Pathology Department, Welsh National School of Medicine, Cardiff, Wales, U.K.
A Fine Structure Study of Human Thymus Eine ultrastrukturelle Studie fiber das menschliche Thymom D. L. JONES, K. THOMAS, and W. JONES WILLIAMS With 10 Figures' Received February 21, 1975 . Accepted in revised form October 10, 1975
Key words: Thymocytes - Lymphocytes - Secretion - Mucoprotein Ultrastructure
Summary A study of the fine structure of human thymus, including normal hyperplastic and with thymoma, is reported in this paper. There is increasing experimental evidence that thymic epithelial cells are a source of hormone. It is, therefore, of interest that we found epithelial cells containing intravesicular cytoplasmic material that reacted with silver methenamine as do mucoproteins. Similar material, possibly extracellular, was seen in close contact with lymphocytes and macrophages in the thymus. The results suggest a possible mechanism for epithelial cell/lymphocyte interaction. Some epithelial cells also showed phagocytic features and all appear to playa structural role. The presence of nuclear pockets was noted within lymphocyte nuclei.
The increasing interest in the immunological role of the thymus, highlighted by Burnet (1962), has encouraged us to examine lymphocytes and epithelial cells in the human thymus. There is increasing evidence that epithelial cells are the source of a hormone, thymosin, responsible for lymphocyte growth and T cell specificity, (Lancet, 1975; Goldstein et al., 1966; Mandi and GIant 1973; Dardenne et al., 1974). It is, therefore, of considerable interest to search for evidence of secretory activity in thymic
388 . D. L. Jones, K. Thomas, and W. Jones Williams
epithelial cells and for possible pathways of products derived from epithelial cells and acting on lymphocytes. Here we illustrate, describe and comment on the following features:the overall and intravesicular mucoprotein content of epithelial cells; the relationship between epithelial cells, macrophages and lymphocytes; the development of macrophages into foam cells and the presence of nuclear pockets in lymphocytes.
Materials and Methods Thymic tissue was obtained from four patients: (r) normal thymus gland from a boy aged nine years undergoing surgery for congenital heart disease, who was otherwise normal; (2) normal thymus from a forty-year-old female undergoing mitral valve replacement. The material consistent of fat-containing islands of thymus tissue; (3) hyperplastic thymus from a female aged nineteen years, suffering from myasthenia gravis; (4) a thymus with a hyperplastic lobe and a thymic mixed tumour in the opposite lobe, removed from a male aged 3 r years with myasthenia gravis. Blocks of tissue approximately I mm 3 were fixed in 0.067 M phosphate buffer pH 7.4 containing 3010 glutaraldehyde, for four hours at 4 () C. After washing over-night in the phosphate buffer the tissue was post-fixed in rOfoMilionig's osmium tetroxide, dehydrated in ethanol and embedded in araldite. I[lm sections were cut on an LKB III ultramicrotome and stained with toluidine blue for selection of suitable areas. Ultrathin sections were stained with uranyl acetate and lead citrate and examined in a Phillips E.M. 300 electron microscope. Tissue blocks from the tumour and hyperplastic thymus were also processed for the silver methenamine technique (Rambourg, r967), to detect I : 2 glycol groups. The processing for this technique was the same as above except that post-fixation in osmium tetroxide was omitted. Ultra-thin sections were oxidised with periodic acid and treated with silver methenamine reagent. Controls included unoxidised sections and blocking with acetic anhyride and pyridine before the silver methenamine staining.
Results Normal and Hyperplastic Glands Light microscopy-the normal thymus of the child showed well demarcated cortex and medulla with prominent epithelial cells and Hassall's corpuscles, in the medulla. In the cortex the lymphocyte/epithelial cell ratio was about 5 : I and in the medulla 3 : I. The normal adult thymus consisted of islands of lymphoid cells with scattered Hassall's corpuscles, but lacked cortico-medullary differentiation. At all ages, both small and large lymphocytes were seen in the cortex, in the ratio of about 5 : I and approximately 2 : I in the medulla. The hyperplastic thymuses showed a loss of cortico-medullary differ-
E. M. Thymus. 389
lA
1
B
Fig. I A and B. Nuclear pockets in small lymphocytes comammg membranes, ribosome sized particles and vesicles. Normal thymus. A X 36,000, B X 30,000.
