Keio J. Med. 20: 57-68. 1971

THYMIC LYMPHOID FOLLICLES IN AUTOIMMUNE DISEASES

‡U. HISTOLOGICAL, HISTOCHEMICAL AND ELECTRON MICROSCOPIC STUDIES

NORIKAZU TAMAOKI, SONOKO HABU and TORU KAMEYA

Department of Pathology, School of Medicine, Keio University, Tokyo

(Received for publication May 8, 1971)

Thymic lymphoid follicles have been known to occur frequently in patients of myasthenia gravis, Graves' disease and other autoimmune diseases, and occa sionally in the young subjects dying by accident.1,2,3,4,5 Spontaneous as well as experimental lymphoid follicles have also been described in the thymus of rodents 6,7,8,9 The presense of a thymic lymphoid follicle has been interpreted as an indication of an abnormal immunological reaction, since the thymus is not a site of antibody production under normal conditions. 10 The thymus is an epithelial organ separated from mesenchymal tissue by an epithelial barrier on the capsular surface and around the blood vessels.11,12,13,14 On the other hand, lymphoid follicles are usually found in the peripheral lymphoid tissue composed of mesenchymal reticular cells, such as lymph nodes, spleen andd lymphoid tissue associated with gut. The present study was intended to describe histological, histochemical and electron microscopic characteristics of thymic lymphoid follicles in order to con firm whether they were related to epithelial or mesenchymal elements and to clarify their histogenesis in the human thymus.

MATERIALS AND METHODS

The materials were the same biospied human thymuses as described in the previous report.5 Serial sections in some cases were made from paraffin blocks and stained alternatively with H & E and silver impregnation. Fresh biopsied thymus and lymph nodes were fixed in cold Baker's calcium-

57 58 NORIKAZU TAMAOKI et al formalin from 18 to 24 hours and then kept in cold gum sucrose for the histo chemical survey. Sections cut at 6 ƒÊ with a cryostat were incubated for alkaline phosphatase,15 ƒÀ-glucuronidase,16 acid phosphatase17 and N-acetyl-ƒÀ-glucosamini dase18 activities using naphthol AS-BI compounds as substrates. Modified

Gomori•Œs acid phosphatase using cytidine-5•Œ-monophosphate was also carried out.19 Fresh thymic tissue was fixed in 1% osmium tetraoxide, dehydrated, and embedded in EPON for the electron microscopic study. Thin sections were stained with uranyl acetate followed by lead citrate and observed with a JEM-100B elec tron microscope.

RESULTS

A. Histological observations The silver impregnation revealed that the perivascular tissue of the inter lobular septa of human thymus was considerably spacious as compared with that of rodents and contained a large number of lymphocytes and occasional plasma cells and granulocytes. These spaces will be termed "perithymic lymphoid tissue" in the present article. Plasma cells were prominent in the thymuses of young people under 20 years of age, which were resected incidentally from the patients of congenital heart diseases and mediastinal tumors. The distribution of plasma cells was usually limited in perithymic lymphoid tissue. Plasma cells were found among epithelial cells within the thymic parenchyma, however, in the thymuses with lymphoid follicle. A few lymphoid follicles with or without germinal centers were observed in the midst of wide interlobular connective tissue, other than in the usual medul lary location in a thymus resected from a 1-year-old girl suspected of possessing mediastinal tumor due to an asymmetrically enlarged thymic shadow. Lymphoid follicles of most other cases were located in the medullary region close to peri thymic lymphoid tissue. It was confirmed by observation on serial sections that a small lymphoid follicle measuring about 70 ƒÊ was located in the small perivas cular space of the medulla (Photo 1) in the case of a 5-year-old girl with myas thenia gravis. Another lymphoid follicle without a germinal center, measuring about 100 ƒÊ, was situated in the medullary parenchyma outside the perivascular space (Photo 2). A survey of serial sections of the thymuses from two cases of myasthenia gravis, 16 and 17 years old, respectively, revealed that the lymphoid follicles measuring over 150 u contained germinal centers. The precise locations of the THYMIC LYMPHOID FOLLICLES ‡U 59 lymphoid follicles were, however, not clear in these cases, because the boundary of reticular fibers between the medullary parenchyma and the perivascular space was obscured in one or more sites around the lymphoid follicles, suggesting that they were located just between parenchyma and perithymic lymphoid tissue (Photo 3). Reconstruction study of serial sections also revealed that the lymphoid follicle was penetrated by one or a few capillaries connected to the interlobular blood vessels (Fig. 1). The venules with tall endothelium similar to those in post capillary venules of lymph nodes were often observed around large lymphoid folli cles with germinal centers. One of the histological changes associated with lymphoid follicle formation was hyalinization in the small perivascular spaces and sometimes in the medullary region. Hyalinization was also observed in the thymuses without lymphoid folli cles in the cases of myasthenia gravis and systemic lupus erythematosus, sug gesting that hyalinization might be a secondary change following lymphoid folli cle formation. In the thymus with a large number of lymphoid follicles, the thymic paren chyma composed of epithelial cells including Hassall's corpuscles was compressed by the area around the follicles densely infiltrated with lymphocytes and plasma cells (Photo 4, Fig. 2). Hassall's corpuscles were well preserved even in such cases, except for some cases of systemic lupus erythematosus, in which they were dissociated into fragments of epithelial cells by the inflammatory infiltration. Hyalinization among epithelial cells were often associated with fragmentation of corpuscles.

