J Neurol (199:1) 238:256-261

Journal of

Neurology © Springer-Verlag 1991

The thymus in seronegative myasthenia gravis patients N. Wiilcox 1'3, Myriam Schluep 13,,, M.A. Ritter 4, and J. Newsom-Davis 1'2'3 1Department of Neurological Science, Royal Free Hospital School of Medicine, and 2National Hospital for Nervous Diseases, London, UK 3Neurosciences Group, Institute of Molecular Medicine, John Radcliffe Hospital, University of Oxford, Headington OX3 9DU, UK 4Department of Immunology, Royal Postgraduate Medical School, DuCane Road, London, UK Received March 28, 1990 / Received in revised form November 2, 1990 / Accepted November 12, 1990

Summary. In 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. Key words: Myasthenia gravis thymus - Thymic hyperplasia - Thymitis - Seronegative myasthenia gravis H u m a n lymphocyte cultures

Introduction Most patients with generalised myasthenia gravis (MG) symptoms have serum autoantibodies to the acetylcholine receptor (AChR) of striated muscle that can be readily detected in a very sensitive radioimmunoassay [8]. Their titres vary greatly and correlate only moderately with the clinical state [2, 16], even though these antibodies are known to cause the patients' weakness by reducing the number of functional AChRs at the motor endplate [3]. * Present address: Department de Neurologie, CHUV, CH-1011 Lausanne, Switzerland Offprint requests to." N. Willcox, Neurosciences Group. Institute of Molecular Medicine, John Radcliffe Hospital, University of Oxford, Headington OX3 9DU. UK

Where the myasthenia is restricted to the extraocular muscles, anti-AChR levels tend to be low (60% of cases) or within the background (40%, [18]). They are also undetectable in 5-10To of patients with (sometimes severe) generalised symptoms whose myasthenia is otherwise typical both electrophysiologically and clinically, though perhaps with some bias towards bulbar and respiratory involvement (N.Arden, A. Vincent, J. Newsom-Davis, in preparation). When immunoglobulin from these patients' plasma is transferred to mice, it causes an increased curare sensitivity at the motor endplate with no loss of AChRs [9]. Thus pathogenic autoantibodies are apparently responsible, even though they are not detected in the radioimmunoassay. In addition, both plasma exchange and immunosuppressive drugs often prove beneficial in this form of myasthenia [10]. In typical seropositive MG, there may be a special relationship with the thymus; about 10% of patients have a thymoma, whereas most cases of young onset ( < 4 0 years) show medullary "thymitis"/"hyperplasia" which is rare in controls. It includes the presence of germinal centres (GC) and T-cell areas, both very similar to those seen normally in peripheral lymphoid tissues [1, 14]. Furthermore, when cell suspensions from these thymuses are cultured, they spontaneously synthesise anti-AChR antibodies at rates that correlate strongly with the serum titre [10, 12, 14, 20]. At the same time, larger amounts of total IgG are produced spontaneously, and these in turn are greatly increased by culture with pokeweed mitogen (PWM) [14, 20]. In a recent survey of 40 cases, we concluded that this IgG production correlated best with GC frequencies, which varied unpredictably between cases, whereas the anti-AChR was apparently made mainly by plasma cells in the T-cell areas that seemed to be the more widespread and MG-specific abnormality [14]. It is important to establish whether the seronegative myasthenics belong to the same spectrum as the typical cases, or form a distinct subpopulation. As they are rare and perhaps heterogeneous, it is hard to draw firm conclusions from immunogenetic studies. However, we now report thymic cell marker and culture data from 12 such cases, and immunohistological findings in 7; both corn-

257 p a r i s o n s s t r o n g l y s u g g e s t a d i s t i n c t i o n f r o m all s e r o p o s i t i v e cases.

Patients, materials and methods

Patients

epithelial cell subsets, to cortical and mature T cells, to B cells and follicular dendritic (FD) cells and to H L A class II, fibronectin and keratin as in Bofill et al. [1] and Schluep et al. [14]. The panel of sections from each seronegative and control case was coded and examined blind by a single observer (N.W.), who scored each of the main abnormalities as described by Schluep et al. [14] (see Results). In addition, a second observer (M.S.) counted the number of GC per measured area of tissue on the sections labelled for FD and B cells [14].