390 . D. L. Jones, K. Thomas, and W. Jones Williams
Fig.
2.
Nuclear bodies (arrows), in epithelial cells. Hyperplastic thymus. X
21,000.
entiation and were characterised by the presence of well developed germinal centres, in the medulla. The lymphocyte/epithelial cell ratio was about 3 : I and small and large lymphocytes occurred in approximately equal numbers. In both normal and hyperplastic glands the majority of epithelial cells were of the "light" variety, with only occasional "dark" cells. Ultra-structure-essentially, the structure of lymphocytes, lymph follicles, epithelial cells and Hassall's corpuscles, were as described by previous authors, (Kameya and Watanabe, 1965; Tamaoki and Habu, 1971). A number of features were, however, noteworthy. Large lymphocytes were distinguished from small lymphocytes by their larger, overall size, 7 [lm diameter, compared to 5 [lm diameter, and increased number of organelles. A few, 10 [lm diameter, larger lymphocytes, containing many polyribosomes, were also seen with equal frequency, in both normal and hyperplastic glands. Occasional small cortical lymphocytes showed the presence of nuclear pockets (Fig. I A and B). These consisted of round, of oval, spaces at the edge of the nuclei, lined by a double membrane, and separated from the nuclear membrane by a thin rim of chromatin. The contents were variable and included ribosomes, small vesicles and sometimes concentric laminated membranes.
E. M. Thymus· 39 1
Occasional lymphocytes, and epithelial cell nuclei, showed nuclear bodies, as described by Henry and Petts (1969). They were approximately 0.5-1 flm in diameter and consisted of fine collections of fibrous material, and surrounding clear spaces (Fig. 2). We noted that the innermost fibre-rich epithelial cells of Hassall's corpuscles showed peculiar, uniform electron-dense projections (Fig. 3). Both, Hassall's corpuscle cells and the degenerate centres stained strongly for mucoprotein. Dark epivhelial cells were distinguished from light epithelial cells by an increased overall density of staining and by the number of cytoplasmic fibrils. Both varieties showed desmosomes, and occasional phagolysosomes (Fig. 4). Fine processes, extending between adjacent lymphocytes, were prominent. As dark epithelial cells were most frequent in the thymoma, further details are included below.
Fig. 3. Central epithelial cell of Hassall's corpuscle with dense tonofibrils (T), and membrane projections (arrows). Normal thymus. X 30,600.
39 2
•
D. L. Jones, K. Thomas, and W. Jones Williams
'
•
.
•
'. Epithelial cell with phagolysosomes (PL), and tonofibrils (T). Normal thymus. .1". -;
Fig. 4.
.
:
•
~
f
• ,. :
,•
X 27,500.
Occasional macrophages, polymorphs and mast cells were also seen. Plasma cells were scanty but were more frequent in the hyperplastic than in the normal glands. Myoid cells (Henry, 1975) were not detected.
Thymoma Light microscopy-the tumour was of mixed type with more lymphocytes than epithelial cells (Fig. 5). Focal areas of macrophages were conspicuous, but Hassall's corpuscles were scanty. As compared to the non-tumour glands, the lymphocytes showed increased mitotic activity, with a predominance of large, as compared to small, lymphocytes, approximately 5 : 1. The majority of epithelial cells were darkly stained, the ratio of dark/light being 2 : I. Ultra-structure-lymphocytes tended to be aggregated into clusters, situated between, and in close contact with, interconnecting fine processes of epithelial cells. Other than the increased number of mitoses the lympho-
E. M. Thymus . 393
cytes appeared essentially similar to those found in the non-tumourous glands. A few showed nuclear bodies, but nuclear pockets were not identified. We wish to draw attention to the possible secretory and phagocytic features of epithelial cells and their intimate relationship to other cells. Evidence of secretory activity was conflicting. Ribosomes and polyribosomes were conspicuous but RER was sparse and not dilated. Golgi complexes and associated vesicles were seldom prominent. Many of the cells contained small (0.1-0.2 ftm diameter) dense vesicles and occasional lipid vesicles (Fig. 6). Rambourg's stain for mucoprotein showed overall staining of the cytoplasm and in the small dense vesicles (Fig. 7). Control sections showed only scattered cytoplasmic and nuclear staining. Occasional epithelial cells showed the presence of phagolysosomes and fragments of degenerate phagocytosed cells. These epithelial cells were distinguished from macrophages by the presence of tonofibrils and desmosomes.
