Myasthenia Gravis Thymus Research Articles

Altered populations of natural killer cell and natural killer T cell subclasses in myasthenia gravis

Since the innate immune system can influence the disease activity of myasthenia gravis (MG), such as during infection, the frequencies of natural killer (NK) cells and NKT cells were analyzed in the blood and thymus. Before therapy (thymectomy plus glucocorticoid), the MG patients with thymic hyperplasia, but not those with thymoma, showed increased frequencies of mature NKT cells (CD3 +TCRV alpha24 +CD161 bright) in the blood, while the frequency of immature NKT cells was unaltered. In the blood of the patients with thymoma, but not those with hyperplasia, the frequency of cytotoxic subclass of NK cells (CD3 −CD16 +CD56 dim) was lower than that of the control. These alterations returned to normal after therapy. The thymic frequencies of NKT cells and NK cells in MG thymuses were unaltered. These results suggest the involvement of both innate and acquired immunity in the disease activity of MG.

Increased Toll-Like Receptor 4 Expression in Thymus of Myasthenic Patients with Thymitis and Thymic Involution

Thymic abnormalities are present in approximately 80% of myasthenia gravis (MG) patients, and the thymus seems to be the main site of autosensitization to the acetylcholine receptor. In view of findings that the innate immune system can generate an autoimmune response, we studied the expression of Toll-like receptors (TLRs) 2 to 5, key components of innate immunity signaling pathways, in 37 thymuses from patients with autoimmune MG. TLR4 mRNA levels were significantly greater in thymitis (hyperplasia with diffuse B-cell infiltration) and involuted thymus than in germinal center hyperplasia and thymoma. By immunohistochemistry and confocal microscopy, cells positive for TLR4 protein were rarely detected in thymoma. However, in thymitis TLR4 protein was mostly found on epitheliomorphic (cytokeratin-positive) cells located in close association with clusters of acetylcholine receptor-positive myoid cells in thymic medulla and also at the borders between cortical and medullary areas. B cells were never TLR4-positive. TLR4 protein was also present in remnant tissue of involuted thymus. This is the first finding of a possible link between innate immunity and MG. We speculate that in a subgroup of MG patients, an exogenous or endogenous danger signal may activate the innate immune system and give rise to TLR4-mediated mechanisms contributing to autoimmunity.

0293 Fas-Fas ligand system emergence and apoptosis in the thymus of myasthenia gravis - Homology among nAChR, fas and T cell epitopes

0293 Fas-Fas ligand system emergence and apoptosis in the thymus of myasthenia gravis - Homology among nAChR, fas and T cell epitopes

Dendritic cells in hyperplastic thymuses from patients with myasthenia gravis.

To investigate the role of dendritic cells (DCs) in the hyperplastic myasthenia gravis (MG) thymus, we studied the frequency and distribution of three mature DC phenotypes (CD83(+)CD11c(+), CD86(+)CD11c(+), and HLA-DR(+)CD11c(+)) in samples from patients with MG whose symptoms dramatically improved following thymectomy and in non-MG control thymuses. In hyperplastic MG thymuses, mature DCs were much more numerous in nonmedullary areas, such as the subcapsular/outer cortex; around the germinal centers; and in extralobular connective tissue, particularly around blood vessels. Mature DCs strongly coexpressed CD44 and appeared to be components of a CD44-highly positive (CD44(high)) cell population migrating from the vascular system. Furthermore, in the hyperplastic MG thymus, the expression of secondary lymphoid-tissue chemokine (SLC) markedly increased especially around extralobular blood vessels, where the CD44(high) cell population accumulated. These findings suggest that DCs may migrate into the hyperplastic thymus from the vascular system via mechanisms that involve CD44 and SLC. DCs may present self-antigens, thereby promoting the priming and/or boosting of potentially autoreactive T cells against the acetylcholine receptor.

NKT-associated markers and perforin in hyperplastic thymuses from patients with Myasthenia gravis.

