The disposition of epithelial cells and extracellular ma- trix, in the thymus of8 cases of myasthenia gravis (MG) and in controls (over a wide age range) was ...
Microenvironments in the Normal Thymus in Myasthenia Gravis
Thymus and
the
From the Departments of Immunology and Neurological Science, Royal Free Hospital School of Medicine, and the National Hospital for Nervous Diseases, London, England
MARGARITA BOFILL, G. JANOSSY, N. WILLCOX, M. CHILOSI, L. K. TREJDOSIEWICZ, and J. NEWSOM-DAVIS
The disposition of epithelial cells and extracellular matrix, in the thymus of 8 cases of myasthenia gravis (MG) and in controls (over a wide age range) was studied. In the controls, the subcapsular epithelium was strongly Leu7-positive in the fetus, negative in childhood, and positive again in adults. Another antibody, RFD4, also labeled the subcapsular epithelium in childhood and adults, but not fetal samples. The samples from MG cases showed the same staining pattern as adult control samples. The medullary epithelium was also RFD4', and at all ages. The most striking changes in the advanced cases of MG were the unusual arrangement and hypertrophic appearance of medullary epithelial cell areas, separated by laminin-positive basement membranes from the alternating multiple bands ofperipheral lymph-node-like areas.
The latter had regions resembling the paracortex of lymph nodes as well as germinal centers (GCs). The T-cell zones contained heavy deposits of fibronectin. These T-cell zones were unique to the thymus in MG and were absent in the two normal thymic samples with isolated GCs. In MG the laminin-containing basement membrane, which separated the medullary epithelial and peripheral lymphnode-like areas, was fenestrated at circumscribed points closest to the GCs, thus apparently permitting communication among the medullary epithelium, the T-cell zones, the GCs and the associated antigen-presenting cells. Large numbers of interdigitating cells and some lymphocytes ofcortical thymocyte phenotype were also found at these special sites, where opportunities for autosensitization may persist in MG. (AmJ Pathol 1985, 119:462-473)
IN MYASTHENIA GRAVIS (MG) autoantibodies to the acetylcholine receptor (AChR) of skeletal muscle damage the motor endplates, which leads to a loss of AChRs and to muscle weakness. There is a special relationship between MG and the thymus. AChR-like material is normally present in the thymus, notably on rare myoid cells1 and perhaps also on other cell types. In about two-thirds of patients the thymus is hyperplastic, with germinal centers (GCs) in the medulla2; and the myasthenia in these patients often improves after thymectomy. In tissue culture, cell suspensions from these thymuses spontaneously synthesize anti-AChR antibodies,3 which appear to be mainly derived from the GCs.4 Specific antigen-presenting cells may also be present, because the same suspensions of thymic cells can also preferentially stimulate anti-AChR production by autologous blood lymphocytes.5 It is also clear that in some patients with MG, lymph node cells spontaneously produce anti-AChR antibodies as well.4 Previous histologic studies on the thymus in MG revealed no gross abnormality in the cortex but frequently showed an expanded medulla with GCs,6 which, like those found in peripheral lymph nodes, accumulated
lacy immunoglobulin deposits on follicular dendritic cells. Medullary epithelial hyperplasia has also been noted in the vicinity of these GCs3' 78; and in some cases HLA-DR' interdigitating reticulum (T-zone dendritic, ID) cells appear to be increased, especially near the corticomedullary junction.6 Furthermore, abundant high endothelial venules9 point to the possibility of increased lymphocyte traffic through these myasthenic medullary areas. Clearly, many of these features are those of peripheral lymph nodes, while cell types peculiar to the thymus such as medullary epithelial cells not only persist but appear to be hyperplastic in MG. During the last 2 years monoclonal antibodies McAbs) to the epithelial and mesenchymal elements of the thymus have become available (reviewed in Janossy Supported by grants from Armour Pharmaceutical Company (to Dr. Bofill), the Medical Research Council of Great Britain, and the Sir Jules Thorn Charitable Trust. Accepted for publication January 31, 1985. Address reprint requests to Prof. G. Janossy, Department of Immunology, Royal Free Hospital School of Medicine, London NW3 2PF, England.
