N. Willcox

Immunological Heterogeneity and Cellular Mechanisms in Myasthenia Gravis

The clinical and immunological heterogeneity that characterizes myasthenia gravis (MG) has implications for etiology, pathogenetic mechanisms, cellular interactions, and treatment. Moreover, it raises the important question of whether MG is a “single” disorder, attributable exclusively to autoantibodies directed against the postsynaptic nicotinic acetylcholine receptor (AChR). In this paper we present evidence that antibodies to determinants other than AChR may impair neuromuscular transmission in some MG patients, show data indicating a diversity of genetic factors influencing susceptibility to MG, and describe recent immunohistological and functional studies of the thymus and of thymic cells.

The thymus in seronegative myasthenia gravis patients.

SummaryIn 5–10% of all patients with typical generalised myasthenia gravis (MG), serum antibody to the acetylcholine receptor (AChR) is undetectable. To determine whether these represent a distinct subgroup, we have compared the thymuses of 14 seronegatives, 70 seropositives and 12 non-myasthenic controls. By quantitative immunohistology on coded sections, the 7 seronegative samples were clearly distinguishable from the controls by the pronounced lymph node-type T-cell areas in the medulla. While these closely resembled those in the seropositive cases, germinal centres were significantly sparser, and total in vitro IgG production was disproportionately low (per B cell) in the 12 cases tested. Furthermore, specific anti-AChR production was never detected in any of these cultures. The data support the view that the medullary T-cell areas are the most consistent abnormalitiy in the MG thymus (though it may not be primary), and they strongly imply that seronegative and seropositive MG are distinct entities.

ANTI‐ACETYLCHOLINE RECEPTOR ANTIBODY SYNTHESIS BY CULTURED LYMPHOCYTES IN MY ASTHENIA GRAVIS: THYMIC AND PERIPHERAL BLOOD CELL INTERACTIONS*

The thymus has long been thought to be involved in the pathogenesis of myasthenia gravis (MG) . Associated histological abnormalities have been recognized since the turn of the and thymectomy has been claimed to benefit some patienb4s5 The part played by the thymus in the disease process is, however, not yet understood. There is general agreement that the principal end-plate abnormality in MG is a decreased number of acetylcholine receptors 6, (AChRs) and that the anti-AChR antibody is directly implicated in the receptor Several lines of evidence suggest that the thymus might be concerned in this auto-antibody response. First, antigen appears to be present. The rat thymus contains small amounts of AChR,10 as shown by immunoprecipitation of the lZ5I-alphabungarotoxin (a-BuTx) labelled receptor using experimentally raised anti-AChR antibodies. Thymic epithelial cells appear to bear AChR l1 and myoid (musclelike) cells grown out in tissue culture from rat and human thymus express AChR.l* Immunofluorescence studies using experimental autoimmune MG sera have shown binding to about 80% of mouse thymocytes, suggesting the presence of an AChR-like antigen on the surface of these cells, although they do not bind a-BuTx.13 There is also evidence that the thymus is a site of antibody production. Thymic germinal centers contain cells exhibiting B cell l5 Mittag et d.16 found that MG thymus contained anti-AChR IgG, and wex7 previously reported that thymic cells from MG patients can spontaneously synthesize anti-AChR antibody in culture, In this presentation, we will describe the characteristics of anti-AChR production by thymic cells and by peripheral blood lymphocytes (PBL) in MG patients, and will show that thymus cells can enhance anti-AChR production by autologous PBL.