B. Histochemical observations

Acid hydrolase activities of lymphocytes :

The human thymic lymphocytes of the cortex and medulla showed no or little activities for ƒÀ-glucuronidase, acid phosphatase and N-acetyl-ƒÀ-glucosamini dase, as revealed by using naphthol AS-BI compounds as substrates (Photo 6).

Most lymphocytes in the paracortical area of the lymph node, on the other hand, showed discrete particulated activity for ƒÀ-glucuronidase, and some of them also showed activities for acid phosphatase and glucosaminidase (Photo 7). The modified Gomori method for acid phosphatase revealed several positive particles within the lymphocytes in the thymus as well as in the paracortical area of the lymph node. The lymphocytes in the dark zone of lymphoid follicles in lymph nodes showed no activity for these enzymes by both Gomori method and naphthol

AS-BI method. 60 NORIKAZU TAMAOKI et at

The lymphocytes except for macrophages in the dark zone and germinal center of thymic lymphoid follicles did not show any activity for acid hydrolases.

Lymphocytes around the thymic lymphoid follicle, however, showed strong activity for ƒÀ-glueuronidase (Photo 5). This finding on the thymic lymphoid follicle coincided with the lymphocytes in the lymph node and differed from that of normal thymic lymphocytes (Photo 6). Alkaline phosphatase activities of reticular cells : The reticular cells of thymic lymphoid follicle showed a strong alkaline phos phatase activity along their cytoplasmic processes which composed a reticular network circumscribing the germinal center of the follicle (Photo 8). These activities of reticular cells were the same as those of reticular cells in the para cortical area of lymph node. The human thymic epithelial cells showed the ac tivity not along the cytoplasmic process, but confined to a localized intracyto plasmic inclusion in the medulla or the cytoplasmic membrane of cells composing Hassall's corpuscle.

C. Electron microscopic observation

The germinal center of a case of systemic lupus erythematosus in a 57-year old woman was observed with the electron microscope. The germinal center was composed of reticular cells, large blastic cells, lymphocytes and plasma cells. The reticular cells were connected to each other with prominent interdigita tions. There were occasionally desmosome-like structures composed of thickened cell membranes (Photo 9). However, these cells contained no filaments terminat ing at the junctional complex. No basement membrane was observed around the reticular cells facing the collagen fibers (Photo 10). The large blastic cells were round-shaped and showed some interdigitation on cell boundary. Abundant polysomes and prominent nucleoli were characteristic for these cells. There were occasional lipid granules in the cytoplasm (Photo 9, 10). Lymphocytes of variable size were scattered throughout the germinal center. Plasma cells contained large granules filled with moderately electron-dense mate rial and enlarged rough-surgaced endoplasmic reticulum. Macrophages were filled with large vacuoles, some of which contained degenerated nuclei. The epithelial cells outside the germinal center were abundant in filaments and connected to each other with desmosomes. Interdigitation of cell membrane was almost absent except for the cells composing Hassall's corpuscle which were filled with bundle of filaments. Basement membrane was found around the cells facing the collagen fibers (Photo 11) . THYMIC LYMPHOID FOLLICLES ‡U 61