Thymus was available from 14 patients with clinical features of generalised MG (Table 1), a positive response to intravenous edrophonium and undetectable serum anti-AChR antibodies in repeated assays (< 0.2nmolll). Only 2 were even marginally seropositive when re-tested months or years later (Table 1). They had all been maintained on anti-cholinesterases prior to thymectomy: only cases 9 had received prednisolone (30 mg alt. die) for the 2 previous weeks, with apparently minimal effects on the thymus (cf. [211). We compare their results below with those from all the nonimmunosuppressed seropositive MG cases we have studied similarly [1, 10, 12-14, 17, 20, 21]. For our immunohistological study [14], 40 of these had been selected to cover a full range of clinical parameters (serum anti-AChR titre, duration of symptoms, age at thymectomy). They included 14 with durations less than 11 months; of the 9 with the lowest titres (2.4-5.8 nmol/1), 2 had very long durations (17 and 25 years) but all were otherwise similar [14]. Samples of control thymus were taken from non-myasthenic subjects undergoing cardiothoracic surgery.

About 85-90% of each entire MG thymus was disrupted mechanically (THYconv) in 4 cases; in the other 8, about 30% of it was dispersed instead with the proteases collagenase plus dispase (THYc+D) [20]. Total cell yields were determined by a single observer. Cells were cultured in 0.2 ml RPMI/15% fetal calf serum, and supernatants sampled and assayed for the total IgG and anti-AChR antibody produced per 106 cells per 7 days [20]. For marker studies, viable cells were labelled in suspension with mAbs to immature cortical (CD1 +) T cells and to H L A class II + cells by indirect immunofluorescence, and analysed on an Ortho Systems 50 Fluorescence-Activated Cell Sorter, or occasionally by fluorescence microscopy by a single observer [20].

Immunohistology

Immunohistological abnormalities

About 5% of the thymus from 12 controls and 7 randomly selected seronegatives (cases 1, 3, 6, 7, 11, 12, 14) was snap-frozen in two blocks in liquid nitrogen; 5-gm cryostat sections were subsequently prepared from them, fixed and stained by indirect immunoperoxidase using the same monoclonal antibodies (mAbs) to thymic

Description

Cell suspension

Results

S e v e r a l m a j o r a b n o r m a l i t i e s w e r e n o t e d in t h e 7 s e r o negative thymus samples studied. In the 5 adults, there was extensive expansion of septal and adipose tissue be-

Table 1. Clinical features at the time of thymectomy of the seronegative myasthenia gravis (MG) cases studied Patient, sex

Age at onset (years)

Myasthenic symptoms Grade a Duration (months)

Seronegativity b persisted for (months)

Electromyography c D J

Thymic d histopathology

1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14,

8 10.7 14 15.1 16 20 21.7 22.4 20.4 32.8 36.7 38.9 34 43

II A II B IIB II A II B II A IIB II A II B IIB IIB III II B IIB

> 4 > 34 > 2 > 46 1 5 > 20 > 0.5 > 1.5 > 13 > 2.5 > 5 > 107 > 4

ND ND ND + + + + + + +

(H) e H Ne H N A~ Ae N N N Ae Ae (H) Ae

M F F F M M M F F M F F F M

4 26 6 4 1.5 14.5 10 14 15 1.7 6 5 114 9

a After Osserman and Genkins [11]; II A, II B, mild, moderate; III, acute severe generalised MG symptoms b Seronegativity has persisted for at least the indicated interval (spanning the time of thymectomy). Case 5 became transiently seropositive 1.2 months post-thymectomy (serum anti-AChR titre 5.6 nmol/1) before starting corticosteroid therapy. By 32 months post-thymectomy, case 6 had a titre of 1.1nmol/l (cut-off= 0.4 nmol/1)