Fig. 5. Light (1), and dark (d), epithelial cells, lymphocytes (L), some in mitosis (arrows), and macrophage/foam cells (M). Thymoma, toluidine blue. X 450.
394 . D . L. Jones, K. Thomas, and W. Jones Williams
Fig. 6. Dark epithelial cell with small dense vesicles (v), lipid droplet (f), and desmosomcs (arrow). Thymoma. X 36,000.
We found that the fine processes of epithelial cells formed a complex interdigitating system with those of adjacent macrophages. At these sites it was often difficult to identify the limiting membranes of each cell. Thus, the interdigitations, even allowing for plane of section artefact, sometimes appeared to be delimited by only a single membrane (Fig. 8). Macrophages were more numerous in the tumour than in the other tissues examined. They often contained complex phagolysosomes, and sometimes "empty" vesicles. Such cells, may therefore, have a "foamy" appearance (Fig. 9). Fine processes of macrophages were also intimately related to dense extracellular material (Fig. 10), which, together with the phagolysosomes, gave the Rambourg stain.
Discussion In their fine structure, the three types of non-follicular lymphocytes were similar to those found in peripheral lymph nodes and in circulating
E. M. Thymus. 395
blood. We expected, but did not find, the large immunoblast-type lymphocytes to be most frequent in the cases suffering from myasthenia. The nuclear pockets found in small lymphocytes have previously been described in normal human foetal thymus (Sebuwufu, 1966), but are seldom mentioned in studies of post-natal human thymus. Mollo and Stramignoni (1967) found nuclear pockets, referred to as nuclear projections, in circulating and lymph node cells in lymphatic and myeloid leukaemia, in Hodgkin's disease (in lymphoid and Reed-Sternberg, cells), and in PHAstimulated, transformed, lymphocytes, They have also been found in Burkitt's lymphoma (Achong and Epstein, 1966). They are not confined to lymphoid cells but have also been recognised in epithelial cells of thyroid carcinoma (Soderstrom and Biorklund, 1973). Our findings, and those of earlier workers, suggest that the pockets are intranuclear cytoplasmic invaginations and occur most frequently at sites of nuclear indentations facing Golgi complexes. The significance of nuclear pockets remains unclear, but may be related to possible nuclear exchange in metabolically active cells.
Fig. 7. Epithelial cell showing methenamine stain. X 20,200 .
mucoprotein
stained
vesicles. Thymoma,
silver
396 . D. L. Jones, K. Thomas, and W. Jones Williams
Fig. 8.
Macrophage (M)/epithelial cell (E), interdigitations. Thymoma. X
34,000.
The role of epithelial cells appears to be multiple. Our finding of numerous tonofibrils with associated desmosomes and ramifying fine processes, supports the view that epithelial cells serve a structural function (Hwang et aI., I974)' We have also shown that some epithelial cells, in addition, are phagocytic. This was most often seen in the tumour specimen presumably under conditions of high cell turnover and cell death. Vetters and Macadam (I973), on the basis of their description of intracytoplasmic vesicles, consider that the cells are secretory, but others disagree (Toker, I968; Kay, 1970). We found that many dark epithelial cells contained vesicles that took the Rambourg stain and also showed diffuse cytoplasmic, Rambourg-positive staining associated with the presence of ribosomes and polyribosomes. Though RER and Golgi complexes were inconspicuous, we consider that our findings support a secretory function. Further, we found that the fine epithelial cell processes were difficult to distinguish from the dark extracellular material. This material was in close contact with adjacent lymphocytes. Could this be a mechanism for possible hormonal epithelial cell/lymphocyte interaction? It may offer an
E. M. Thymus· 397
Fig 9.