DISCUSSION

Histological survey showed that small lymphoid follicles without germinal center were found in the perivascular space or in the medulla very close to this space, while well-developed lymphoid follicles with germinal centers were situated between the medullary parenchyma and the perithymic lymphoid tissue. Con sidering cyclic changes of germinal center,20 the process of formation of thymic lymphoid follicles appears to be as follows. A small aggregation of lymphocytes begins to proliferate in association with capillaries, in the perivascular space or nearby medulla, and gradually develops into a larger lymphoid follicle with a germinal center. The blood vessels originate from those in the perivascular space, some of which become equipped with tall endothelium of the post-capillary-venule type. Infiltration of lymphocytes, plasma cells and exudation around the follicle follows these changes. Hyalinization or fibrosis may remain after the disappear ance of the germinal center. Presence of post-capillary venules around the thymic lymphoid follicles sug gests active immigration of lymphocytes from the blood stream 21 Histochemical findings on acid hydrolase activity in lymphocytes supported this assumption. The lymphocytes in the medulla around the lymphoid follicles showed a strong ƒÀ-glucuronidase activity similar to the recirculating lymphocytes in the para cortical area.19 The absence of activity in the lymphocytes in the dark zone of the thvmie lvmnhoid follicle also coincided with those in the lvmnh node. It is, however, also possible that the lymphocytes of the peripheral type may be derived from the thymic lymphocytes in the medulla. Therefore, it remained to be resolved whether "mutation" of thymic lymphocytes or invasion of peri

pheral lymphocytes triggers the formation of lymphoid follicles in the thymus. Reticular cells with strong alkaline phosphatase activity along their cyto plasmic processes have been described in the paracortical area of the lymph node of the rabbit under antigenic stimulation.22 Since these reticular cells are not found in or around the human thymus under normal conditions, they would have originated from mesenchymal cells in the perivascular space. The reticular cells of a thymic germinal center were characterized by pro minent interdigitation of cell membrane and desmosome-like attachments as seen in electron micrographs. The latter finding coincides with the description on reticular cells of primary and secondary nodules including germinal centers of human and rodent lymph nodes.23,24 No conclusion was reached as to whether the reticular cells were epithelial or mesenchymal in the report on the fine struc ture of thymic germinal center,25 The authors could not find typical desmosomes, 62 NORIKAZU TAMAOKI et al connected with tonofilaments in the thymic lymphoid follicle, while they were abundant between epithelial cells of the thymic parenchyma. Reticular cells of the thymic germinal center were different from thymic epithelial cells, in addi tion to the absence of typical desmosomes, in the paucity of filaments and in lacking a basement membrane around the collagen fibers. The large blast cells with abundant polysomes and prominent nucleoli were identical with cells in the germinal center termed "germinoblast" by Lennert,26 which had not been observed in the thymus. These findings indicated that the thymic germinal centers were identical with those in the peripheral lymphoid tissue.

SUMMARY

Both histochemical and electron microscopic findings confirmed that thymic lymphoid follicles were identical in structure with lymphoid follicles in peripheral lymphoid tissues and not related to thymic epithelial cells. Histochemical demon stration of ƒÀ-glueuronidase and other acid hydrolases showed that thymic medulla around the lymphoid follicles was infiltrated with the lymphocytes of the peri pheral type corresponding to recirculating lymphocytes in paracortical areas of lymph nodes. Reticular cells around the thymic germinal center showed the same activity of alkaline phosphatase as those in lymph nodes. Electron micrographs revealed that the thymic germinal center contained reticular cells connected to each other with prominent interdigitation and desmo some-like structures. These cells differ from thymic epithelial cells in the absence of typical desmosomes, abundant tonofilaments and basement membranes. It could be assumed that reticular cells of thymic lymphoid follicles arise from mesenchymal cells of the perithymic tissue, and that the lymphocytes of the peripheral type occur in the thymus by mutation of thymic lymphocytes or by immigration of circulating lymphocytes.

