periodic-acid Schiff (PAS) reaction. For immunohistochemical staining, dewaxed sections were incubated for 30 rain in 0.3% H202-methanol and then in 2% normal goat serum for 30 rain at room temperature. Sections were then incubated overnight at 4 °C with rabbit antiserum against keratin from bovine muzzle epidermis (diluted 1:2,000; Dako, USA). The antiserum recognized keratin subunits, mainly of 52, 56 and 58 kD (Steinert, 1975). After three washes (5 rain each) in 0.01 M phosphate-buffered saline (PBS, pH 7.4), the sections were incubated for 30 rain at room temperature with biotinylated goat anti-rabbit IgG (diluted 1:500; Vector Laboratories, USA). Then the sections were rinsed three times (5 rain each) in PBS and incubated for 30 min at room temperature with avidin-biotinylated peroxidase complex (ABC; Vector Laboratories). The peroxidase activity was visualized by incubating sections in 0.05 M TrisHC1 buffer (pH 7.4) that contained 3,3'-diaminobenzidine tetrahydrochloride (20mg/100ml) and 0.003% H202. The sections were lightly counterstained with hematoxylin. In the histochemical controls, the primary antiserum was replaced by non-immune rabbit serum as appropriate. For electron microscopy, small tissue samples from two chicks were postfixed for 2 h at 4 °C in a solution of 1% osmium tetroxide in 0.1 M phosphate buffer (pH 7.3) and embedded in epoxy resin. Ultrathin sections were stained with uranyl acetate and lead citrate, and observed with an electron microscope (H-8100; Hitachi, Japan).
Results Light microscopy The medulla of the pigeon thymus was composed of various types of cells and cellular groups: epithelial cells, leu-
kocytes, myoid cells, lymphocytes, Hassall's corpuscles, cysts, and blood vessels (Fig. 1 A). There were two types of intracellular and intercellular cyst, both of which were dispersed through the thymic medulla. The intracellular cysts were found in large epithelial cells with light cytoplasm and often contained PAS-positive substance. The diameter of the cysts measured up to 4 gm. The intercellular cysts were surrounded by a wall which consisted of large and pale epithelial cells. The cells were cuboid, oval or spindle-shaped with attenuated cytoplasm and contained a regularly oval nucleus with a conspicuous nucleolus. The intercellular cysts were round or oval in contour, ranged from 10 gm to 33 gm in diameter, and contained leukocytes which were composed of heterophils, eosinophils and lymphocytes. The numbers of cells varied from cyst to cyst, but a majority of them were filled with leukocytes. No PAS reactivity was seen in the intercellular cysts. Occurrence of the intercellular cysts was high (595 + 47/mm 2, n = 5), while that of the intracellular cysts was very low (22 + 4/ram 2, n = 5). Immunohistochemical staining for keratin showed moderately positive at the edges of the intercellular cysts, and weakly positive in the epithelial cells around the cysts and Hassall's corpuscles (Fig. 1 B).
Electron microscopy Ultrastructurally, the intracellular cysts were located in large electron-lucent epithelial cells, which contained a regular or often irregular shaped nucleus. The cells were divided into two types. Type I was composed of cells in which organelles such as the Golgi apparatus, short profiles of flattened rough endoplasmic reticulum (RER), glycogen particles, small vacuoles, and a few bundles of keratin filaments were conspicuous in the cytoplasm (Fig. 2A). Type II was characterized by the presence of dilated cisternae of R E R with a few ribosomes, thick bundles of keratin filaments, and large vacuoles containing fibrous degenerated material (Fig. 2 B). The nucleus, Golgi apparatus and mitochondria were similar to those of
Fig. 1. Light micrographs of the intercellular cyst. A: Five intercellular cysts (arrows) in the medulla. Arrowheads indicate light epithelial cells in walls of cysts. Bar = 20 gm. B: Edges of a cyst (arrowheads) and epithelial cells (arrows) are positive with immunohistochemical staining for keratin. Bar = 10 gin.