ND + ND + ND + + + + + + +

° D, Abnormal decrement on repetitive stimulation; J, abnormal jitter a Routine histopathology report; (H), (mild) hyperplasia; N, normal for age; A, atrophic e Used for immunohistological studies: cell suspension was prepared from all thymus samples except nos. 7 and 12

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tween and sometimes into the thymic lobules, and frequently some lymphocyte necrosis in the cortex as well. This combination probably accounts for the "atrophic" description often reported (Table 1), and was only conspicuous in 2 of the 40 seropositive samples. As was typical of the latter, an expanding medulla often disrupted and eroded the cortex internally and sometimes showed mild cystic changes too. Very small (but otherwise typical) GC were generally much less prominent than in seropositive cases (see below), and so this medullary expansion primarily reflected the presence of lymph node-type T-cell zones. These were the most striking and consistent feature and were very similar to those we have described in seropositive cases [1, 14]; they contained no epithelial cells and were evidently extraparenchymal expansions of septallperivascular areas. They showed many fibronectin + blood vessels, including high endothelial venules, and much more intense infiltration by mature T cells', interdigitating ("dendritic") cells' and B cells - i.e. "thymitis" - than in the normal thymus or in the residual true medulla nearby. This latter was relatively normal and was rarely compressed into the bands and arches so characteristic of seropositive cases with abundant GC [1, 14]. The features italicized above were all scored on a scale from 0 to 6; their sum was the thymitis score. Quantitation of thymic changes

Total cell yields'. In seropositive MG, the total yield of thymic cell suspension declines significantly with age and varies over about a tenfold range [14, 21]. In 10 of the 12 seronegative cases analysed, it was well within this "hyperplastic" range (not shown), and only 2 fell near its lower border. The same applied both to total CD1 ÷ (cortical) and medullary ( C D 1 ) cell yields (Fig. 1), consistent with the medullary expansion noted above. Histological abnormalities. All the sections were coded and scored (blind) for the principal thymitic features listed above, including cortical erosion but excluding GC. The values were summed to give an overall "thymitis score", which correlated very well with the similar T-

Fig.1. Total yield of CDI (medullary) thymus cells plotted against age at thymectomy. The scale is logarithmic, a value of 3.0 ~ 1000 x 106 cells. • Typical seropositive M G cases, plus the regression line; r = - 0 . 4 8 , P < 0.001. • Seronegative myasthenia gravis (MG) cases

zone score (and the hyperplastic index, which also includes GC) of Schluep et al. [14] (r = 0.89 and 0.80, P <

o.ool). Regardless of age, most of the 12 controls gave very low scores, which barely overlapped with those of the seropositive or negative cases (Fig. 2a), from both of which they differed highly significantly (Table 2). There was a single exceptional control sample (from a 35-yearold donor) with modest hyperplasia (including GC) very similar to that in typical M G (Fig. 2b). Of the 40 seropositives, 29 showed advanced changes (thymitis score > 14), and 10 fell closer to the control borderline regardless of serum anti-AChR titre: 1 was very atrophic. The results were very similar in the 7 seronegative samples studied, with 4 clearly showing advanced and 3 intermediate abnormalities; furthermore, these were evidently not simply a series of normal age changes (Fig. 2a). in seropositive MG, thymitis scores correlated with GC frequencies (Fig. 2b, r = 0.55, P < 0.001), which were medium or high even in 4 of the 9 cases with the lowest positive serum anti-AChR titres (Fig. 2b). In contrast, GC frequencies were very low in 5 of the 7 seronegatives, as well as in almost all the controls (both differing significantly from the seropositives; Table 2, Fig. 2b). This thymitis with minimal GC distinguished the seronegative samples both from the controls and from most of the seropositives; this latter distinction is reinforced by the cell culture data given below.