REFERENCES

1. Sloan, H. E., Jr. (1943) Thymus in myasthenia gravis with observation on the normal anatomy and histology of the thymus. Surgery 13: 154-174. 2. Gunn, A. and Michie. W. (1965) Biopsy of the thvmus. Brit. J. Sure. 52: 957-963. 3. Middleton, G. (1967) The incidence of follicular structure in the thymus at autopsy. Aust. J. Exp. Biol. Med. 45: 189-199. 4. Okabe, H. (1967) Thymic lymph follicles: A histopathological study of 1356 autopsy cases. Acta Path. Jap. 16: 109-130. 5, Habu, S., et al (1971) Thymic lymphoid follicles in autoimmune diseases. I. Quantitative studies with special references with myasthenia gravis. Keio J. Med. 20: 45-56. THYMIC LYMPHOID FOLLICLES ‡U 63

6. Nishizuka, T. and Nakakuki, K. (1963) Significance of the thymus and virus in mouse leukemia. ‡V. Histology of the thymus in preleukemic and early. leukemic AKR mice. Acta Hemat. Jan. 26: 386 7. Burnet, F. M. and Holmes, M. C. (1964) Thymic change in the mouse strain NZB in relation to autoimmune state. J. Path. Bact. 88: 229-241. 8. Marshall, W. H. and White, R. G. (1961) The immunological reactiivty of the thymus. Brit. J. Exp. Path. 42: 379-385. 9. Kotani, M. et al (1966) Experimental formation of lymphatic nodules in the thymus of guinea-pig after injection of histamine. J. Anat. 100: 585-591. 10. Burnet, F. M. and Mackay, I. R. (1965) Histology of the thymus removed surgi cally from a patient with severe untreated systemic lupus erythematosus. J. Path. Bact. 89: 263-270. 11. Hoshino, T. (1963) Electron microscopic study of the epithelial reticular cells of the mouse thymus. Z. Zellforsch. 59: 513-529. 12. Clark, S. L., Jr. (1963) The thymus in mice of strain 129/J studied with electron microscope. Am. J. Anat. 112: 1-33. 13. Weiss, L. (1963) Electron microscopic observations on the barrier of the cortex of thymus of the mouse. Anat. Rec. 145: 413-438. 14. Kameya, T. and Watanabe, Y. (1965) Electron microscopic observations on human thymus and thvmoma. Acta. Path. 15: 223-246. 15. Burstone, M. S. (1958) Histochemical comparison of naphthol AS phosphate for the demonstration of phosphatases. J. Natl. Cancer Inst. 20: 601-615. 16. Hayashi, M. et al (1963) The cytologic demonstration of ƒÀ-glucuronidase em

ploying naphthol AS-BI glucuronide and hexazonium pararosanilin; a prelimi nary report. J. Histochem. Cytochem. 12: 293-297. 17. Barka, T. and Anderson, P. J. (1962) Histochemical method for acid phosphatase using pararosanilin as coupler. J. Histochem. Cytochem. 10: 741-753. 18. Hayashi, M. (1965) Histochemical demonstration of N-acetyl-ƒÀ-glucosaminidase employing the naphthol AS-BI N-acetyl-ƒÀ-glucosaminidine as substrate. J. Histo chem. Cytochem. 13: 335-360. 19. Tamaoki, N. and Essner, E. (1969) Distribution of acid phosphatase, ƒÀ-glucuro nidase and N-acetyl-ƒÀ-glucosaminidase activities in lymphocytes of lymphatic tissue of man and rodents. J. Histochem. Cytochem. 17: 238-243. 20. Conway, E. A. (1937) Cyclic changes in lymphatic nodules. Anat. Rec. 69: 487 513. 21. Gowans, J. L. and Knight, E. J. (1964) The route of recirculation of lymphocyte in the rat. Proc. Rov. Soc. (B) 159: 257-282. 22. Watanabe, K. and Masubuchi, S. (1969) Enzyme cytochemical study on alkaline phosphatase in reticulum cells of lymph nodes, with special reference to antigen and antibody localization. Proc. 7th. International Congress of Electron Micro sconv. nn. 553-554. 23. Mori, Y. and Lennert, K. (1969) Electron microscopic atlas of lymph node cytology and pathology. Springer-Verlag. Heiderberg and New York. 24. Kojima, M. et al (1968) Electro-microscope study on the cells of secondary nodules of the rat. Recent advances in R. E. S. research. 8: 119-142. 25. Goldstein, G. et al (1968) An electron microscopic study of the human thymus: Normal appearances and findings in myasthenia gravis and systemic lupus erythe matosus. J. Path. Bact. 95: 211-215. 26. Lennert, K. et al (1967) Electron microscopic studies of germinal centers in man, Germinal Centers in Immune Responses. eds. Cottier, H., Odartchenko, N., 64 NORIKAZU TAMAOKI et al

