Journal of Neuroimmuno/ogy, 4 (1983) I 17- i 27 I l '/ Elsevier Science Publishers

Thymic Involvement in Myasthenia Gravis

Study by I~amunofluorescent Itttd Immunoperoxidase Staining

S, PizzigheUa, A.P. Riviera and G. Tridente Cattedra di lmmunopatolo$;.a, ,rJni~ersit~ di Verona, V~rona (Italy)

(Received 211 May, 1982) (Revised, receivexl 16 November, 1982)

(Accepted 16 November, 1982~

Sunnnary

Changes in the thymus are often encountered in myasthenia gravis (MG), together with neuromuscular patholo~;, l~.n the present study, the cell }~)pulation of 28 thymuses from patients with MG we]re analyzed by immunofluore,eent and 49 thymuses by immunoperoxidase technique,;. We were able to show the presence of slg receptor positive, SpA reactive medium eLnd large B lymphocytes of light specific density in the majority of thymuses, and to demonstrate a characterislic pattern of distribution in tl.e gland. PAP analysis showed that the infiltrating B cells appeared in the interlobular septa, then reached the medulla, occasic>nally the cortex which mostly revealed signs of atrophy.

These findings are consistent with an autoimmune reaction occurria;~ against a thymic antigen; such an antigen could be acetylcholine receptor (AchR} of the thymic structures, since anti-AchR antibody or serum from patients with MG wifl react with 60~ thymocytes and some epithe]iiai cells and Hassalrs corpuscles.

Key words: Acetylcholine receptor - A n t i - ~ c h R antibodies - B lymphocytes - B

mi tosens - Densi ty gradient - 11;'- Myasthenia grat~iz -- P A P - Thymus

- Velocity cell sedimentation

This work was supported in part by grants frnm CNR, Rome, Finalized Project 'Control of Neoplastic Growth' n.81.01481.96, from MPl, ROme, ~md from ARBI, Verona.

Correspondence :o: Dr. G. Tl"idente, Cattedra di lmml,nopatoh)gia, Policlinico di Borgo Roma, 37100 Verona, Italy.

0165.5728/83/0(0)0-0000/$03.00 ¢~ 1983 Elsevier Science Publishers

118

Introduction

Thymic involvement in myasthenia gravis (MG) is an outstanding feature of the disease and hypcrplasia, follicular hyperplasia and epithelial ~hymoma are frequently encountered (Castleman 1966).

As for the pathogenetic niechanism in MG, the presence of autoantibodies to muscular acetylcholine receptor (AchR) is the most striking feature (Lindstrom 1979). The role of thymus in this conte~.t is poorly understood, although a finkage between the thymic and the neuromuscular changes has been sugges~.zl (Aharonov et al. 1975; Kao and Drachman 1977; Wekerle et al. 1977). The recent finding that AchR exists on epithelial (Eng,;! et al. 1978) and myoid 0Vekerle et al. 1975) thymus cells, and that an AchRolike stmc~t~re (Tri~entc et al. 1978; Fuchs et al. 1980; Ueno et al. 1980) is detectable on thymocyte plasma membrane, does provide support for such a unifying hypothesis in MG.

Although the pathogenetic importance of anti-AchR, autoantibodies has been demonstrated, no strict correlation has been found between clinical status, thymic pathology and serum antibody titre in patients with MG (Lindstrom 19/9) or in animals with experimental autoimmune myastbenia gravis, EAM(3 (Berman and Patrick 1980). Lysr, phoid follicles in the thymus in MG often show large germinal centers, suggesting the presence of proliferating, antigen-triggered B lymphocytes, as in peripheral lymphoid tissue, although such follicles are occasionally found al~o in the normal thymus (Levine and Rosai 19/8).

We undertook morphological and functional studies of the characteristics and pattern of distribution of B lymphocy~es and immunoglobulins (lgs) within tbe thymus in MG. We used immunofluorescent and immunoperoxidase techniques, to identify surface Ig receptors (slgs), Igs and Is-Positive cells, together with a method to estimate B cell mitogen reactivity. Moreover, we evidentiated thymic structures which bind lgs from homologous MG serum or antiserum to T. caiifornica AchR.