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Table 1-MG Patients Studied Age (years) at Sex thymectomy Case
6 7 8
0.6 1.0 4.0 0.8
22 21 29 24 34 22 27 28
F F F F M F F F
2 3 4 5
Duration of symptoms (years)
1.0 1.0 8.0 5.0
Clinical grade*
Anti-AChR titret
IIA III III IIA IIB IIA IIB IIB
5.0 22.3 23.7 32.8 187.0 258.0 1190.0 1740.0
463
Thymic histologyt Hyperplasia Hyperplasia Hyperplasia Hyperplasia Hyperplasia + Hyperplasia + Hyperplasia + Hyperplasia +
+ + + +
* Modified after Osserman and Genkins.2 IIA, mild; IIB, moderate; III, severe, generalized symptoms. t Measured as described by Scadding et al3 and expressed in nanomoles per liter. t As reported independently from the routine pathology department. § Nine months previously, this patient underwent an incomplete transcervical thymectomy.
et al'0). The structural elements of peripheral lymph nodes have also been defined, and not only the T- and B-cell distribution but also the fibronectin network and vasculature can now be studied with these antibodies. In this study we have analyzed the different subsets of thymic epithelial cells (subcapsular, cortical, and medullary) identified by these McAbs, as well as the extracellular matrix and the peripheral lymph-node-like features using both immunoperoxidase and double-immunofluorescence analysis. The aim has been the definition of characteristic micro-environments where autosensitization may occur in MG.
Materials and Methods Patients and Controls
MG Donors Eight MG thymectomy samples were studied, and the relevant clinical features of the patients are summarized
in Table 1. Four cases were selected for their prominent medullary hyperplasia and GCs. The others each showed two to four smaller GCs per section, plus substantial areas of GC-free medulla. There was no sign of thymoma in any. Controls Five thymic samples were obtained from fetuses aborted with prostaglandins at the 14th to 17th weeks of gestation (as determined by crown-rump length). The nonmyasthenic thymus donors also included 8 children aged 6-12 years and 7 adults aged 16-44, all undergoing cardiac surgery. Two of the juvenile specimens contained single small GCs in the medulla, which enabled us to study these structures in the non-MG thymus. Six samples of normal tonsil removed from patients treated with antibiotics for tonsillitis, and two mesenteric lymph node biopsies, taken during diagnostic studies for cancer but found to be uninvolved, served as controls.
Table 2-Immunologic Reagents for Analyzing Thymic Microenvironments Type Monoclonal antibodies or Cell types reactive of reagent hetero-antisera to HLA-DR
H
HLA-DR HTA-1 (NA1/34)
M
RFD-4
M
Keratin
H
HNK-1 (Leu-7) RFD-3
M M H
IgM Laminin Fibronectin
H H
M, monoclonal antibody; H, heterologous antiserum.
Selected references
Cortical epithelium, interdigitating reticular cells in the medulla
14
Cortical thymocytes, Langerhans cells, and some ID cells Medullary epithelium and subcapsular cortex (infant thymus) Medullary epithelium, Hassall bodies, and subcapsular epithelium strong staining; cortical epithelium weak staining Subcapsular cortex in fetus Follicular dendritic (FD) cells in germinal center Membrane immunoglobulin on B cells and antibody complexes in germinal center Compartmental boundaries, basement membrane Traffic areas in the T-cell zone, blood vessels (similar to reticulin pattern)
15, 29
This study 25 30 31 11 13 12
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Methods Preparation of Samples Tissue samples were divided in two. One part was processed for conventional histologic studies after formalin fixation, and the other was frozen and stored at -70 C until the sections were cut in a cryostat. These were air-dried for 1-4 hours, fixed in acetone for 30 minutes, lyophilized, and stored in sealed containers at -70 C until they were used for immunohistologic study.