366
Fig. 2. Electron micrographs of the intracellular cysts. A: An epithelial cell of type I contains a cyst (asterisk) in the cytoplasm. The cell morphological characteristics are similar to those of adjacent epithelial cells which participate in formation of an intercellular cyst. Bar = 2 btm. B: An epithelial cell of type II has a cyst (asterisk). The cell contains much balloon-like RER, thick bundles of filaments (arrowheads) and a large vacuole (arrow). Bar = 2 jim.
type I. The cysts of both types were lined with microvilli and frequently contained flocculent or fibrillar substances. Both types of epithelial cells bound surrounding cells by interdigitation and desmosomes. In the intercellular type of cysts, epithelial cells which made up the walls were essentially similar to cells of type I, which contained the intracellular cysts (Fig. 3 A). Microvilli covered the cysts which contained homogeneous substance or cells (Fig. 3 B). Large epithelial cells in the walls were connected to each other by zonula occludens, desmosomes and interdigitation of cell membranes (Fig. 3C). Small vesicles measuring 150nm to 200 nm in diameter were frequently found in the cytoplasm beneath the microvilli (Fig. 3 D). The cells in the cysts were identified as heterophils, eosinophils, lymphocytes, and macrophages (Fig. 4 A - D ) . Most of them showed a morphologically well-preserved state of the organelles, and the remainder looked to be disintegrated. No morphological connections between cells in cysts and epithelial cells in walls were seen. In the medulla outside the intercellular cysts, leukocytes such as heterophils, eosinophils and basophils (probably the some as the mast cells present in the extravascular tissues) were numerous.
Discussion There are two types of cysts in the avian thymus, namely intracellular and intercellular cysts. Only a single type of epithelial cell containing intracellular cysts has been reported in other species of birds (Frazier 1973; Kendall and Frazier 1979; Isler 1976; Hashimoto et al. 1982). In the present study, we demonstrated two types of epithelial cells which contained intracellular cysts. Whether these two types clearly differ or are merely variations of a single type of cell remains unknown. Frazier (1973) pointed out that epithelial cells bearing intracellular cysts have many morphological characteristics in common with the undifferentiated epithelial cells in the medulla of the chicken thymus. In the present study, there are major morphological differences between the two types of epithelial cells containing intracellular cysts in the pigeon thymus, namely, the shape of the cisternae of the RER, the size of the bundles of keratin filaments, and the presence of large vacuoles containing fibrillar material. The dilated cisternae of the R E R are very similar to those found in the reticuloepithelial cells which are seen in the 13-embryonic thymus of the chicken (Sugimoto et al. 1977), but not in the postnatal chicken (Frazier 1973). On
367
Fig. 3. Walls of the intercellular cysts. A: A large, pale epithelial cell with an indented nucleus containing a thick bundle of filaments (arrow), lipid-like vacuoles, slender mitochondria and glycogen particles. Bar = 2 ktm. B: Homogeneous materials stored in a cyst. Note the single type of epithelial cells forming the cyst. Bar = 2 gm. C: Epithelial cells are bound to each other by desmosome (large arrow), zonula occludens (small arrow) and interdigitation (arrowheads). Bar = i gin. D: Numerous small vesicles are seen beneath microvilli. Thick bundles of filaments (arrows) are also noted. Bar = 1 I~m.
368
Fig. 4. Leukocytes and macrophages in the intercellular cysts. A: A heterophil (H), eosinophils (E) and macrophages (M) in a cyst. A degenerated cell (asterisk) is present. A basophil (B) is seen outside the cyst. Bar = 2 ~tm. B: A n eosinophil (E) and lymphocytes (L) in a cyst. Bar = 2 gin. C: A macrophage (M), a heterophil (H) and an eosinophil (E) in a cyst. The macrophage is characterized by a large phagosome, phagocytosis (probably of an eosinophil) and a long string of RER. Bar = 2 ~tm. D: Enlargement of a macrophage which contains myelin-like structures, a long string of R E R and long cytoplasmic processes. Bar = 2 gm.