Macrophage/toam cell. Phagolysosomes (PL), vacuole (V). Thymoma. X 23,400.
explanation for the apparent discrepancy between the experimental evidence for epithelial cell secretion and the lack of agreement on morphological secretory hallmarks. The relationship of the extracellular material to macrophages is also of interest. The macrophage processes appeared to be in contact with extracellular material and the cells contained mucoprotein phagolysosomes that took the Rambourg stain. Further, macrophages appeared to develop into foam cells. It is, therefore, likely that they ingest extracellular material Could this be a hormone storage mechanism? The apparent similarity of cells in Hassall's corpuscles to those in epidermal cells forming keratin did not suggest to earlier workers, any functional role. Blau (I967, I969), however, has suggested a more dynamic role, by the demonstration of antigen/antibody concentration and uptake of particulate material, in the corpuscles. We have demonstrated curious short, blunt processes on the inner aspect of the central epithelial cell which may imply transposition across the cell membrane. 27 Beitr. Path. Bd. 156
398 . D. L. Jones, K. Thomas, and W. Jones Williams
Fig. ro. Macrophage (M), with fin e processes related (arrow). Thymoma. X 22,000.
to
dense extracellular material
In conclusion, though these morphological findings are not proof that epithelial cells and macrophages are concerned with hormone production and storage, they suggest that further experimental work should consider the role of both these intimately related cells. Despite the increasing interest and possible importance of myoid cells in the aetiology of myasthenia gravis (Henry, 1975), we were unable to find such cells in our material. A cknow ledgements. Mr. D. L. Jones is supported by an M. R. C. Grant.
Zusammenfassung Es wird iiber die Feinstruktur des menschlichen Thymus im Normalzustand sowie im Falle eines Thymoms berichtet. Es liegen zahlreiche Hinweise dafii r vor, daB die Thymozyten hormonproduzierend sind. Mit der Methenamin-Silber-Reaktion wurden Muko-
E. M. Thymus . 399 proteine in Zytoplasmavesikel gefunden. Khnliches Material wurde im engen Kontakt mit Lymphozyten und Makrophagen gesehen. Diese Resultate sprechen fur eine mogliche Thymozyten-Lymphozyten-Interaktion. Einige Thymozyten zeigten auch morphologische Hinweise einer Phagozytose. In einigen Lymphozyten wurden Kernmembraneinstiilpungen beobachtet.
References Achong, B. G., Epstein, M. A.: Fine structure of the Burkitt tumour. J. Nat. Cancer Inst. 36, 877-897 (1966) Blau, J. N.: Antigen and antibody localisation in Hassall's corpuscles. Nature (London) 21 5, 1073-1075 (19 67) Blau, ]. N.: The localisation of BCG in the guinea-pig thymus with special reference to Hassall's corpuscles. Immunology 17, 995-998 (1969) Burnet M.: Role of the thymus and related organs in immunity. Brit. Med. J. ii, 807-8 II (1962) Dardenne, M., Papiernik, M., Bach, J" and Stutnan, 0.: Studies on thymic products. III Epithelial origin of the serum thymic factor. Immunology 27, 299-304 (1974) Goldstein, A. L., Slater, F. D., and White, A.: Preparation, assay and partial purification of a thymic lymphocytopoietic factor (thymosin). Proc. Nat. Acad. Sci. U.S.A. 56, 1010-1017 (1966) Henry, K., and Petts, V.: Nuclear bodies in human thymus . .J. Ultrastruct. Res. 27, 33 0-343 (19 69)