Immunohistology The antibodies and antisera used are listed in Table 2. In essence, the McAbs reacted with 1) various thymic epithelial cells (subscapular, cortical, and medullary); 2) bone-marrow-derived dendritic cells (ID cells and Langerhans-type cells); and 3) cell types which are normally restricted to lymph nodes the follicular dendritic reticulum (FDR) cells. These reagents were culture supernatants used at 1:4 or 1:8 dilution as first layers on sections. Peroxidase-conjugated rabbit (R) anti-mouse (M) Ig second layers (Dakopatts; catalog number P260) were applied as described previously.1' These sections were counterstained with Haemalum. The same antibodies were also used in parallel indirect immunofluorescence studies; we used goat (G) anti-M Ig-TRITC (tetramethyl rhodamine isothiocyanate) as a second layer in combination with rabbit (R) -
antisera to cytokeratin (Dakopatts), fibronectin,'2 and
laminin,'3 followed by G anti-R IgG-FITC (fluorescein isothiocyanate) as a second layer, as described previously.'4 Two further combinations were also applied; M antibodies (using G anti-M Ig-FITC) were combined with a chicken (C) anti-human HLA-DR antibody (labeled with sheep anti-C Ig-TRITC). In some cases, antibodies were used together after conjugation with biotin and arsanilic acid (ars), respectively. The second layers for these were avidin-FITC and anti-ars-TRITC. Finally, goat anti-human IgM-FITC (Kallestad, catalog number 148) was used in a direct immunofluorescence assay to visualize B cells (membrane staining), plasma cells (cytoplasmic staining), and Ig complexes (lacy deposits). Immunofluorescence preparations were viewed in a Zeiss microscope with epifluorescence illumination.
Preparation and Testing of McAbs BALB/C mice were immunized by intraperitoneal injection of 106 thymic cells. The mice were boosted 8 days later by an intravenous injection of the same cell dose. Spleen cells, removed 4 days later, were fused with P3-NS1/l Ag4-1 myeloma cells with the use of PEG 1500. The fused cells were plated into Linbro plates at 106 cells/ml concentration. Hybrids were selected in HAT medium, and the culture supernatants from cultures were screened on sections of normal human tonsil, thymus, kidney, and skin. RFD4 strongly stained thymic medullary and subcapsular epithelium, as well as the basal layer of the epidermis and the tonsillar crypts, but was negative on all other cell types. After having been cloned twice by limiting dilution, the RFD4 clone has produced antibody for longer than 9 months.
Definition of Cortical and Medullary Areas The cortex and medulla were distinguished by immunohistologic study. In the cortex the lymphoid elements were tightly packed and contained >70%/6 terminal transferase-positive (TdT+) cells expressing thymocyte antigen (CD1, detected by NA1/34 OKT6like antibody"5), and strongly HLA-DR+ epithelial cells. 14 In the medulla >90% of lymphocytes were TdT-, NA1/34-, and interdigitating (ID) cells"4 as well as an epithelial population reacting with RFD4 antibody were also present (see Haynes et al16 and below). In addition, the perivascular space around the thymic vessels is extraparenchymal and separated from the parenchyma by a basal lamina.17'l8 These areas are known to contain, even in the normal thymus, a relatively increased number of Ig-containing B cells and plasma cells. 18
Results The Subcapsular Thymic Epithelium The subcapsular epithelium contained large amounts of cytokeratin but was clearly HLA-DR-negative in all samples tested (eg, Figure Ic, f, and i). In the control thymus it varied strikingly, both in thickness and in Leu7 and RFD4 staining patterns, according to donor age. In the fetus, subcapsular epithelial cells were strongly Leu-7-positive. Some of these cells penetrated deeply
Figure 1-Reactivity of subcapsular epithelium of the fetal (a, b, and c), child (3-year-old) (d, e, and f), and MG (adult Case 2) thymus (g, h, and I) with monoclonal antibodies Leu-7 (HNK-1 Ab; a, d, and g), RFD-4 (b, e, and h), and HLA-DR (c, f, and i). The subcapsular epithelium is Leu-7+, RFD4in the fetus, Leu-7-, RFD4+ in the child, and Leu-7+, RFD4+ in the adult (MG and control). It is also cytokeratin-positive (not shown), but HLA-DR- (asterisks in a-c and arrows in e and f). The cortical epithelium is Leu-7-, RFD4-, HLA-DR+. Sections b-c, e-f, and g-h were double-labeled by immunofluorescence with FITC or TRITC and photographed with selective filters; a and d are immunoperoxidase preparations. co, cortical areas; is, interlobular septa.