Schindler, R. and Congdon, C. pp. 49-55. Springer-Verlag. Berlin, Heiderberg and New York.

EXPLANATION OF PLATES AND FIGURES

Photo 1. Small lymphoid follicle without germinal center measuring 70ƒÊ in diameter located in the perivascular space of the thymus. F: follicle. H: Hassall's corpuscle. PS: perivascular space. Myasthenia gravis, 5-year-old female.

Silver impregnation. Photo 2. Small lymphoid follicle without germinal center measuring l00ƒÊ in diameter located in the thymic medulla. The same case as photo 1. Silver impregna tion. Photo 3. Lymphoid follicle with germinal center measuring 2O0ƒÊ in diameter situated between thymic medulla and perivascular space. Myasthenia gravis, 16-year old female. Fig. 1. Reconstruction of thymic germinal center (GC), which is penetrated with capillaries (C) connected with blood vessel in the perivascular space (right). There are several post-capillary venules (V) around germinal center. H: Hassall's corpuscles. Photo 4. The thymus with many lymphoid follicles with germinal centers found in a case of systemic lupus erythematosus. 57-year-old female. Fig. 2. Trace of figure 5 shows that the medullary parenchyma including Hassall's corpuscles (M) are compressed by the dotted areas containing germinal center (GC) and dense infiltration of lymphocytes and plasma cells. The cortex (C) remains at the margin of thymus. Photo 5. Thymic medulla near the lymphoid follicle with germinal center, which is not shown in this picture. The lymphocytes showing strong activity for ƒÀ-glucuronidase are scattered around a Hassall's corpuscle . Autoimmune hemolytic anemia, 16-year-old female. Photo 6. Thymic lymphocytes show no activity for ƒÀ-glucuronidase in the medulla of the thymus without germinal center, while macrophage is positive for the reaetion. Parathyroid adennma, 22-year-old female.

Photo 7. The lymphocytes in the germinal center and dark zone (left) are negative for ƒÀ-glucuronidase, while those in paracortical area (right) show activity. Human perigastric lymph node removed at the operation for peptic ulcer. Photo 8. The reticular cells around the thymic lymphoid follicle show strong activity for alkaline phosphatase along cytoplasmic processes. The thymic paren chyma including a Hassall's corpuscle shown in upper left is negative and compressed by germinal center. The same case as shown in Photo 5. Photo 9. Electron micrograph of thymic germinal center. The reticular cells (R) connecting with marked interdigitation compose matrix of germinal center.

Desmosome-like structure is indicated by arrow . B: large blastic cell. P: plasma cell. L: lymphocyte. •~4250. Systemic lupus erythematosus, 57 year-old female. Photo 10. The reticularr cells in germinal center has no basement membrane around the collagen fibers (C) •~ 5100. The same case as Photo 9 . Photo 11. The thymic parenchyma around the Hassall's corpuscle . Epithelial cells (E) with abundant tonofilaments have desmosome (arrow.) and-basement mem brane around the colagen fibers (C) . •~ 8500. The same case as Photo 9 and 10. 65

Fig. 1 Photo 1

Photo 4 Photo 2

Fig. 2 Photo 3 66

Photo 5 Photo 6

Photo 7 Photo 8 67

Photo 9 68

Photo 10

Photo 11