Genetic Susceptibility to Myasthenia Gravis Studied by DNA Hybridization

The susceptibility to myasthenia gravis (MG) is known to be associated with certain genetic polymorphisms. These include major histocompatibility complex (MHC) antigens and in certain populations immunoglobulin (Ig) heavy chain allotypes (Gm). To further investigate the immunogenetic background of MG, we have utilized the Southern blotting technique in conjunction with DNA probes that recognize various genes intricately involved in the immune response, that is, Ig constant region genes and T-cell receptor (TCR) genes. In this study we have used a probe that recognizes the Ig C p gene switch region (Sp). This probe detects restriction fragment length polymorphisms (RFLP) on either side of the gamma-chain genes (which code for Gm allotypes). Using the restriction endonuclease Sacl in conjunction with this probe, RFLPs are detected within the Sp region. TABLE 1 shows the incidence of these RFLPs in MG and the Lambert-Eaton myasthenic syndrome (LEMS). In the MG patients (both thymoma and nonthymoma), there was a significant association between the incidence of the homozygote 2.6-kilobase (kb) phenotype and normal healthy controls (50% versus 18.4%, respectively, p = 0.001). In addition, this phenotype was associated with the LEMS patients (70% versus 18.4%, p = 0.002). No overall difference was found between either thymoma and nonthymoma MG patients, or the age onset of disease (patients developing the disease after the age of 40 were categorized as “old onset,” and those before as “young onset”). Patients were also investigated for TCR-constant beta-chain polymorphisms using the restriction endonuclease BglII. This probe and enzyme combination has previously been shown to detect RFLPs within this region. Overall, no significant association was found between either MG, LEMS, or any of the MG subgroups, although the 10.Q9.2-kb phenotype was increased in each of the groups.

Isolation of Germinal Centre (Gc) Cells is Greatly Improved by using the Protease Dispase to Prepare Cell Suspensions

The characteristic muscle weakness in myasthenia gravis (MG) is caused by autoantibodies to the acetylcholine receptor (AChR) of the motor endplate. They can be measured in a very sensitive radio-immunoassay (using detergent extracts of muscle, labelled with l25I-a-bungarotoxin1). There appears to be a special relationship with the thymus in MG. About 10% of cases have thymoma, and, in about two thirds of the remainder, there is thymic hyperplasia with medullary germinal centres (GC, reviewed in 1). Cell suspensions prepared by chopping and sieving these thymi (THYconv) spontaneously synthesise anti-AChR antibodies in culture.1 That is greatly increased if the tissue is dispersed instead by using the proteases collagenase and, especially, dispase2 (THYC+D) Dispase is a neutral protease that is apparently essential for isolating mouse follicular dendritic (FD) cells.3 We now report experiments probing the histological and functional basis of this striking bonus.

IMMUNOPATHOLOGICAL STUDIES ON LYMPHOCYTE SUBPOPULATIONS INVOLVED IN AUTOANTIBODY PRODUCTION IN MY ASTHENIA GRAVIS

We have studied the frequencies of B-lymphocytes and of T-cell subsets in blood and thymus of myasthenia gravis patients by immunofluorescent staining. We find striking variability in the ratios of T-lymphocytes bearing the OKT4 “helper”/ “inducer,” and the OKT8 “suppressor”/ “cytotoxic” markers. In three of our ten cases, there appeared to be an increase in “suppressor” T-cells in the blood, and only two cases showed an obvious decrease. The ratio of “helpers” to “suppressors” did not correlate with the production of anti-AChR antibody by blood, thymic or lymph node lymphocytes in culture. The ratio also failed to predict total IgG production in response to pokeweed mitogen, though most low responders did have a low ratio. However, when irradiated thymocytes were cocultured with autologous blood lymphocytes, they enhanced the production of anti-AChR antibody by the latter, and this enhancement did correlate with the “helper”/“suppressor” ratio in the thymus in the seven informative cases. That may imply the presence of an activated helper cell type in these thymuses, and/or of AChR-like material. In tissue sections of thymus from nine myasthenics, we have studied the disposition of cells bearing the cortical thymocyte antigen HTA-1, Ia antigen, or immunoglobulins M, D, G or A. In six cases, the medulla was markedly expanded at the expense of the cortex (which was packed with HTA-1 positive thymocytes), and the extent of this distortion reflected to some extent the duration/severity of the disease. Solitary B-cells were seen in the cortex in all cases, and medullary B-cell follicles in eight out of nine. The extent of these B-cell follicles, and the number of plasma cells in the germinal centers and interlobular septa again correlated with disease duration/severity. When germinal centers occurred they were typical of those in other lymphoid tissues, with a surrounding corona of B-cells (? memory cells) bearing IgM and IgD. The B-cells showed a normal (polyclonal) ratio of about 2 ~ : l A. Finally, large irregular, strongly Ia positive cells, the interdigitating reticular cells, were often prominent or increased in the medulla of the myasthenic thymuses. In conclusion, there was greater B-cell and plasma cell involvement in the thymus in late or severe myasthenia: the lesser extent of these changes in early cases suggests that they do not play a primary r61e in the pathogenesis of the disease.