369
the other hand, the large vacuoles containing fibrillar material are similar to those seen in epithelial cells which participate in the formation of the Hassall's corpuscles in the adult chicken (Frazier 1973). To clarify a relationship between epithelial cells, types I and II, morphological studies on the differentiation and classification of medullary epithelial cells in the pigeon thymus are needed. The intercellular cysts are further divided into two types on the basis of the cellular components of the walls of cysts in the chicken, quelea, sparrow, and starling (Isler 1976; Kendall and Frazier 1979). One is made up of a single type of large and columnar epithelial cell. Such cells contain a regular oval nucleus, slender mitochondria, glycogen particles, free ribosomes and bundles of tonofilaments. Another type contains two types of cell. In addition to the cells of the former type, mucous cells participate in the formation of the cyst walls. The mucous cells are filled with large vacuoles with finely filamentous material of varying density. In the duck, the intercellular cysts consist only of a single type of large epithelial cell with apical microvilli (Hashimoto et al. 1982). In the present study, walls of the intercellular cysts are composed of a single type of epithelial cell and no mucous cells. The presence of mucous cells in the walls of intercellular cysts is probably based on the species difference. The significance of cysts is still a subject for speculation. The first possibility is a "graveyard" of leukocytes within the thymus. The aggregation of leukocytes in cysts has been found in this type of intercellular cysts in birds. Types of leukocytes in the intercellular cysts are granulocytes in the quelea, sparrow, and starling (Kendall and Frazier 1979), lymphocytes in the chicken (Frazier 1973), and heterophils and lymphocytes in the duck (Hashimoto et al. 1982). In the present study, heterophils, eosinophils, lymphocytes and macrophages were observed in the cysts. Neither erythrocytes nor thrombocytes occurred in the intercellular cysts of any birds examined. It is conceivable that the accumulation of leukocytes in cysts differs among species. However, the importance is the presence of macrophages and breakdown of leukocytes in the cyst. Most granulocytes or lymphocytes in thymic cysts of the chicken, duck, quelea, sparrow, and starling are disintegrated, disrupted or have the appearance of cellular debris (Frazier 1973; Isler 1976; Kendall and Frazier 1979; Hashimoto et al. 1982). In the present study, most cells in cysts appeared to be viable, since their fine structure was well preserved, but some granulocytes were disintegrated and others had been phagocytosed by macrophages. These findings suggest a breakdown of granulocytes and lymphocytes in the cysts. The second possibility is that cysts serve to store the secretions of epithelial cells. Small vesicles beneath the microvilli of the intercellular cysts of the chicken have been described as pinocytotic (Isler 1976). This evidence indicates the presence of soluble materials in cysts, because pinocytosis means reabsorption of
contents from cysts. In the present study, the cysts contain homogeneous materials, the walls of cysts are constantly shielded by junctional apparatus, and there are numerous small vesicles (150 nm-200 rim) in the apical cytoplasm of epithelial cells. This would at least suggest exocytosis, which plays a role in the secretion into cysts by epithelial cells.
References Anderson WL (1970) Seasonal changes in thymus weights in ring-necked pheasants. Condor 72:205-208 Atoji Y, Yamamoto Y, Suzuki Y (1996) Neurotensin-containing endocrine cells and neurotensin receptor mRNA-expressing epithelial cells in the chicken thymus. Arch Histol Cytol 59: 197-203 Atoji Y, Yamamoto Y, Suzuki Y (1997) Neurotensin pathway in the chicken thymus. Acta Anat Nippon 72:181-186 Campbell F (1967) Fine structure of the bone marrow of the chicken and pigeon. J Morphol 123:405-440 Estes MK, Cohen J (1989) Rotavirus gene structure and function. Microbiol Rev 53:410-449 Frazier JA (1973) Ultrastructure of the chick thymus. Z Zellforsch Mikrosk Anat 136:191-205 Gulati R Chan AS, Leong SK (1995) Ultrastructural localisation of NADPH-diaphorase in the chick thymic medulla. Cell Tissue Res 279:405-409 Hadden JW (1992) Thymic endocrinology. Int J Immunopharmacol 14:345-352 Hapak RC, Stanley CM, Henzl MT (1996) Intrathymic distribution of the two avian thymic parvalbumins. Exp Cell Res 222: 234-245 HashimotoY, KitagawaH, KudoN, SugimuraM (1982) Fine structural and immunohistochemical studies