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RFD-4
Leu-7 (HINK-1)
.
.
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co II0.
I1
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Leu-7 (HNK-1)
is 9 S
S 9
co
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Figure 2-Expression of cytokeratin (a), RFD4 (b) and HLA-DR (c) on cortical epithelium in MG thymus (Case 3). The epithelial network is weakly cytokeratinpositive and strongly HLA-DR+ (eg, the lower part) but virtually RFD4-. Nevertheless, in some areas modest expression of RFD4 is seen (arrows) which is not observed in control thymus samples (eg, Figure le). In the inset in a the cortical thymocyte nuclei are strongly labeled by anti-terminal deoxynucleotidyl transferase (anti-TdT) antibody. co, cortical areas; is, interlobular septa.
into the thymus but were RFD4- (Figure la and b). In the children the subcapsular epithelium was Leu7-negative but strongly RFD4+ (Figure Id and e), an opposite of the fetal pattern. In addition to the different expression of these antigens, in children, as opposed to fetus, the subcapsular epithelial cells remained fully subcapsular and did not penetrate into the cortex. In normal adults the subcapsular epithelium was thick and now strongly expressed both Leu-7 and RFD4; in the MG thymic samples the same reactivity (Leu-7+, RFD4+) was observed (Figure Ig and h). Cortical Areas In the control thymus of all age groups, cortical thymocytes showed membrane/ring staining with NAI/34 (OKT6-like) antibody."5 In the infants and adults, but not in the fetuses,14 these contained the enzyme terminal deoxynucleotidyl transferase (TdT). In the adult samples a variable degree of involution was seen, but NA1/34+, TdT+ thymocytes were observed in all eight samples. The normal cortical epithelium was Leu7- (Figure Id), RFD4- (Figure le) and strongly HLADR+ (Figure If) in all age groups. In the MG thymus, there were lobules of cortex appended to an enlarged medulla; the cortex contained many NA1/34+, TdT+ thymocytes (Figure 2a, inset) enmeshed by the strongly HLA-DR+ epithelial cells. In a few areas, these showed a moderate increase in labeling for cytokeratin and RFD4. This is demonstrated in Figure 2a and b (arrows in cortical areas). The appropriate control in Figure le shows the lack of RFD4 posi-
tivity in the cortical areas. There was no evidence for cortical involution in these examples (results not shown). Medullary Epithelial Cells In the control medulla (at all ages) there were elongated "fishlike" cells which did not form an extensive branching network, but tended to collect in ductlike structures around Hassall bodies, particularly in the childhood thymus (Figure 3a). These cells had plentiful cytokeratin (Figure 3a) but were Leu 7-: double staining showed conclusively that these keratin-containing epithelial cells were HLA-DR- (Figure 3a and c). The positive labeling of medullary epithelial cells with RFD4 (but not with anti-HLA-DR) is shown with higher magnification in Figure 4a and b. Interspersed among the RFD4+, keratin' medullary epithelium very strongly HLA-DR+ ID cells were seen (Figures 3c and 4b), which were, on the other hand, totally RFD4- (Figures 3b and 4a) and cytokeratin-negative (Figure 3a). In some parts of the sections HLA-DR labeling can be abundantly extracellular and diffusely distributed, but again shows no relationship to RFD4 positivity and to the distribution of medullary epithelium. The appearance of the medulla in MG was relatively normal in some cases, but there were abundant GCs in others. When studied immunohistologically the medullary epithelial cells were hyperplastic in all 8 MG samples and retained the RFD4+ (Figures 3e and 4c), HLA-DR- (Figures 3f and 4d) and intensely keratinpositive phenotype (Figure 3d). In the more advanced
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Figure 3-Expression of cytokeratin (a and d), RFD4 (b and e), and HLA-DR (c and f) in medullary epithelium in the thymus of a control (12-year-old) (a-c) and of MG Case 2 (d-f). The medullary epithelial cells are cytokeratin-positive (a and d), RFD4+ (b and e), but HLA-DR- (c and f). Nonepithelial interdigitating cells in the medulla are RFD4- and strongly HLA-DR+. a and c and d-e are the same areas labeled with FITC and TRITC. In section f, double labeling (not shown) identified the edge (broken line) of the medullary epithelial area (M). gc, germinal centre; Hb, Hassall body; T, T-cell area.