Immunohistochemical expression of natural killer T (NKT) cell-associated markers (Valpha24 and CD56) and perforin in relation to CD44-highly positive (CD44(high)) cells was studied in hyperplastic thymuses from patients with myasthenia gravis (MG) whose symptoms dramatically improved after thymectomy and compared with non-MG control thymuses. In the control thymuses, Valpha24-positive (Valpha24(+)) and CD56-positive (CD56(+)) cells were sparsely distributed in the medullary area only. In contrast, in hyperplastic MG thymus, Valpha24(+) and CD56(+) cells were more frequent in connective tissue, appeared to have penetrated the thymic parenchyma, and most coexpressed CD44(high). Perforin-positive cells were not present in the control thymus, but were in the connective tissue and perilobular cortical areas in the hyperplastic MG thymus. Most of these perforin-positive cells were CD44(high) and were located near blood vessels. They appeared to have migrated directly from the vascular system and penetrated the thymic parenchyma. Some perforin-positive cells coexpressed Valpha24, CD56, or both. These findings suggest that in this particular type of MG thymus, NKT-like cells may have increased via a CD44- and perforin-mediated mechanism, leading to an imbalance in the immune system that favored an antibody-mediated autoimmunity against the acetylcholine receptor.

Decreased expression of c- myc family genes in thymuses from myasthenia gravis patients

The thymus is a critical organ for the elimination of autoreactive T cells by apoptosis. We studied the expression of apoptosis-associated genes, bcl-xL, bad, caspase-3, and c- myc family genes in myasthenia gravis (MG) thymuses. We observed that the mRNA levels of myc family genes, c- myc and max, were markedly reduced in MG thymuses. These results indicate that c- myc-mediated signaling is abnormal in MG thymuses. The levels of molecules whose expressions are associated with myc, such as STAM, prothymosin-alpha, and NFkappaB, were also analyzed.

True epithelial hyperplasia in the thymus of early-onset myasthenia gravis patients: implications for immunopathogenesis

The early-onset myasthenia gravis (EOMG) thymus shows characteristic medullary epithelial bands (MEB), greatly expanded perivascular infiltrates and fenestrations of the intervening basement membranes. We now compare epithelial expression of epidermal growth factor receptor (EGFR) and many integrins in EOMG and control samples. The main differences are striking/consistent thickening (in MEB) of what is normally a monolayer of perivascular epithelium, with focal protrusion into the infiltrates. This evidently hyperplastic epithelial subpopulation also strongly expresses EGFR and certain integrins. We suggest that its enhanced interactions with the locally increased extracellular matrix protein deposits may play an important role in autosensitization.

Expression of OX40 and OX40 ligand (gp34) in the normal and myasthenic thymus

To examine the expression of OX40, an activated memory T-cell marker, and its ligand (OX40L), a set of molecules for T-cell-B-cell interaction, and other lymphocyte activation markers in the thymuses of myasthenia gravis (MG) and controls. We studied the expression of OX40, OX40L, IL-2Ralpha and HLA-DR in the thymic tissues of MG and controls using immunocytochemistry and flowcytometry. In both hyperplastic thymus of MG and control thymus, OX40+ cells were scattered mainly in the medulla with much fewer OX40L+ cells being distributed in the corticomedullary junctions. IL-2Ralpha and HLA-DR were expressed in the medulla at higher frequencies as compared with OX40 in controls as well as MG. In contrast, the numbers of OX40+ cells around the germinal centers (GC) were significantly greater than those of control thymuses, and some mononuclear cells in GC were OX40L+. A considerable number of OX40+ cells were seen in the thymic tissues adjacent to thymomas. OX40+ cells were CD4+ CD8- or CD4+ CD8+ and were mostly HLA-DR-. (The coexpression of OX40 and IL-2Ralpha on activated CD4+ T cells was previously reported.) OX40, expressed in a fraction of activated CD4+ T cells, may be upregulated in thymic tissues adjacent to GC and thymoma in MG, and OX40 may interact with OX40L in GC to enhance anti-acetylcholine receptor antibody production in MG.

Marked increase in CD44-highly positive cells in hyperplastic thymuses from patients with myasthenia gravis

To investigate the role of the thymus in the pathogenesis of myasthenia gravis (MG), immunohistochemical expression of CD44, CD45R0, B7-1, and IL-2 was studied in: (1) hyperplastic thymuses of patients with MG whose symptoms markedly improved after thymectomy, (2) remnant thymuses of patients with MG whose symptoms did not respond to thymectomy, and (3) non-MG control thymus. Lymphocytes strongly expressing CD44, a marker for homing lymphocytes and activated memory lymphocytes in adults, were much more frequently observed in hyperplastic MG thymuses than in remnant thymuses and non-MG control thymuses. These CD44-highly positive cells in hyperplastic MG thymuses were for the most part located in the subcapsular and cortical areas but also occasionally in medullary areas. Some of these CD44-highly positive cells coexpress CD45R0. CD44-highly positive cells were located in the vicinity of blood vessels and thus appeared to have migrated directly from extralobular blood vessels. B7-1-positive cells and interleukin (IL)-2-positive cells were also more abundant in the MG patients than in controls and were localized in the proximity of CD44-highly positive cells. These findings suggest that mature T and B cells recirculate into hyperplastic MG thymus via CD44-associated mechanisms and are activated there.