Henry, K.: Myoid cells, myasthenia gravis, and thymomas of myoid cell origin. Patho!' Soc. Gt. Brit., Abstract 36 (1975) Hwang, W. S., Ho, T. Y., Luk, S. c., and Gerard, T. S.: Ultrastructure of the rat thymus. A transmission, scanning electron microscope, and morphometric study. Lab. Invest. p, 473-487 (1974) Kameya, T., and Watanabe, Y.: Electron microscopic observations on human thymus and thymoma. Acta Path. lap. 15,223-246 (1965) Kay, S.: Comparative ultrastructural studies on three thymic lesions. Arch. Path. 90, 4 16-4 22 (197 0) Lancet (ed.): Thymus hormones. Lancet i, 314-315 (1975) Mandi, B., and Giant, T.: Thymosin producing cells of the thymus. Nature (New Bio!.) 24 6,25 (1973) Mollo, F., and Stramignoni, A.: Nuclear projections in blood and lymph node cells of human leukaemics and Hodgkin's disease and in lymphocytes cultured with phytohaemagglutinin. Brit. J. Cancer 21, 519-523 (1967) Rambourg, A.: An improved silver methenamine technique for the detection of periodic acid-reactive complex carbohydrates with the electron microscope. J. Histochem. Cytochem. 15,409-412 (19 67) Sebuwufu, P. H.: Nuclear blebs in the human foetal thymus. Nature (London) 212, 1382-1383 (19 66 ) Soderstrom, N., and Biorklund, A.: Intranuclear cytoplasmic inclusions in some types of thyroid cancer. Acta Cytol. 17, 191-197 (1973) Tamaoki, N., Habu, S., and Kameya, T.: Thymic lymphoid follicles in autoimmune disease. II Histological, histochemical and electron microscopic studies. Keio J. Med. 26, 57-68 (1971)
400 .
D. L. Jones, K. Thomas, and W. Jones Williams
Toker, c.: Thymoma, An ultrastructural study. Cancer 21, II57-II63 (1968) Vetters, J. M., and Macadam, R. F.: Fine structure evidence for hormone secretion by the human thymus. J. Clin. Path. 26, 194-197 (1973) D. L. Jones, K. Thomas, W. Jones Williams, Pathology Department, Welsh National School of Medicine, Cardiff, Wales, U.K.
A Fine Structure Study of Human Thymus Eine ultrastrukturelle Studie fiber das menschliche Thymom D. L. JONES, K. THOMAS, and W. JONES WILLIAMS With 10 Figures' Received February 21, 1975 . Accepted in revised form October 10, 1975
Key words: Thymocytes - Lymphocytes - Secretion - Mucoprotein Ultrastructure
Summary A study of the fine structure of human thymus, including normal hyperplastic and with thymoma, is reported in this paper. There is increasing experimental evidence that thymic epithelial cells are a source of hormone. It is, therefore, of interest that we found epithelial cells containing intravesicular cytoplasmic material that reacted with silver methenamine as do mucoproteins. Similar material, possibly extracellular, was seen in close contact with lymphocytes and macrophages in the thymus. The results suggest a possible mechanism for epithelial cell/lymphocyte interaction. Some epithelial cells also showed phagocytic features and all appear to playa structural role. The presence of nuclear pockets was noted within lymphocyte nuclei.
The increasing interest in the immunological role of the thymus, highlighted by Burnet (1962), has encouraged us to examine lymphocytes and epithelial cells in the human thymus. There is increasing evidence that epithelial cells are the source of a hormone, thymosin, responsible for lymphocyte growth and T cell specificity, (Lancet, 1975; Goldstein et al., 1966; Mandi and GIant 1973; Dardenne et al., 1974). It is, therefore, of considerable interest to search for evidence of secretory activity in thymic
388 . D. L. Jones, K. Thomas, and W. Jones Williams
epithelial cells and for possible pathways of products derived from epithelial cells and acting on lymphocytes. Here we illustrate, describe and comment on the following features:the overall and intravesicular mucoprotein content of epithelial cells; the relationship between epithelial cells, macrophages and lymphocytes; the development of macrophages into foam cells and the presence of nuclear pockets in lymphocytes.