on the thymic cysts in ducks. Jpn J Vet Sci 44:597-605 Islet H (1976) Fine structure of chicken thymic epithelial vesicles. J Cell Sci 20:135-147 Kendall MD, Ward P (1974) Erythropoiesis in an avian thymus. Nature 249:366-367 Kendall MD (1994) The endocrine thymus. Endocrine J 2: 333339 Kendall MD, FrazierJA (1979) Ultrastructural studies on erythropoiesis in the avian thymus. I. Description of cell types. Cell Tissue Res 199:37-61 Marsh JA, Scanes CG (1994) Neuroendocrine-immune interactions. Poultry Sci 73:1049-1061 Minamoto N, Oki K, Tomita M, Kinjo T, Suzuki Y (1988) Isolation and characterization of rotavirus from feral pigeon in mammalian cell cultures. Epidemiol Infec 100:481-492 Steinert PM (1975) The extraction and characterization of bovine epidermal c~-keratin. Biochem J 149:39-48 Sugimoto M, Yasuda T, Egashira Y (1977) Development of the embryonic chicken thymus. II. Differentiation of the epithelial cells studied by electron microscopy. Develop Biol 56:293-305 Ward R D'Cruz D (1968) Seasonal changes in the thymus gland of a tropical bird. Ibis 110:203-205 Accepted November 4, 1998
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Thymic epithelial cysts in the pigeon Yasuro Atoji, Yoshio Yamamoto and Yoshitaka Suzuki Laboratory of Veterinary Anatomy, Faculty of Agriculture, Gifu University, Yanagido, Gifu 501-1193, Japan
Summary. Medullary epithelial cells forming cysts in the thymus of pigeon chicks were examined in order to clarify these morphological characteristics by immunohistochemical and electron microscopic techniques. Light microscopically, cysts were divided into intracellular and intercellular types. Intracellular cysts were positive for the periodic-acid Schiff reaction, while the intercellular type was not. With immunohistochemical staining for keratin, the edges of the intercellular cysts were moderately stained, whereas the cytoplasm of epithelial cells was weakly positive. Heterophils, eosinophils and lymphocytes were included in most of the intercellular cysts. Ultrastructurally, there were two types of epithelial cells which contained intracellular cysts densely packed with microvilli. They differed with regard to the shape of the rough endoplasmic reticulum and bundles of keratin filaments, and in the presence of large vacuoles. The walls of the intercellular cysts were composed of a single type of epithelial cell with a pale nucleus and bundles of keratin filaments. Small vesicles measuring 150 to 200 nm in diameter were numerous in the apical cytoplasm beneath the microvilli. In intercellular cysts, there were abundant heterophils, eosinophils, lymphocytes and macrophages which mostly appeared to be living cells. This evidence suggests that the intercellular cysts might provide a specific microenvironment for leukocytes in the pigeon thymus. Key words: Epithelial cell - Cyst - Leukocytes - Thymus - Ultrastructure
increasing evidence that epithelial cells also serve an endocrine function (Hadden 1992; Kendall 1994). In birds, epithelial cells play an important role, as they do in mammals. In the chicken, this is particularly so for the production of thymic hormones and the expression of peptide receptors (March and Scanes 1994; Atoji et al. 1996, 1997; Hipak et al. 1996). One type of epithelial cell in the thymic medulla is characterized by the formation of cysts lined with microvilli. Two types of cysts have been observed in the thymic medulla of birds (Frazier 1973; Isler 1976; Kendall and Frazier 1979; Hashimoto et al. 1982). One is an intracellular cyst formed in a single cell, a phenomenon also observed in mammals. Another type is an intercellular cyst formed by two or more epithelial cells, which is unique to birds. In cysts of the latter type, in addition to homogeneous material, there are sometimes lymphocytes and granulocytes which appear to have degenerated (Kendall and Frazier 1979; Gulati et al. 1995). The function of both types of cyst remains unknown. Recently, a virus infection in the pigeon has attracted attention in veterinary medicine (Minamoto et al., 1988; Estes and Cohen, 1989). To clarify the immune response against the virus, it is very important to understand the mechanism of the central lymphoid tissues of the thymus and bursa of Fabricius in the pigeon. However, there is, unfortunately, no histological description of them to our knowledge. Therefore we investigated the structure of the lymphoid tissues of the pigeon. In the present study, we examined mainly the ultrastructure of epithelial cysts in the normal pigeon thymus with special reference to the aggregation of leukocytes within them.