Materials and Methods

Immunofluorescent studies and eslimation of mitogenic response Twenty-eight fresh thymuses, from 20 females and 8 males with MG, thymecto-

mized at between 15 and 60 (mean 32) years of age, were observed by direct immunofluorescence staining (slF) for slg-pcr~itive cells. Briefly, 50 ~i of a sospen.. sion of thymocytes, containing i0 ~ cells, were incubated for 30 rain at room temperature, with an equal volume of FITC rabbit-anti-human ig, directed against both heavy and light Ig chains, diluted in phosphate-buffered s,~,line (PBS), 5~$ feral calf serum (FC$) and sodium azide 0.02%. After 3 washings, the cells were examined by incident fluorescent light microscopy (Ploem system), Specificity for slg was checked by restaining after capping and membrane resynthesis, after mild enzymatic stripping, using F(ab)2 antibody (Ferrarini et al. 19"/6).

Six normal fresh thymuses° obtained from children undergoing cardiac surgery., were used as a source cf controls. These cells were also used for indirect slF analysis,

!1 ~)

by incubation in dilutions of sen,m ftom myastherdc patients or in arAi-AchR polyclonal anti-serum, which had beam raised against T. californiccr ek~ctric organ receptor (Fuchs et al. 1980). Linear density gradients (Shortman 1c.~8) or velocity sedimentation (Miller and Pllillips 1969), techniques of cell separat/o~a v,c~e used for B cell characterization, as previous'y de~a:ribed (Tridente et al. 1978). ,

The mitogenic response of B -ymphocytes was evaluated in miciocuhures of unfractionated or fractionated thymus ,(106 cell in 0.25 rid), sfin~zlated with 15 I~l/ml Staphylococcus aureus protein A (SPA, sepharose bound, Pharmacia, Uppsala. Sweden), 0.5% w/v (Forsgren et ;~I. 1976) in MEM to which 10% FCS bad added (both from Gibco NY, U.S.A.). After 72 hours' incubation at 3"1°C in 5% CO~ atmosphere, tritiated thymidine ([~H]TdR, 2.0 mCi/mM, Amersham, U.K.) uptake was measured by liquid scintillation (LKB, mod. 1215 Rack Beta).

Immunoperoxidase staining procedure Fourty-nine thymuses, from 32 feraales and 17 males with MG, ,vho had been

thymectomized between 1 and 59 0.qean 30.5) years of age, were studied by means of formalin-fixed paraffin-embedded t.;_~.~e sections, using the unlal~elled antibody peroxidas¢ (peroxidase-anti-peroxidase, PAP) method (Sternberger et al. 1970; Wagener et al. 1979). Six o~' these patiemts were ,~ncluded in the previous gr¢,up of 28 fresh thymuses.

Paraffin sections were kept in xylene ,avernight, then passed thrc,uch decreasing ethanol solutions in water, each immersion lasting about three mic~utes, and then placed in 0.05 M Tris-HCI buffer pH "7.6 at 4c'C. The sections were subsequently covered with some drops of the following solutions: (i) H202 1~[ in Tris-HCI (alternatively H202 1~ in methanol). (ii~ Normal swine serum (NSS). undiluted, for 30 rain. Then, without washing, (iii) rabbit antiserum (lgG fraction of anti-human light or heavj chains, diluted in NSS, either 1 : 40 dilution with 30 mia incubation at room temperature or 1 : 1000 dilution ore.might at 4°C. (iv) After 3 washin#s with Tris-HC! buffer in 1 : I0 in saline, swine-anti-rabbit Ig, dilution 1 :dO in NS.g. was added for 30 rain at room temperature. The ne~t wash with 'Iris bufh;r was I~lowed by: (v) PAP, diluted 1 : g0 in NSS for 30 rain a~. room teraperature. Wash again with Tris buffer. (vi) Diaminobenzidine (DAB) tetrahydrochloride 0.5 mg/ml in Tris-HCi HzO 2 0.01-0.05% about 10 min in the dark, at room temperature.