Culture responses In 12 of the cases, blood mononuclear cells and cell suspension prepared from 85-90% of the entire thymus were cultured in the presence and absence of PWM, and anti-AChR and total IgG were assayed in the supernatants [20]. Anti-AChR production was never detectable with any seronegative patient. Spontaneous IgG production In seropositive MG thymus cell cultures, basal IgG synthesis by T H Y .... correlates with frequencies of class II + cells (mainly B cells: r = 0 . 5 8 , P < 0 . 0 0 1 ) . Both values

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F i g . 2 . a Thymitits score plotted against age at thymectomy, b Thymitis score versus germinal centre (GC) frequency. • Typical seropositive MG cases; ® seropositive MG cases with low serum anti-AChR titres (2.4-6 nmol/1); • seronegative MG cases; ×, ~* non-myasthenic controls; in samples marked x in b, the GC frequency was less than the indicated value. The seronegatives with GC frequencies of 0.64 and 0.34 were cases 11 and 6 respectively

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Table 2. Histological scores and culture responses of seronegative MG thymus. * Versus (ii), P = 0.00001: versus (iii), P < 0.001 (MannWhitney U test). ** Versus (ii) P < 0.00001: versus (iii), P = 0.1. IgG values are in gg/ml per 106 cells cultured for 1 week Patient group

Thymitis score

GC/mm 2

(n)

% class II-- cells

(n)

Total IgG production by THY¢o~ -- PWM

THYc +D + PWM

(n)

-

PWM

+

PWM

(n)

Median (range) (i) Non-MG control

7.1" (5.7-16.7)

0.0"* (0.0-0.16)

(12)

1.9 (15) (0.4- 8.8)

ND

(ii) Seropositive MG

17.2 (9.5-25.0)

0.31 (0.0-2.0)

(40)

4.5 (61) (0.4-26.0)

0.20 (0-1.9)

22.1 (0.08-139)

(41)

2.30 (0.1 -5.9)

52.2 (0-315)

(iii) Sero-nega- 15.7 tive MG (11.5-20.5)

0.052 (0.0-0.64)

(7)

3.4 (0.3-8.9)

0.092 (0-0.16)

3.8 (0.0 - 49.4)

(12)

0.58 (0.17-6.0)

5.41 (0- 19.2)

(ii) versus (iii) P value

0.024

0.016

0.002

0.66

(12)

0.1

were low in all 12 seronegative cases tested, and I g G p r o d u c t i o n was significantly higher in the seropositives overall, especially in 15 of the 41 cases (Table 2, P < 0.01). W h e n the cell suspensions are p r e p a r e d instead with the proteases collagenase and dispase, basal I g G production is consistently about tenfold higher with seropositive cases [20], but it only rose c o m p a r a b l y in 4 of the 8 seronegatives tested, and r e m a i n e d significantly lower overall (Table 2).

0.007

ND

0.0054

(35)

(8)

T h e I g G response to P W M With seropositive M G t h y m u s cells, P W M - i n d u c e d I g G p r o d u c t i o n is also loosely related to class II ÷ frequencies (Fig. 3). A g a i n , responses w e r e lower in 11 o f the 12 seronegatives ( P < 0 . 0 1 , Table 2), w h e r e a s they were higher even in 4 of the 6 low titre seropositives (Fig. 3). F u r t h e r m o r e , they were scarcely e n h a n c e d in T H Y c + D c o m p a r e d with THYconv cultures (Table 2).

260 160

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Finally, this generally lower total IgG productivity in the seronegatives seems out of proportion to the marginal difference in class II + cell frequencies (Table 2).