Introduction Thymic epithelial cells are associated with a thymic microenvironment, in particular the differentiation and maturation of T lymphocytes. Recently there has been
Materials and methods
Correspondence to: Y. Atoji
Seven chicks of the pigeon, Columba livia, (2 to 4 weeks old, 3 males and 4 females) were used in the present study. Food and
Ann Anat (1999) 181: 365-370 © Urban & Fischer Verlag http:llwww.urbanfischer.deljournalslannanat
0940-9602/99/181/4-365 $12.00/0
water were given ad libitum. They were anesthetized with sodium pentobarbital (25 mg/kg, i.v.) and then perfused transcardially with Ringer's solution followed by Bouin's fixative for light microscopy or by a mixture of 2% paraformaldehyde and 2% glutaraldehyde in 0.1 M phosphate buffer (pH 7.3) for electron microscopy. After perfusion, the thymuses were removed and fixed in the respective solutions. For light microscopy, thymuses from five chicks were embedded in paraffin, and thin sections (4 gm thickness) were stained with hematoxylin and eosiu, and for the periodic-acid Schiff (PAS) reaction. For immunohistochemical staining, dewaxed sections were incubated for 30 rain in 0.3% H202-methanol and then in 2% normal goat serum for 30 rain at room temperature. Sections were then incubated overnight at 4 °C with rabbit antiserum against keratin from bovine muzzle epidermis (diluted 1:2,000; Dako, USA). The antiserum recognized keratin subunits, mainly of 52, 56 and 58 kD (Steinert, 1975). After three washes (5 rain each) in 0.01 M phosphate-buffered saline (PBS, pH 7.4), the sections were incubated for 30 rain at room temperature with biotinylated goat anti-rabbit IgG (diluted 1:500; Vector Laboratories, USA). Then the sections were rinsed three times (5 rain each) in PBS and incubated for 30 min at room temperature with avidin-biotinylated peroxidase complex (ABC; Vector Laboratories). The peroxidase activity was visualized by incubating sections in 0.05 M TrisHC1 buffer (pH 7.4) that contained 3,3'-diaminobenzidine tetrahydrochloride (20mg/100ml) and 0.003% H202. The sections were lightly counterstained with hematoxylin. In the histochemical controls, the primary antiserum was replaced by non-immune rabbit serum as appropriate. For electron microscopy, small tissue samples from two chicks were postfixed for 2 h at 4 °C in a solution of 1% osmium tetroxide in 0.1 M phosphate buffer (pH 7.3) and embedded in epoxy resin. Ultrathin sections were stained with uranyl acetate and lead citrate, and observed with an electron microscope (H-8100; Hitachi, Japan).
Results Light microscopy The medulla of the pigeon thymus was composed of various types of cells and cellular groups: epithelial cells, leu-
kocytes, myoid cells, lymphocytes, Hassall's corpuscles, cysts, and blood vessels (Fig. 1 A). There were two types of intracellular and intercellular cyst, both of which were dispersed through the thymic medulla. The intracellular cysts were found in large epithelial cells with light cytoplasm and often contained PAS-positive substance. The diameter of the cysts measured up to 4 gm. The intercellular cysts were surrounded by a wall which consisted of large and pale epithelial cells. The cells were cuboid, oval or spindle-shaped with attenuated cytoplasm and contained a regularly oval nucleus with a conspicuous nucleolus. The intercellular cysts were round or oval in contour, ranged from 10 gm to 33 gm in diameter, and contained leukocytes which were composed of heterophils, eosinophils and lymphocytes. The numbers of cells varied from cyst to cyst, but a majority of them were filled with leukocytes. No PAS reactivity was seen in the intercellular cysts. Occurrence of the intercellular cysts was high (595 + 47/mm 2, n = 5), while that of the intracellular cysts was very low (22 + 4/ram 2, n = 5). Immunohistochemical staining for keratin showed moderately positive at the edges of the intercellular cysts, and weakly positive in the epithelial cells around the cysts and Hassall's corpuscles (Fig. 1 B).