Marked increase in CD44-highly positive cells in hyperplastic thymuses from patients with Myasthenia gravis.

To investigate the role of the thymus in the pathogenesis of myasthenia gravis (MG), immunohistochemical expression of CD44, CD45R0, B7-1, and IL-2 was studied in: (1) hyperplastic thymuses of patients with MG whose symptoms markedly improved after thymectomy, (2) remnant thymuses of patients with MG whose symptoms did not respond to thymectomy, and (3) non-MG control thymus. Lymphocytes strongly expressing CD44, a marker for homing lymphocytes and activated memory lymphocytes in adults, were much more frequently observed in hyperplastic MG thymuses than in remnant thymuses and non-MG control thymuses. These CD44-highly positive cells in hyperplastic MG thymuses were for the most part located in the subcapsular and cortical areas but also occasionally in medullary areas. Some of these CD44-highly positive cells coexpress CD45R0. CD44-highly positive cells were located in the vicinity of blood vessels and thus appeared to have migrated directly from extralobular blood vessels. B7-1-positive cells and interleukin (IL)-2-positive cells were also more abundant in the MG patients than in controls and were localized in the proximity of CD44-highly positive cells. These findings suggest that mature T and B cells recirculate into hyperplastic MG thymus via CD44-associated mechanisms and are activated there.

Leukemia inhibitory factor, oncostatin M, IL-6, and stem cell factor mRNA expression in human thymus increases with age and is associated with thymic atrophy.

The roles that thymus cytokines might play in regulating thymic atrophy are not known. Reversing thymic atrophy is important for immune reconstitution in adults. We have studied cytokine mRNA steady-state levels in 45 normal human (aged 3 days to 78 years) and 34 myasthenia gravis thymuses (aged 4 to 75 years) during aging, and correlated cytokine mRNA levels with thymic signal joint (sj) TCR delta excision circle (TREC) levels, a molecular marker for active thymopoiesis. LIF, oncostatin M (OSM), IL-6, M-CSF, and stem cell factor (SCF) mRNA were elevated in normal and myasthenia gravis-aged thymuses, and correlated with decreased levels of thymopoiesis, as determined by either decreased keratin-positive thymic epithelial space or decreased thymic sjTRECs. IL-7 is a key cytokine required during the early stages of thymocyte development. Interestingly, IL-7 mRNA expression did not fall with aging in either normal or myasthenia gravis thymuses. In vivo administration of LIF, OSM, IL-6, or SCF, but not M-CSF, i.p. to mice over 3 days induced thymic atrophy with loss of CD4+, CD8+ cortical thymocytes. Taken together, these data suggest a role for thymic cytokines in the process of thymic atrophy.

Analysis of the human thymic perivascular space during aging.

The perivascular space (PVS) of human thymus increases in volume during aging as thymopoiesis declines. Understanding the composition of the PVS is therefore vital to understanding mechanisms of thymic atrophy. We have analyzed 87 normal and 31 myasthenia gravis (MG) thymus tissues from patients ranging in age from newborn to 78 years, using immunohistologic and molecular assays. We confirmed that although thymic epithelial space (TES) volume decreases progressively with age, thymopoiesis with active T-cell receptor gene rearrangement continued normally within the TES into late life. Hematopoietic cells present in the adult PVS include T cells, B cells, and monocytes. Eosinophils are prominent in PVS of infants 2 years of age or younger. In the normal adult and the MG thymus, the PVS includes mature single-positive (CD1a(-) and CD4(+) or CD8(+)) T lymphocytes that express CD45RO, and contains clusters of T cells expressing the TIA-1 cytotoxic granule antigen, suggesting a peripheral origin. PBMCs bind in vitro to MECA-79(+) high endothelial venules present in the PVS, suggesting a mechanism for the recruitment of peripheral cells to thymic PVS. Therefore, in both normal subjects and MG patients, thymic PVS may be a compartment of the peripheral immune system that is not directly involved in thymopoiesis.