cases (notably in Patients 2, 3, and 7) they accumulated in closely packed bands or "whorls," often with a multilayered "onionlike" appearance (Figure 3d and e). In these bands, there were also many T-lymphocytes and ID cells (Figure 3f).
The Distribution of Laminin In the control thymus, labeling for laminin (one constituent of basement membrane) was restricted to the sheaths running along the septa and to the blood vessels. No penetration into the substance of cortex or medulla was noticed (Figure 4a). In normal lymph nodes, too, no laminin boundaries were observed between the anatomically different areas, such as paracortical zones and GCs.
In contrast, in the MG thymus, a laminin-positive basement membrane ensheathed the medullary epithelial bands and separated them from the epithelium-free T-cell zones (Figure 5b; see also arrows in Figure 5d). This laminin layer became fenestrated only in circumscribed sites where the bands came closest to the GCs (eg, Figure 5c and d; see also below). T Cells and Fibronectin In control thymus fibronectin staining was likewise confined to the septa and blood vessels and appeared to be otherwise absent from the substance of the cortex and medulla (Figure 6a). By contrast, in MG, linear deposits of fibronectin formed a coarse network in the T-cell zones (Figure 6c). Intermingled with the
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Figure 4-Expression of RFD4 reactivity (a and c) and HLA-DR (b and d) in the normal thymus (a and b) and in MG thymus (c and d). The same areas were photographed with selective filters for FITC and TRITC. The medullary epithelial cells (asterisk) are "fishlike" cells with long protruding processes. These cells are strongly RFD4- but HLA-DR-. The ID cells (see arrows) exhibit "veils" and give a more "fluffy" impression when stained for HLA-DR (arrows). These cells are RFD4-. The dotted line is the corticomedullary boundary.
fibronectin were many T cells which were mainly OKT3+, NA1/34-, but included 2-5% of NA1/34+ thymocytes as well. These NA1/34+ (CD1+) cells with ringlike membrane staining contained TdT enzymes but expressed no HLA-DR. These lymphoid cells are also distinguishable by morphology from a NA1/34+ population of larger nonlymphoid cells which appear to be a small subset of the HLA-DR+ ID cells. Some Blymphocytes, abundant HLA-DR+ ID cells, and many blood vessels with high endothelium (bv in Figure 6c) were observed as well (see also Soderstrom et al9). This constellation is very similar to that in the paracortical areas of lymph node and tonsil (eg, Figure 5b), except that in the peripheral lymphoid tissues no NA1/34+ thymocytes are seen. These T-cell zones were not observed in any control thymic samples. Notably, these zones are absent around the GCs that are rarely seen in normal thymic tissue (see also below).