A bright brown staining revealed the presence of the antigen in the tissue :~ecfions. After counterstaining with hematoxylin, sections were dehydrated by 2 passages in absolute ethanol and the slides were mourtted in Cedax. Five normal thymlx~es were used as cc~trols, The specificity of:..h.e PAP method was controlled by trciting aH ~ctions with normal rabbit serum (NI~:S), in the place of the inmmne serum. L~ ~ome thymuses which were positive an additional control was obtai~aed by treating the sections with antisera which had been previously absorbed with the conespond- ing Ig chain.

i I I ig

l

121

Results

The clinical staging and thymic pathology of the two grout's under study are shown in Table 1. Most patients fell into class !I of Osserrnan's classification. Histological examination reuealed follicular hyperpla, ia in n~ore than 50% of thymuses, associated with thymc,ma in 3 cases.

Direct immunofluorescent staining for the major immunogiobutin classes revealed a variable number of Ig positive lymphocytes (Fig. la, left) and stromal celJs in the :hymuses of patients with MG. The structural and functi,mai characterization of the lymphocyte cell populat ion is shown in Table 2. In all 17 positive cases I:Imphocyt_es were also stained with antisera to mu and delta chains. In 7 out of 15 cases, a fe~ IgG- or lgA-positive lymphocytes were also detected.

Kinetic experiments, after capping and membrane resynthesis ha¢l ~ c u r r e d , revealed that only l g M / I g D surfilce positivity on B cells was of the receptor type. In 3 cases slg positive cells were evaluated in thymuses from myasthenic patients alter density gradient centrifugation, and in 5 cases evaluation was carried out after velocity sedimentation f~mctionalion. Most slg (mu, deha)-positive cells were de- tected at 1.058 g / c m 3 density and in 2 fractions which had approximately 3.5 and 4.5 m m / b velocity sediment~:tJion. The latter fractions con~tained a higher percentage of slg-positive cells as revealed by s lF (C.F. = 2.4-3.2) and the response to SpA mitogen (C.F. = 3.3-8.6).

lg-positive cells were detect,~d in 31 out of 49 thymuses from myasthenic patients by PAP, and in 17 out of 17 cases by s lF (Table 3). When tested for lg light chains. cells positive for either kappa or lambda ( ~ / h = 3: I) were present in the same spe ~imen, clearly indicating the policlonal nature of the positivity detected. Plasma cells were seen, but mc,st of the positive cells were medium to large iymph(~-ytes (Fig. Ib). PAP staining was detected on Hassall 's corpuscles, as superficial caps or diffuse granular positivity, and in the extracellular spaces, often localized at the interlobular septa (Fig. Ic, d). Ig-positive cells were present within the cortex (Fig. le, f), but more frequently thes, e cells grouped in the deep medulla to form pseudofoilicular structures pressing onto the surrounding thymic parenchyma. Be- side these areas a line of Hassall 's corpuscles was often seen (Fig. Ig). In cases which showed large numbers of lg..positive cells, the cortex appeared reduced to small

Fig. I. C?,tological and histological pattea'm 3f ~g-positive cells in myasthenic thym.~s. a: Left: cappin~ lympho~:ytes stained by FITC-anti-lgM; right: normal epithelial cells and lympho.~t¢

incubate, d in homologous MG scram and stained by FITC-anti-igG. b: Mononuclear cells arm plasma cells i~a biG thymus stained by PAP-anti-kappa chains. ,:: Epithelial cells within Hassall's corpuscle stained by PAP-and-kappa chains. J: Septal invasion of Ig and of lg.positive cells stained by PAP-anti-13G. e: Ig-positive cells lining around a cortical nodule, stained by PAP-anti-kappa chains. f: invasion of a cortical nodule frcm septal lg-positive cells, stained by PAPoanti.-kappa chains. Note .l

plasma cell (blue) negative for such light chains. g: Lining of Hassall's corpuscles a~ound a large Ig-positive medullar:¢ nodule, stained by PAF-anti-

kappa chains. h: Atrophic cortex surrounded by Ills and Ig-positive cells stained by PAP-and-kappa chains.