Discussion Our main findings are that the seronegative MG thymus could clearly be distinguished from that of controls by its pronounced T-cell areas, and (still more so) from that of seropositives by its minimal GC involvement and IgG production in culture. Further, in vitro anti-AChR antibody production was undetectable with either PBL or thymic cells from seronegatives in contrast with the report of Lefvert et al. [7], but in agreement with its strong correlation with the serum anti-AChR titre in seropositires [10, 12, 14]. Thus seronegative and positive MG are largely separate entities, and not just part of the same spectrum. Even in the seropositives with low anti-AChR titres, GC frequencies and total IgG production were higher than in the seronegatives; indeed, in the seropositives overall, neither parameter correlates significantly with the serum titre [14]. Similarly, others have also reported prominent thymic GC in myasthenics with low positive titres and pure ocular symptoms [15], as well as in occasional controls with apparently typical hyperplasia [14]. In keeping with these lower GC frequencies, total IgG production was also significantly lower in the seronegatives, even after enzymic dispersion of the cells. As total B-cell frequencies were only marginally lower, this is consistent with our previous conclusion that IgG production - either in the presence or absence of PWM mainly depends on the B cells in the GC rather than on those in the surrounding T-cell areas [14]. Furthermore, it is based on cell suspension derived from almost the entire thymus in a larger sample (of 12 cases), and thus provides strong independent support for the conclusions from the histological studies. In seropositive MG, thymic GC frequencies correlate poorly with the patient's age, serum anti-AChR titre and especially symptom duration [14]. Hence we do not believe that the relatively short durations in several of the present series are the explanation for these low GC frequencies. Rather, as we suggested previously [14], their

25

capricious variability in the seropositives might reflect differences in the circulating immune complexes that are known to evoke strong GC responses in experimental animals [6]. If so, our present results imply that these complexes must be less prevalent (or less stimulatory) in seronegative cases - perhaps because of the nature of the target antigens or of the response to them. The target antigens might also vary between these cases, and some of them might be recognised by typical seropositives too, perhaps explaining some of the heterogeneity observed here. Likewise, the occasional seroconversions (Table 1) may be a sign of broadening specificities as the response progresses. In contrast with the variable GC, medullary T-cell areas were apparently the most consistent feature in the seropositive MG thymus and also proved to be the most conspicuous abnormality in the seronegatives. With their scattered B cells and extra-parenchymal fibronectin labelling, they are very reminiscent of the "thymitis with diffuse B-cell infiltration" described by Kirchner et al. [4] in some seronegative and corticosteroid-pretreated myasthenics. These features were also much more prominent than in controls, whereas GC were again smaller and rarer than in seropositives [4]. It remains highly debatable whether the T-cell areas are (a) the sites where the autoimmune response is initiated [19], or whether (as we favour [14, 17]), they are secondary changes reflecting either (b) an attack on medullary autoantigens (e.g. A C h R on myoid cells [5]) by cells initially sensitised elsewhere or (c) a non-specific breakdown in the blood/ thymus barrier. If alternatives (a) or (b) are indeed correct, then the target antigens in seronegative MG must also be expressed in the thymus, in addition to AChR. Whatever the case, our data are not evidence that the disease is purely T-cell mediated: on the contrary, it is clearly ameliorated by plasma exchange and is transferable to mice with serum Ig [9]. While seronegative MG responds well to corticosteroid therapy, we have scant evidence for its amelioration by thymectomy in these cases. Perhaps that is a further sign that it is a distinct entity from seropositive MG.

Acknowledgements. We thank Messrs P. B. Deverall, A. K. Yates, S. C. Lennox, and M. Sturridge and Professor M. H. Yacoub and Dr. L. Loh for help in obtaining thymus samples, Professors A.J. McMichael and G. Janossy for generous gifts of mAbs, Dr. A. C.

261 Vincent for critical review of the typescript and Dr. B. Ong for help with plotting. This work was supported by the Medical Research Council, the Sir Jules Thorn Charitable Trust and the Muscular Dystrophy Group of Great Britain.