Materials and Methods Thymic tissue was obtained from four patients: (r) normal thymus gland from a boy aged nine years undergoing surgery for congenital heart disease, who was otherwise normal; (2) normal thymus from a forty-year-old female undergoing mitral valve replacement. The material consistent of fat-containing islands of thymus tissue; (3) hyperplastic thymus from a female aged nineteen years, suffering from myasthenia gravis; (4) a thymus with a hyperplastic lobe and a thymic mixed tumour in the opposite lobe, removed from a male aged 3 r years with myasthenia gravis. Blocks of tissue approximately I mm 3 were fixed in 0.067 M phosphate buffer pH 7.4 containing 3010 glutaraldehyde, for four hours at 4 () C. After washing over-night in the phosphate buffer the tissue was post-fixed in rOfoMilionig's osmium tetroxide, dehydrated in ethanol and embedded in araldite. I[lm sections were cut on an LKB III ultramicrotome and stained with toluidine blue for selection of suitable areas. Ultrathin sections were stained with uranyl acetate and lead citrate and examined in a Phillips E.M. 300 electron microscope. Tissue blocks from the tumour and hyperplastic thymus were also processed for the silver methenamine technique (Rambourg, r967), to detect I : 2 glycol groups. The processing for this technique was the same as above except that post-fixation in osmium tetroxide was omitted. Ultra-thin sections were oxidised with periodic acid and treated with silver methenamine reagent. Controls included unoxidised sections and blocking with acetic anhyride and pyridine before the silver methenamine staining.
Results Normal and Hyperplastic Glands Light microscopy-the normal thymus of the child showed well demarcated cortex and medulla with prominent epithelial cells and Hassall's corpuscles, in the medulla. In the cortex the lymphocyte/epithelial cell ratio was about 5 : I and in the medulla 3 : I. The normal adult thymus consisted of islands of lymphoid cells with scattered Hassall's corpuscles, but lacked cortico-medullary differentiation. At all ages, both small and large lymphocytes were seen in the cortex, in the ratio of about 5 : I and approximately 2 : I in the medulla. The hyperplastic thymuses showed a loss of cortico-medullary differ-
E. M. Thymus. 389
lA
1
B
Fig. I A and B. Nuclear pockets in small lymphocytes comammg membranes, ribosome sized particles and vesicles. Normal thymus. A X 36,000, B X 30,000.
390 . D. L. Jones, K. Thomas, and W. Jones Williams
Fig.
2.
Nuclear bodies (arrows), in epithelial cells. Hyperplastic thymus. X
21,000.
entiation and were characterised by the presence of well developed germinal centres, in the medulla. The lymphocyte/epithelial cell ratio was about 3 : I and small and large lymphocytes occurred in approximately equal numbers. In both normal and hyperplastic glands the majority of epithelial cells were of the "light" variety, with only occasional "dark" cells. Ultra-structure-essentially, the structure of lymphocytes, lymph follicles, epithelial cells and Hassall's corpuscles, were as described by previous authors, (Kameya and Watanabe, 1965; Tamaoki and Habu, 1971). A number of features were, however, noteworthy. Large lymphocytes were distinguished from small lymphocytes by their larger, overall size, 7 [lm diameter, compared to 5 [lm diameter, and increased number of organelles. A few, 10 [lm diameter, larger lymphocytes, containing many polyribosomes, were also seen with equal frequency, in both normal and hyperplastic glands. Occasional small cortical lymphocytes showed the presence of nuclear pockets (Fig. I A and B). These consisted of round, of oval, spaces at the edge of the nuclei, lined by a double membrane, and separated from the nuclear membrane by a thin rim of chromatin. The contents were variable and included ribosomes, small vesicles and sometimes concentric laminated membranes.
E. M. Thymus· 39 1
Occasional lymphocytes, and epithelial cell nuclei, showed nuclear bodies, as described by Henry and Petts (1969). They were approximately 0.5-1 flm in diameter and consisted of fine collections of fibrous material, and surrounding clear spaces (Fig. 2). We noted that the innermost fibre-rich epithelial cells of Hassall's corpuscles showed peculiar, uniform electron-dense projections (Fig. 3). Both, Hassall's corpuscle cells and the degenerate centres stained strongly for mucoprotein. Dark epivhelial cells were distinguished from light epithelial cells by an increased overall density of staining and by the number of cytoplasmic fibrils. Both varieties showed desmosomes, and occasional phagolysosomes (Fig. 4). Fine processes, extending between adjacent lymphocytes, were prominent. As dark epithelial cells were most frequent in the thymoma, further details are included below.
