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