Acetylcholine receptor alpha subunit mRNA expression in human thymus: augmented expression in myasthenia gravis and upregulation by interferon-gamma.

Previous studies by us and others have demonstrated the expression of acetylcholine receptors on epithelial cells in the thymus of myasthenia gravis (MG) and control subjects. In the present experiments, we used a reverse transcription-polymerase chain reaction (RT-PCR) to analyze the profile of the two major isoforms of the alpha chain of these receptors (AChRalpha), P3A- and P3A+, in thymus tissue obtained from MG and control subjects and a human thymic epithelial cell line (TEC9). In addition, using a semiquantitative RT-PCR, we compared the amounts of P3A- and P3A+ mRNA expressed in thymic tissue obtained from these two sources and determined if their expression in TEC9 is modulated by cytokines. We found that mRNAs encoding P3A- and P3A+ are expressed at approximately a 5:1 ratio in both MG and control thymus tissue. This contrasts with skeletal muscle where mRNAs encoding these isoforms are expressed equally. A pattern of preferential P3A- vs P3A+ mRNA expression was also observed in TEC9. We observed 2.8-fold greater expression of both isoforms in MG than in control thymus. Expression of both isoforms in TEC9 was enhanced significantly by treatment with interferon-gamma whereas IL-1alpha, IL-4, and IL-6 had no effect. Thus, there is differential regulation of AChRalpha variants in thymus and TEC relative to muscle and interferon-gamma represents a novel regulator of AChRalpha mRNA expression. MG thymus is distinguished by increased expression of both isoforms of this autoantigen, a finding that may reflect enhancement of transcription by local microenvironmental factors.

Functional Expression of Receptors for Calcitonin Gene-Related Peptide, Calcitonin, and Vasoactive Intestinal Peptide in the Human Thymus and Thymomas from Myasthenia Gravis Patients

The molecular and functional expression of serpentine membrane receptors for vasoactive intestinal peptide (VIP), calcitonin gene-related peptide (CGRP), and calcitonin (CT) were characterized in human thymus and thymomas from myasthenia gravis (MG) patients and thymic epithelial cells either in primary culture (PTEC) or transformed by the simian virus 40 large T (SV40LT) oncogene (LT-TEC). Using RT-PCR combined with Southern analysis, we identified the PCR products corresponding to the receptor (-R) transcripts for VIP, CGRP, and CT in thymus from control subjects and MG patients with either hyperplasia or thymoma. Similar expressions of the VIP- and CGRP-R transcripts were observed in PTEC, whereas the CT-R message was not detected. In LT-TEC, the signals for VIP-R, CGRP-R, and CT-R transcripts were seen with a lower intensity than those in control and MG thymus. In agreement with our molecular analysis, 1) VIP was the most potent peptide among VIP-related peptides (VIP > PACAP > PHM > PHV) to stimulate cAMP production through specific type 1 VIP receptors in both PTEC and LT-TEC; 2) cAMP generation was induced by CGRP in PTEC and by CT in LT-TEC; 3) in frozen thymic sections and by flow cytometry, type 1 VIP-R, CGRP-R, and CT-R were localized in epithelial cells; and 4) in parallel, the transcription of the acetylcholine receptor alpha subunit (the main autoantigen in MG) was induced by CGRP and CT in PTEC and LT-TEC, respectively. Our data suggest that the neuroendocrine peptides VIP, CGRP, and CT may exert functional roles during MG and malignant transformation of the human thymus.

An acetylcholine receptor alpha subunit promoter confers intrathymic expression in transgenic mice. Implications for tolerance of a transgenic self-antigen and for autoreactivity in myasthenia gravis.

Myasthenia gravis (MG) is an autoimmune disease targeting the skeletal muscle acetylcholine receptor (AChR). Although the autoantigen is present in the thymus, it is not tolerated in MG patients. In addition, the nature of the cell bearing the autoantigen is controversial. To approach these questions, we used two lineages of transgenic mice in which the beta-galactosidase (beta-gal) gene is under the control of a 842-bp (Tg1) or a 3300-bp promoter fragment (Tg2) of the chick muscle alpha subunit AChR gene. In addition to expression in muscle cells, thymic expression was observed in both mouse lines (mainly in myoid cells in Tg1 and myoid cells and epithelial cells in Tg2). After challenge with beta-gal, Tg1 mice produced Th2-dependent anti-beta-gal antibodies, while Tg2 mice were almost unresponsive. By contrast, in a proliferation assay both Tg lines were unresponsive to beta-gal. Cells from Tg1 mice produce Th2-dependent cytokine whereas cells from Tg2 mice were nonproducing in response to beta-gal. These data indicate that the level of expression in Tg1 mice could be sufficient to induce tolerance of Th1 cells but not of Th2 cells, while both populations are tolerated in Tg2 mice. These findings are compatible with the hypothesis that AChR expression is not sufficiently abundant in MG thymus to induce a full tolerance.