Electron microscopy Ultrastructurally, the intracellular cysts were located in large electron-lucent epithelial cells, which contained a regular or often irregular shaped nucleus. The cells were divided into two types. Type I was composed of cells in which organelles such as the Golgi apparatus, short profiles of flattened rough endoplasmic reticulum (RER), glycogen particles, small vacuoles, and a few bundles of keratin filaments were conspicuous in the cytoplasm (Fig. 2A). Type II was characterized by the presence of dilated cisternae of R E R with a few ribosomes, thick bundles of keratin filaments, and large vacuoles containing fibrous degenerated material (Fig. 2 B). The nucleus, Golgi apparatus and mitochondria were similar to those of
Fig. 1. Light micrographs of the intercellular cyst. A: Five intercellular cysts (arrows) in the medulla. Arrowheads indicate light epithelial cells in walls of cysts. Bar = 20 gm. B: Edges of a cyst (arrowheads) and epithelial cells (arrows) are positive with immunohistochemical staining for keratin. Bar = 10 gin.
366
Fig. 2. Electron micrographs of the intracellular cysts. A: An epithelial cell of type I contains a cyst (asterisk) in the cytoplasm. The cell morphological characteristics are similar to those of adjacent epithelial cells which participate in formation of an intercellular cyst. Bar = 2 btm. B: An epithelial cell of type II has a cyst (asterisk). The cell contains much balloon-like RER, thick bundles of filaments (arrowheads) and a large vacuole (arrow). Bar = 2 jim.
type I. The cysts of both types were lined with microvilli and frequently contained flocculent or fibrillar substances. Both types of epithelial cells bound surrounding cells by interdigitation and desmosomes. In the intercellular type of cysts, epithelial cells which made up the walls were essentially similar to cells of type I, which contained the intracellular cysts (Fig. 3 A). Microvilli covered the cysts which contained homogeneous substance or cells (Fig. 3 B). Large epithelial cells in the walls were connected to each other by zonula occludens, desmosomes and interdigitation of cell membranes (Fig. 3C). Small vesicles measuring 150nm to 200 nm in diameter were frequently found in the cytoplasm beneath the microvilli (Fig. 3 D). The cells in the cysts were identified as heterophils, eosinophils, lymphocytes, and macrophages (Fig. 4 A - D ) . Most of them showed a morphologically well-preserved state of the organelles, and the remainder looked to be disintegrated. No morphological connections between cells in cysts and epithelial cells in walls were seen. In the medulla outside the intercellular cysts, leukocytes such as heterophils, eosinophils and basophils (probably the some as the mast cells present in the extravascular tissues) were numerous.
Discussion There are two types of cysts in the avian thymus, namely intracellular and intercellular cysts. Only a single type of epithelial cell containing intracellular cysts has been reported in other species of birds (Frazier 1973; Kendall and Frazier 1979; Isler 1976; Hashimoto et al. 1982). In the present study, we demonstrated two types of epithelial cells which contained intracellular cysts. Whether these two types clearly differ or are merely variations of a single type of cell remains unknown. Frazier (1973) pointed out that epithelial cells bearing intracellular cysts have many morphological characteristics in common with the undifferentiated epithelial cells in the medulla of the chicken thymus. In the present study, there are major morphological differences between the two types of epithelial cells containing intracellular cysts in the pigeon thymus, namely, the shape of the cisternae of the RER, the size of the bundles of keratin filaments, and the presence of large vacuoles containing fibrillar material. The dilated cisternae of the R E R are very similar to those found in the reticuloepithelial cells which are seen in the 13-embryonic thymus of the chicken (Sugimoto et al. 1977), but not in the postnatal chicken (Frazier 1973). On
367
Fig. 3. Walls of the intercellular cysts. A: A large, pale epithelial cell with an indented nucleus containing a thick bundle of filaments (arrow), lipid-like vacuoles, slender mitochondria and glycogen particles. Bar = 2 ktm. B: Homogeneous materials stored in a cyst. Note the single type of epithelial cells forming the cyst. Bar = 2 gm. C: Epithelial cells are bound to each other by desmosome (large arrow), zonula occludens (small arrow) and interdigitation (arrowheads). Bar = i gin. D: Numerous small vesicles are seen beneath microvilli. Thick bundles of filaments (arrows) are also noted. Bar = 1 I~m.