Fig. 3. Central epithelial cell of Hassall's corpuscle with dense tonofibrils (T), and membrane projections (arrows). Normal thymus. X 30,600.
39 2
•
D. L. Jones, K. Thomas, and W. Jones Williams
'
•
.
•
'. Epithelial cell with phagolysosomes (PL), and tonofibrils (T). Normal thymus. .1". -;
Fig. 4.
.
:
•
~
f
• ,. :
,•
X 27,500.
Occasional macrophages, polymorphs and mast cells were also seen. Plasma cells were scanty but were more frequent in the hyperplastic than in the normal glands. Myoid cells (Henry, 1975) were not detected.
Thymoma Light microscopy-the tumour was of mixed type with more lymphocytes than epithelial cells (Fig. 5). Focal areas of macrophages were conspicuous, but Hassall's corpuscles were scanty. As compared to the non-tumour glands, the lymphocytes showed increased mitotic activity, with a predominance of large, as compared to small, lymphocytes, approximately 5 : 1. The majority of epithelial cells were darkly stained, the ratio of dark/light being 2 : I. Ultra-structure-lymphocytes tended to be aggregated into clusters, situated between, and in close contact with, interconnecting fine processes of epithelial cells. Other than the increased number of mitoses the lympho-
E. M. Thymus . 393
cytes appeared essentially similar to those found in the non-tumourous glands. A few showed nuclear bodies, but nuclear pockets were not identified. We wish to draw attention to the possible secretory and phagocytic features of epithelial cells and their intimate relationship to other cells. Evidence of secretory activity was conflicting. Ribosomes and polyribosomes were conspicuous but RER was sparse and not dilated. Golgi complexes and associated vesicles were seldom prominent. Many of the cells contained small (0.1-0.2 ftm diameter) dense vesicles and occasional lipid vesicles (Fig. 6). Rambourg's stain for mucoprotein showed overall staining of the cytoplasm and in the small dense vesicles (Fig. 7). Control sections showed only scattered cytoplasmic and nuclear staining. Occasional epithelial cells showed the presence of phagolysosomes and fragments of degenerate phagocytosed cells. These epithelial cells were distinguished from macrophages by the presence of tonofibrils and desmosomes.
Fig. 5. Light (1), and dark (d), epithelial cells, lymphocytes (L), some in mitosis (arrows), and macrophage/foam cells (M). Thymoma, toluidine blue. X 450.
394 . D . L. Jones, K. Thomas, and W. Jones Williams
Fig. 6. Dark epithelial cell with small dense vesicles (v), lipid droplet (f), and desmosomcs (arrow). Thymoma. X 36,000.
We found that the fine processes of epithelial cells formed a complex interdigitating system with those of adjacent macrophages. At these sites it was often difficult to identify the limiting membranes of each cell. Thus, the interdigitations, even allowing for plane of section artefact, sometimes appeared to be delimited by only a single membrane (Fig. 8). Macrophages were more numerous in the tumour than in the other tissues examined. They often contained complex phagolysosomes, and sometimes "empty" vesicles. Such cells, may therefore, have a "foamy" appearance (Fig. 9). Fine processes of macrophages were also intimately related to dense extracellular material (Fig. 10), which, together with the phagolysosomes, gave the Rambourg stain.
Discussion In their fine structure, the three types of non-follicular lymphocytes were similar to those found in peripheral lymph nodes and in circulating
E. M. Thymus. 395
blood. We expected, but did not find, the large immunoblast-type lymphocytes to be most frequent in the cases suffering from myasthenia. The nuclear pockets found in small lymphocytes have previously been described in normal human foetal thymus (Sebuwufu, 1966), but are seldom mentioned in studies of post-natal human thymus. Mollo and Stramignoni (1967) found nuclear pockets, referred to as nuclear projections, in circulating and lymph node cells in lymphatic and myeloid leukaemia, in Hodgkin's disease (in lymphoid and Reed-Sternberg, cells), and in PHAstimulated, transformed, lymphocytes, They have also been found in Burkitt's lymphoma (Achong and Epstein, 1966). They are not confined to lymphoid cells but have also been recognised in epithelial cells of thyroid carcinoma (Soderstrom and Biorklund, 1973). Our findings, and those of earlier workers, suggest that the pockets are intranuclear cytoplasmic invaginations and occur most frequently at sites of nuclear indentations facing Golgi complexes. The significance of nuclear pockets remains unclear, but may be related to possible nuclear exchange in metabolically active cells.