Germinal Centers and Their Environs Among the 17 control samples studied from children with no autoimmune disorders two contained a single GC each. These GCs had typical FDR cells labeled with the RFD3 antibody (Figure 7a). Deposition of extracellular IgM, a characteristic feature of GCs in peripheral lymph nodes, was also seen (Figure 7c). These GCs were embedded within normal medulla (M in Figure 7a), and no surrounding fibronectin- and T-cell-rich zones (Figure 7d) or laminin boundaries (Figure 7b) were detected. In the MG samples the GCs showed RFD3 and Ig staining similar to that in the lymph node samples (as also seen in Figure 4c and d). As mentioned above, the GCs in the MG thymus were surrounded by T-cell areas. The relationships of GCs with T-cell T zones and medullary epithelial bands (M) are summarized in the two
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Figure 5-Labeling of basement membranes with anti-laminin in a 12-year-old control thymus (a) and in MG thymus (b-d; Case 2). In the control (a), laminin is confined to blood vessels. The corticomedullary (C-M) junction and medulla are unstained. In MG (b) a thin laminin layer ensheathes the medullary epithelial bands (M; immunoperoxidase labeling). The panels of c show two sections co-stained for follicular dendritic cells (RFD3+ in the GCs) plus epithelial cells (RFD4- in M) using TRITC. In d the same sections are stained for laminin-FITC. Where the epithelial bands come close to the GCs, the laminin sheath is perforated (white line), permitting communication between T cells, medullary epithelial cells, GCs, and associated antigen presenting cells.
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Figure 6-Fibronectin deposition in a control (8-year-old) thymus (a), in a normal peripheral lymph node (b), and in MG Case 2 (c). In a fibronectin is confined to the interlobular septa (is) and the sheaths of blood vessels. In b and c, it is abundant in the paracortical T zone (T). The germinal centers (GC) are virtually unstained (except for blood vessels (bv)).
panels of Figure 5c and d and in Figure 8. The laminin boundaries encapsulating the M bands were broken up where the bands approached the GCs (large arrows). Here an open communication is possible (broken white lines) between Ms and GCs through the T zone. Finally, in one MG thymus (Patient 3) only two GCs were seen: one was surrounded by medullary epithelium (as seen in nonmyasthenic control thymus; Figure 7), and the other showed the features seen in MG (Figure 5c and d) and was surrounded by a fibronectin-rich T zone.
Discussion This study was made possible by the availability of McAbs that discriminate between the various populations of thymic epithelial cells16 (reviewed by Janossy et all') and between the accessory cells of the immune system (Table 2). The thymic cortical epithelium develops from the ectoderm of the third pharyngeal cleft19.20 and the medullary epithelium derives from the endoderm of the third pharyngeal pouch.20 The origin of the superficial subcapsular epithelium is unknown, and these cells' phenotypic features vary, depending
upon the age of the individual (Figure 1). These epithelial elements may have different functions and may also be involved in the developing myasthenic process. As indicated above and by the morphometric analysis of our samples (results not shown), in MG the thymic cortical epithelium and TdT+ NA1/34+ cortical thymocytes are both present, and the cortex does not appear to be involuted when compared with age-matched controls.2,22 These findings confirm the results of biochemical assays for TdT which also show that in MG this enzyme activity is maintained.23 In addition, NAl/34+ cortical thymocytes are observed in the medulla of MG thymus, suggestive of continued cortical output. The medullary epithelium in MG is hyperplastic (Figure 3; see also Henry7), and the production of thymic hormones is increased.8 We have now also established the HLA-DR negativity of medullary epithelium in MG. In the normal thymus, the lack of HLA-DR expression on medullary epithelial cells could only be ascertained by careful double-labeling studies using two-color immunofluorescence for RFD4 (red) and HLA-DR (green in Figures 3 and 4; see also Janossy et al10). This was because the HLA-DR staining on the RFD4- interdigitating reticular cells was very strong and appeared
Figure 7-Germinal center in a control infant thymus (patient is aged 6 and has no autoimmune disease). In a the section was co-stained for medullary epithelium (RFD4+; M) plus follicular dendritic cells (RFD3+; GC-both TRITC). The corresponding figure for MG is shown in Figure 5c. In b the same section is labeled for laminin (FITC). Another section was stained for IgM (in c, showing extracellular deposits) and for fibronectin (in d). The corresponding figure in MG thymus is in Figure 6c.