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TABLE 2 . . . . ....

STRUCTURAL AND FUHCTIOI~IAL CHARACTERISTICS OF B LYMPHOCYTES IN HUMAN THYMUS : .,. ~ :,

Number o f siF 3H.TdR V.S," S.D. b cases (7o) (cpm

: : :' X 10 -3 ) mm/h CF g~/cm ~ CF

MG thymus tt 17

B 17

y 15

a 15

SpA 5

Normal thymus I~, B 6 y,a 6 SpA 4

5.17+4.36

5.37 + 5.70

1.44+2.20

0.56+,1.20

~.2±2,0

0.35 :E 0.~[) 0.10:[:0.12

0,2+0.1

4.5 + 0.20 2A 3.6 + 0.25 1.0.58 + 0.002 3.2

4.4+0.15 2.3 3.5 + 0.36

4.5+0.10 1.048 c 1.6 3.3+0.20

5.6+0.95 I.! no peaks 3.8+0.50 4.9:/:0.30 3.3 1.062+0.001 8.6

no peals no peaks rLo peaks • no p . h n.d. n.d.

s lF-surface immunofluorescence; 3H.TdR- net tritiatecl thymidine tJptake in SpA-sliamlal~ dmn terra cultures; V.S. - velocity cell sedimentation; S.D. - specific cell density: CF= coacentratioa f=ctm; SpA - protein A, StophyJ. aureus, sephar0se-bound, n.d. ~ not done. • Discrete peaks obtained in 5 different eXl~'riments. b Peaks obtained in 3 different experimentaL. " Peak detectable only in one experintont.

TABLE 3

PRESENCE OF Is-POSITIVE CELLS IN MG THYMUSES A~, DEFECTED BY IM,- MUNOPEROXIDASE (PAP) AND $:URFACE IMMUNOFLUORESCF, N CE (silF)

PAP performed on titme sections, slF on fresh cel| suspensions.

PAP slF

Tes ted P~sitive Tested t~osid',,e

Total 49 31 F0B 30 18 1' 30 18 a 30 14 x 49 31

49 31 NRS" 49 0

28 17 17 "7 15 7 15 7 13 13 I! ll _

" Nmmai rabbit scram.

124

residual islets, surrounded by such positive cell.,; merging from an enlarged medulla (Fig, lh).

Normal thyw..~ses in either IF or PAP studies, rarely showed lg-positive lympho- cytes. On the other hand, incubation with either autologous or heterologous serum from myastheni¢ patients, or with polyclonal anti-Torpedo californica AchR anti- body, produced a spot-like positivity and capping appearance in about 60% normal thymocytes, on slg-negative cells from thymuses of subjects with MG, and showed a diffuse surface positivity on epithelial thymic stroma (Fig. la, right).

Discussion

Staining for slg receptors by immunofluorescence has revealed a variable number of B lymphocytes within the thymus of myasthenic patients, even when no histologi- cal lesions were detectable. These lymphocytes reacted in vitro to the B cell mitogen, SpA. The polyclonal nature of the cells was ascertained by staining for Ig light chains and was confirmed by PAP tests, which showed intracytoplasmatic Ig of the major classes. These cells were not numerous in the thymus in the absence of follicular hyperplasia of the medulla. Attempt~ to separate and concentrate them by density gradients or sedimentation velocity, revealed a relative heterogeneity in cell volume and specific density which grossly corresponded (Shortman 1977) to those of medium-light density and medium-large, activated lymphocytes.

PAP staining on tissue sections, as compared to sIF results, revealed the con- sistent presence of polyclonal lg secreting lymphocytes, mainly medium to large size, including some plasma cells and some positive .~tromal cells. This difference reflects technic~l features in the two staining methods employed in the study: while slF detects membrane Ig, mainly of the receptor type after capping and stripping, PAP staining on sections detects antibody producing cells (intracytoplasmatic lg), as well as Ig-coated cells and extra-cellular lg. Therefore, one expacts to detect a higher number of Ig-positive cells using the latter method, which identifies not only antibody producers but also free antibodies and antibody-coated targets.