Fig. 7. Epithelial cell showing methenamine stain. X 20,200 .
mucoprotein
stained
vesicles. Thymoma,
silver
396 . D. L. Jones, K. Thomas, and W. Jones Williams
Fig. 8.
Macrophage (M)/epithelial cell (E), interdigitations. Thymoma. X
34,000.
The role of epithelial cells appears to be multiple. Our finding of numerous tonofibrils with associated desmosomes and ramifying fine processes, supports the view that epithelial cells serve a structural function (Hwang et aI., I974)' We have also shown that some epithelial cells, in addition, are phagocytic. This was most often seen in the tumour specimen presumably under conditions of high cell turnover and cell death. Vetters and Macadam (I973), on the basis of their description of intracytoplasmic vesicles, consider that the cells are secretory, but others disagree (Toker, I968; Kay, 1970). We found that many dark epithelial cells contained vesicles that took the Rambourg stain and also showed diffuse cytoplasmic, Rambourg-positive staining associated with the presence of ribosomes and polyribosomes. Though RER and Golgi complexes were inconspicuous, we consider that our findings support a secretory function. Further, we found that the fine epithelial cell processes were difficult to distinguish from the dark extracellular material. This material was in close contact with adjacent lymphocytes. Could this be a mechanism for possible hormonal epithelial cell/lymphocyte interaction? It may offer an
E. M. Thymus· 397
Fig 9.
Macrophage/toam cell. Phagolysosomes (PL), vacuole (V). Thymoma. X 23,400.
explanation for the apparent discrepancy between the experimental evidence for epithelial cell secretion and the lack of agreement on morphological secretory hallmarks. The relationship of the extracellular material to macrophages is also of interest. The macrophage processes appeared to be in contact with extracellular material and the cells contained mucoprotein phagolysosomes that took the Rambourg stain. Further, macrophages appeared to develop into foam cells. It is, therefore, likely that they ingest extracellular material Could this be a hormone storage mechanism? The apparent similarity of cells in Hassall's corpuscles to those in epidermal cells forming keratin did not suggest to earlier workers, any functional role. Blau (I967, I969), however, has suggested a more dynamic role, by the demonstration of antigen/antibody concentration and uptake of particulate material, in the corpuscles. We have demonstrated curious short, blunt processes on the inner aspect of the central epithelial cell which may imply transposition across the cell membrane. 27 Beitr. Path. Bd. 156
398 . D. L. Jones, K. Thomas, and W. Jones Williams
Fig. ro. Macrophage (M), with fin e processes related (arrow). Thymoma. X 22,000.
to
dense extracellular material
In conclusion, though these morphological findings are not proof that epithelial cells and macrophages are concerned with hormone production and storage, they suggest that further experimental work should consider the role of both these intimately related cells. Despite the increasing interest and possible importance of myoid cells in the aetiology of myasthenia gravis (Henry, 1975), we were unable to find such cells in our material. A cknow ledgements. Mr. D. L. Jones is supported by an M. R. C. Grant.
Zusammenfassung Es wird iiber die Feinstruktur des menschlichen Thymus im Normalzustand sowie im Falle eines Thymoms berichtet. Es liegen zahlreiche Hinweise dafii r vor, daB die Thymozyten hormonproduzierend sind. Mit der Methenamin-Silber-Reaktion wurden Muko-
E. M. Thymus . 399 proteine in Zytoplasmavesikel gefunden. Khnliches Material wurde im engen Kontakt mit Lymphozyten und Makrophagen gesehen. Diese Resultate sprechen fur eine mogliche Thymozyten-Lymphozyten-Interaktion. Einige Thymozyten zeigten auch morphologische Hinweise einer Phagozytose. In einigen Lymphozyten wurden Kernmembraneinstiilpungen beobachtet.
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