368
Fig. 4. Leukocytes and macrophages in the intercellular cysts. A: A heterophil (H), eosinophils (E) and macrophages (M) in a cyst. A degenerated cell (asterisk) is present. A basophil (B) is seen outside the cyst. Bar = 2 ~tm. B: A n eosinophil (E) and lymphocytes (L) in a cyst. Bar = 2 gin. C: A macrophage (M), a heterophil (H) and an eosinophil (E) in a cyst. The macrophage is characterized by a large phagosome, phagocytosis (probably of an eosinophil) and a long string of RER. Bar = 2 ~tm. D: Enlargement of a macrophage which contains myelin-like structures, a long string of R E R and long cytoplasmic processes. Bar = 2 gm.
369
the other hand, the large vacuoles containing fibrillar material are similar to those seen in epithelial cells which participate in the formation of the Hassall's corpuscles in the adult chicken (Frazier 1973). To clarify a relationship between epithelial cells, types I and II, morphological studies on the differentiation and classification of medullary epithelial cells in the pigeon thymus are needed. The intercellular cysts are further divided into two types on the basis of the cellular components of the walls of cysts in the chicken, quelea, sparrow, and starling (Isler 1976; Kendall and Frazier 1979). One is made up of a single type of large and columnar epithelial cell. Such cells contain a regular oval nucleus, slender mitochondria, glycogen particles, free ribosomes and bundles of tonofilaments. Another type contains two types of cell. In addition to the cells of the former type, mucous cells participate in the formation of the cyst walls. The mucous cells are filled with large vacuoles with finely filamentous material of varying density. In the duck, the intercellular cysts consist only of a single type of large epithelial cell with apical microvilli (Hashimoto et al. 1982). In the present study, walls of the intercellular cysts are composed of a single type of epithelial cell and no mucous cells. The presence of mucous cells in the walls of intercellular cysts is probably based on the species difference. The significance of cysts is still a subject for speculation. The first possibility is a "graveyard" of leukocytes within the thymus. The aggregation of leukocytes in cysts has been found in this type of intercellular cysts in birds. Types of leukocytes in the intercellular cysts are granulocytes in the quelea, sparrow, and starling (Kendall and Frazier 1979), lymphocytes in the chicken (Frazier 1973), and heterophils and lymphocytes in the duck (Hashimoto et al. 1982). In the present study, heterophils, eosinophils, lymphocytes and macrophages were observed in the cysts. Neither erythrocytes nor thrombocytes occurred in the intercellular cysts of any birds examined. It is conceivable that the accumulation of leukocytes in cysts differs among species. However, the importance is the presence of macrophages and breakdown of leukocytes in the cyst. Most granulocytes or lymphocytes in thymic cysts of the chicken, duck, quelea, sparrow, and starling are disintegrated, disrupted or have the appearance of cellular debris (Frazier 1973; Isler 1976; Kendall and Frazier 1979; Hashimoto et al. 1982). In the present study, most cells in cysts appeared to be viable, since their fine structure was well preserved, but some granulocytes were disintegrated and others had been phagocytosed by macrophages. These findings suggest a breakdown of granulocytes and lymphocytes in the cysts. The second possibility is that cysts serve to store the secretions of epithelial cells. Small vesicles beneath the microvilli of the intercellular cysts of the chicken have been described as pinocytotic (Isler 1976). This evidence indicates the presence of soluble materials in cysts, because pinocytosis means reabsorption of
contents from cysts. In the present study, the cysts contain homogeneous materials, the walls of cysts are constantly shielded by junctional apparatus, and there are numerous small vesicles (150 nm-200 rim) in the apical cytoplasm of epithelial cells. This would at least suggest exocytosis, which plays a role in the secretion into cysts by epithelial cells.
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