References 1. Bofill M, Janossy G, Willcox N, Chilosi M, Trejdosiewicz LK, Newsom-Davis J (1985) Microenvironments in the normal thymus and the thymus in myasthenia gravis. Am J Pathol 119: 462-473 2. Drachman DB, Adams RN, Josifek LF, Self SG (1982) Functional activities of autoantibodies to acetylcholine receptors and the clinical severity of myasthenia gravis. N Engl J Med 307 : 769-775 3. Engel AG, Lambert EH, Howard FM (1977) Immune complexes (IgG and C3) at the motor endplate in myasthenia gravis. Ultrastructural and light microscopic localisation and electrophysiologic correlations. Mayo Clin Proc 52 : 267-280 4. Kirchner T, Schalke B, Melms A, Kugelgen T von, MiillerHermelink HK (1986) Immunohistological patterns of nonneoplastic changes in the thymus in myasthenia gravis. Virchows Arch [B] 52 : 237-257 5. Kirchner T, Hoppe F, Schalke B, Mt~ller-Hermelink HK (1988) Microenvironment of thymic myoid cells in myasthenia gravis. Virchows Arch [B] 54 : 295-302 6. Kunkl A, Klaus GGB (1981) The generation of memory cells IV. Immunisation with antigen-antibody complexes accelerates the development of B memory cells, the formation of germinal centres and the maturation of antibody affinity in the secondary response. Immunology 43 : 371-378 7. Lefvert A-K, Sunden H, Holm G (1986) Acetylcholine receptor antibodies and anti-idiotypic antibodies produced in blood lymphocyte cultures from patients with myasthenia gravis. Scand J Immunol 23 : 655-662 8. Lindstrom JM, Lennon VA, Seybold ME, Whittingham S (1976) Experimental autoimmune myasthenia gravis and myasthenia gravis: biochemical and immunochemical aspects. Ann NY Acad Sci 274 : 254-274 9. Mossman S, Vincent A, Newsom-Davis J (1986) Myasthenia gravis without acetylcholine receptor antibody: a distinct disease entity. Lancet I : 116-118

10. Newsom-Davis J, Willcox N, Schluep M, Harcourt G, Vincent A, Mossman S, Wray D, Burges J (1987) Immunological heterogeneity and cellular mechanisms in myasthenia gravis. Ann NY Acad Sci 505 : 12-26 11. Osserman KE, Genkins G (1971) Studies in myasthenia gravis: review of a twenty year experience in over 1200 patients. Mt Sinai J Med 38 : 497-537 12. Scadding GK, Vincent A, Newsom-Davis J, Henry K (1981) Acetylcholine receptor antibody synthesis by thymic lymphocytes: correlation with thymic histology. Neurology 31:935-943 13. Schluep M, Willcox N, Vincent A, Dhoot GK, Newsom-Davis J (1987) Acetylcholine receptors in human thymic myoid cells in situ: an immunohistologicalstudy. Ann Neurol 22 : 212-222 14. Schluep M, Willcox N, Ritter MA, Newsom-Davis J, Larch6 M, Brown AN (1988) Myasthenia gravis thymus: clinical, histological and culture correlations. J Autoimmun 1 : 445-467 15. Schumm F, Wietholter H, Fateh-Moghadam A, Dichgans J (1985) Thymectomy in myasthenia with pure ocular symptoms. J Neurol Neurosurg Psychiatry 48 : 332-337 16. Seybold ME, Lindstrom JM (1981) Patterns of acetylcholine receptor antibody fluctuation in myasthenia gravis. Ann NY Acad Sci 377 : 292-305 17. Sommer N, Willcox N, Harcourt GC, Newsom-Davis J (1990) Myasthenic thymus and thymoma are selectively enriched in AChR-reactive T cells. Ann Neurol 28 : 312-319 18. Vincent A, Newsom-Davis J (1985) Acetylcholine receptor antibody characteristics in myasthenia gravis. III. Patients with low anti-AChR antibody levels. Clin Exp Immunol 60: 631636 19. Wekerle H, Ketelsen U-P (1977) Intrathymic pathogenesis and dual genetic control of myasthenia gravis. Lancet I : 678-680 20. Willcox HNA, Newsom-Davis J, Calder L (1983) Greatly increased auto-antibody production in myasthenia gravis by thymocyte suspensions prepared with proteolytic enzymes. Clin Exp Immunol 54: 378-386 21. Willcox N, Schluep M, Sommer N, Campana D, Janossy G, Brown AN, Newsom-Davis J (1989) Variable corticosteroid sensitivity of thymic cortex and medullary peripheral-type lymphoid tissue in myasthenia gravis patients; structural and functional effects. Q J Med 73 : 1071-1087