Ig-positive cells were found, by the PAP technique, within septa and close to the perivaseular spaces in the thymus from myasthenic patients. In some cases this was the only detectable abnormality. When these cells were numerous, they were detected not only in the same sites, but isolated l~sitive ceils were also seen in the thymic parenchyma. In thymuses which contained large numbers of Ig-positive cells, there was obvious change in the medulla and the cortex was also affected, either by cell 'spillage', or from compression by the enlarged medulla.

A dynamic interpretation of such patterns suggests that B cells of extrathymic origin may invade the thymus of myasthenic vatients, passing through sepia and blood vessel walls. From the ~ites of these initial lesions, the cells pass into the medulla; the cortex appears to be infiltrated late from isolated groups of lg-positive cells and shows signs of atrophy (Fig. 2).

Hassalrs corpuscles were numerous in the thymuses studied and showed a pattern of polyclonal PAP positivity, suggesting a progressive centripetal involvement.

|2~

Interestinl;'Jy, Engel et al. ,(1978), using peroxidas¢-labeiled ~ l p h ~ - b ~ c ~ r o x i ~ showed that AchR was present on the outer cells of lta.,~sali's co,qmscles.

The prq:sence of Ig-stain,.~d Hassall 's corpuscles and numer4ms B cells /n thymuses of these patients sutggest that there are Sl~-~cific targets within the organ. Therefore, the pathogenetic linka~ge between the t~ymu,,:; and ~the neuromusc,.gar

Fi$. 2. Schematic dynamic representation of thymic lesions in MG (frmn top to bottom). Left: PAP-i~sitive material and ~;lls initially appear in septa, spreading in laedul~y clara.like

formatiom and pseudo-f,~llicular ~oduh:s prer, sin$ onto the cortex. Right: small Hal~lFs bodies show some PAP-positive epithelial .caps and ~u~ted pmitive

PAP.po~itiv~ material is more frequent in large~r Hassalrs bodies and in intracystic

126

junction may be due to the fact that an antigenically similar structure is the corm~aon target of an autoimmune attack. Preliminary data given by Aharonov et al. (1975), Wekerle et al. (1975), Kao and Drachman (1977), Engel et aL (1978), the work of Tridente et al. (1978), Fuchs et al. 0980) and Ueno et aL 0980), which b,s shown the presence of AchR, or AchR-like structures, on the plasma membrane of different thymic cell populations, support this hypothesis.

Our observation that 60~ of normal thymocytes stain for lg in slF after incubation in MG serum and anti-AchR antiserum also supports this view. Simi- larly, the production of anti-AchR antibody in thymus from myasthenic patients grown in vitro, as reported by Vincent et al. (1978), is in line ~vith the afore~men- tioned proposition.

On the other hand, the small number of slg-positive cells shown by direct slF within the thymus, indicates also that autologous anti-AchR antibodies eventually present in myasthenic patients are not revealed by this technique on thymocytes. However, the higher number of Ig-posifive ce~ls seen by PAP staining, including epithelial cells and Hassairs corpuscles, show that by this technique it is possible to detect also antibody-coated targets within the thymus.

Whether the Achg found in thymus is a non-fanctioning membrane structure or a functioning receptor, remains to be clarified. If it is the former (Fuchs et al. 1980), it may be important as the specific thymic target in MG. In the case of the latter (Ueno et al. 1980), it may be that the first pathogenetic change in MG may be a functional alteration. Preliminary functional studies in an in vitro model do suggest that thymic AchR has a physiological role (Pizzighella and Tridente 1981; Piz- zighella et al. 1982).

Acknowledgements

We thank Prof. S. Fuchs for kindly supplying antiserum to Torpedo californica AchR, Prof. A. Micoli for providing some thymus specimen, and Mrs. L. Pascoli for drawings and photographs.

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