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Figure 8-Unique microenvironments of MG thymus. The thymic cortex is not involuted, and some cortical thymocytes apparently even reach deeper medullary areas. In the medulla a special microenvironment is formed by the juxtaposition of hyperplastic medullary epithelium (thymic feature), T-cell zone (7) with interdigitating cells (not shown, but see Figures 3f and 4d) and the dendritic reticulum of the germinal center (GC; lymph node features). The thymic and lymph node elements in these areas intermingle as the laminin-containing basement membranes are fenestrated (e,).
to diffuse into the immediate vicinity of these cells but did not coincide with membrane staining on RFD4+
medullary epithelial cells (Figures 3 and 4). In the MG samples, however, the medullary epithelium is more abundant, and the HLA-DR negativity of these cells can be readily established even with immunoperoxidase staining for HLA-DR alone. It is interesting to note that in inflamed tissue epithelial keratinocytes frequently express HLA-DR (reviewed by Lampert24), but this is clearly not the case in the MG medulla. Our analysis confirms and extends previous morphologic findings6,7"18 that in the myasthenic thymus lymphnode-like features are established which can be regarded as a dysplasia or intrusion by an essentially "alien" peripheral lymphoid tissue. Even in the most developed samples we studied, the hyperplastic compact bands of medullary epithelium were found to be largely (but not fully) separated by a laminin-containing basement membrane from the intruding peripheral lymph-node tissue. This confirms the ultrastructural observations of Levine and Rosai.'8 In the intruding tissue, T-cell zones containing channels marked by fibronectin deposition, and GCs with B-cell accumulation can be distinguished. The apparent absence of such T-cell zones around the rare GCs in control nonmyasthenic thymus suggests that the structurally well-developed T-cell traffic area may be more relevant to the development of MG than the GCs
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themselves. Bierman"7 and Levine and Rosai'8 have already pointed out that even in normal thymus the perivascular spaces are extraparenchymal. According to this view, which is confirmed in this study, the hyperplasia of the myasthenic thymus partly results from the expansion of this extraparenchymal tissue. The cell traffic between the intruding "perivascular" (lymphnode-like) bands and the hypertrophic medullary epithelial areas then becomes an important issue. Perhaps one of our most intriguing observations (Figure 5) is that at specific points the laminin-positive boundaries between the lymph-node-like areas and the medullary thymic cords break up, and the resulting composite micro-environment is unique to the myasthenic thymus. It is found neither in control thymus nor in normal lymph nodes (Figure 8). These areas are close to the tips of GCs, and the cell types observed in the vicinity include the following: 1) hyperplastic medullary epithelium, presumably synthesizing thymic hormones8'26; 2) interdigitating cells (HLA-DR+) and their Langerhans-type variants (HLA-DR+, NA1/34+, OKT6+), both of which may function as antigenpresenting cells to the nearby T-lymphocytes -these areas are rich in T cells and include some NA1/34+ cortical thymocytes; 3) B-lymphocytes scattered among T cells in the fibronectin-rich zones.6 Previous studies have shown many high endothelial venules,9 indicating active lymphoid traffic in this area. This environment might be particularly conducive to the initiation of immune responses to intact or altered self-antigens. In the mouse it appears that T cells in the medulla are normally Mel-14-negative and may be unable to recirculate27 (I. Weissman, personal communication). Hence, the medulla may not normally be a site of lymphocyte recirculation, and therefore the changes we now describe in MG may be important in the pathogenesis of this form of the disease. Myasthenia gravis is an antigen-specific immune deviation, although the exact nature of the relevant AChR determinants and their presence on other cell types is not yet known. Further studies are therefore in progress on their localization in these various thymic
microenvironments. Might there be some common origin for the multiple abnormalities in MG? One possibility is that AChR from thymic myoid cells' is taken up by ID cells and provokes developing T cells to react against it rather than become tolerant. These then initiate chain reactions, leading to entry of peripheral mature T and B cells, high endothelial venule formation, and the development of areas which resemble the T-cell zones in peripheral lymph nodes. Our view is that the development of GCs is a secondary phenomenon and perhaps a response to local polyclonal stimulation. Where the
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medullary epithelial cell hyperplasia belongs in this scheme is not clear. Could it be involved in the initial breaking of self-tolerance, or simply be a reaction to some of the other changes? A systematic study over the course of the disease is in progress; a study of very early cases may help to answer these questions.
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