Respective role of thymus and muscle in autoimmune myasthenia gravis.

Respective role of thymus and muscle in autoimmune myasthenia gravis.

Muscle nicotinic acetylcholine receptor mRNA expression in hyperplastic and neoplastic myasthenia gravis thymus.

Muscle nicotinic acetylcholine receptor mRNA expression in hyperplastic and neoplastic myasthenia gravis thymus.

Thymic myoid cell turnover in myasthenia gravis patients and in normal controls.

Thymic myoid cells (TMC) are sparse muscle-like cells in the thymic medulla, which are believed to trigger the autoimmune response in myasthenia gravis (MG). Ultrastructural investigations have revealed mature, degenerating, and immature TMC, but the number of TMC in MG patients does not differ from that in controls. We examined the turnover of TMC at the subcellular level, performing an immunocytochemical study with muscle-specific anti-desmin labelling of 10 thymuses derived from MG patients with lymphofollicular hyperplasia and from 8 normal controls. All thymuses examined revealed mature, immature, and degenerating TMC. Mature TMC contained desmin filaments in between Z-discs provided the sarcomeres were arranged in register. Morphological features of degenerating TMC included hypercontracted sarcomeres, cytoplasmic granular debris, chromatin clumping and, occasionally, membrane-bound bodies. Macrophages were not involved in the process. Immature TMC were of small diameter and contained myofilaments not arranged in myofibrils. In an MG thymus, small immature TMC were found clustered with dying TMC. It may be that degeneration of TMC is a stimulus for the generation of new TMC with faster turnover. This mechanism may mean that more antigen is available in MG patients than in normal controls, despite constant TMC numbers.

Expression of CD23 in the germinal center of thymus from myasthenia gravis patients

In order to investigate a pathogenic role of germinal centers which appear in the hyperplastic thymus of myasthenia gravis (MG) patients, we performed an immunohistochemical study using various monoclonal antibodies including CD23. In contrast with tonsilar germinal centers from non-MG individuals, CD23 was strongly and diffusely expressed in the whole area of germinal centers of MG thymi, including the outer zone. In addition, we measured the serum level of soluble CD23 (sCD23) in MG patients at various clinical stages. The high serum sCD23 levels, which were noted in the unthymectomized patients, fell to within normal range over 5 years after thymectomy, and the decline of serum sCD23 correlated well with clinical improvement. CD23 is thought to be responsible for preventing unselected germinal center B cells from entering apoptosis and, in turn, leads to the survival of auto-reactive B cell clones.

Failure to down-regulate Bcl-2 protein in thymic germinal center B cells in myasthenia gravis.

The most unusual characteristic of myasthenia gravis (MG) is that the thymus has germinal centers (GC). Cultured thymic lymphocytes from MG patients spontaneously produce anti-acetylcholine receptor antibodies, indicating that autoreactive B cells have escaped negative selection. To investigate the underlying mechanism, we examined the expression of the apoptosis-related protein Bcl-2 in GC B cells (defined as CD19+ CD38+ cells) in the thymus in 14 MG patients using three-color flow cytometry. GC in MG patients did not show the normal down-regulation of Bcl-2 (the frequency of Bcl-2+ GC B cells in the MG thymus and in control tonsils 54.3 +/- 16.2% versus 20.6 +/- 8.0%; mean +/- SD. p < 0.0001). In contrast, Bcl-2 in GC in the mediastinal lymph nodes from four patients was down-regulated to a relatively normal level. Using the terminal deoxynucleotidyl transferase-mediated dUTP-biotin nick end-labeling (TUNEL) method to detect DNA fragmentation in situ, the frequency of TUNEL+ cells in GC in the MG thymus was lower than in control tonsils. These results suggest that autoreactive B cells which normally undergo apoptosis in GC may survive because of Bcl-2 up-regulation in this unusual location.