experimental autoimmune myasthenia gravis: cellular and humoral immune responses

EXPERIMENTAL AUTOIMMUNE MYASTHENIA GRAVIS: CELLULAR AND HUMORAL IMMUNE RESPONSES *

Vanda A. Lennon and Jon M. Lindstrom Salk Institute for Biological Studies

San Diego, California 92112

Marjorie E. Seybold Veterans Administration Hospital

and University of California

San Diego, California 92037

INTRODUCTION

Immunization of animals with acetylcholine receptor protein ( AChR) from the electric organ of Electrophorics electricus and Torpedo californica induces an autoimmune response to the AChR of mammalian skeletal m ~ s c l e . l - ~ We have named the ensuing disease experimental autoimmune myasthenia gravis (EAMG) because of its clinical, pharmacological, and electrophysiological similarity to spontaneous myasthenia gravis (MG) of man.2

Evidence implicating abnormality of the immune system in patients with MG has been extensively documented. For example, thymic hyperplasia and tumors are often found in association with MG,4 thymectomy5 and immuno- suppressive drug therapy can be beneficial, and circulating autoantibodies are frequently found.4, 7 , 8 The incidence of other putative autoimmune diseases in patients with MG or in their relatives is greater than would be expected by c h a n ~ e . ~ The most compelling evidence for the validity of EAMG as a model for MG has been the demonstration of antibody to skeletal muscle AChR in both EAMG and MG.g--" We found that this antibody, in both M G l O * l1

and EAMG,2 is directed to sites on the AChR molecule other than the ACh binding site. Since the role of antibody in the pathogenesis of MG is not yet known, study of EAMG allows systematic analysis of cellular and humoral immune responses to AChR.

The present study was undertaken to investigate the roles of thymus-derived (T) and bone-marrow-derived ( B ) lymphocytes in the pathogenesis of EAMG.

MATERIALS AND METHODS

Preparation of AChR

AChR protein was purified from the main electric organ of Electrophorus electricus as previously described.l2 Further details and the method of prepar- ing an extract of rat muscle AChR for antibody studies are presented at this meeting."

special grant from the California Chapter of the Myasthenia Gravis Foundation. :$This work was supported by NIH Grant No. NS-A1-11719-01 and by a

3117

284 Annals New York Academy of Sciences

Inoculation of Rats

Female Lewis rats aged 8-1 2 weeks (obtained from Microbiological Asso- ciates, Inc., Bethesda, Md.) were injected once with eel AChR. Each dose, in 0.1 ml of phosphate buffered saline (PBS), was emulsified with an equal volume of complete Freund's adjuvant [CFA = 2 parts Marcol-52 (Humble Oil and Refining Co., Los Angeles, Calif.) to 1 part Aquaphor (Duke Laboratories, Inc., So. Norwalk, Conn.) with 2.7 mg/ml M . butyricum (Difco Laboratories, Detroit, Mich.)]. The emulsion was divided intradermally (i.d.) among four sites (hind footpads and shoulders). As additional adjuvant, 0.5 X 1 O 1 O B. pertussis organisms (special vaccine product of Eli Lilly and Co.) were injected subcutaneously into the dorsum of each hind foot, over the sternum and between the scapulae. Doses of AChR ranged from 1 1 to 350 pmole, and the clinical dose response was as predicted from our previous study.'

Clinical Observations

Rats were weighed three times weekly (weights at the beginning of experiments ranged from 140 to 200 g ) and observed daily for signs of muscular weakness which was graded 0 to +++ as previously described (0 = no definite weakness; + = weak grip or cry with fatigability; ++ = hunched posture with lowered head, flexed forelimb digits and uncoordinated movement; ++$- = severe generalized weakness, tremulous, moribund) .2 Signs of adjuvant induced arthritis also were recorded as previously described.'3

Electromyograms

Electromyographic studies were performed as described previously (and also in this volume1+) and curare sensitivity was tested by recording the response to repetitive stimulation after injecting 8 pg of curare intraperitoneally (i.p.).

Skin Tests

Antigens were injected i.d. into the shaven middorsal region. Eel AChR (17 pmole = 5.0 pg) and PPD (50 pg) of human tuberculin (kindly provided by the Ministry of Agriculture, Fisheries and Food, Weybridge, England) were given in 0.1 ml of saline and saline alone was used as a control test. Sites were examined at 1, 4, 24, and 48 hr, and the maximum diameter of induration was recorded.

Blood Samples

These were obtained by venesection of the tail or jugular vein under ether anesthesia. Serum samples were stored at -20" C.

Lennon et al.: Immune Responses 285

Antibody Assays

Sera were tested for antibodies to eel and syngeneic rat muscle AChR by immunoprecipitation using 1'51-labeled-cobra-toxin-AChR as antigens.', lo, l1. l5

This assay, in which rat antibody bound to 1251-toxin-AChR is precipitated by rabbit anti-rat-IgG, measures antibody directed against sites on the AChR molecule other than the ACh binding site.

Characterization of Antibody Class

Serum from a rat in the acute phase of EAMG (10 days postinoculation) and in the chronic phase (35 days) were fractionated by sucrose gradient ultracentrifugation.16 Tritiated mouse myeloma proteins (both IgM and IgG, kindly provided by Dr. R. Coffman) were used as markers. Gradient fractions were tested for antibody to AChR.

Antithymocyte Serum

To raise antithymocyte serum (ATS) for in vivo treatment of rats, rabbits were injected, according to the protocol of Levy and Medawar,17 twice intravenously (i.v.) with lo9 rat thymocytes. To minimize toxicity, pools of ATS and normal rabbit sera (NRS) were held at 56" C for 30 min, then absorbed at 4" C with a 25% suspension of washed rat red blood cells. ATS used for in vitro treatment of spleen cells was raised in rabbits according to the protocol of Reif and Allenls by repeated inoculation i.p. with rat thymocytes and B. pertussis vaccine. This ATS was heat inactivated and absorbed sequentially with rat red blood cells, liver, kidney, and bone marrow.

Thy mectomies

Adult rats were thymectomized under ether anesthesia by vacuum suction with direct visualization through a midsternal incision. Incisions were closed with metal clips. Sham-operated rats were similarly subjected to midsternal incision with thymic exposure. In one experiment, 11 rats were operated upon early in the chronic phase of EAMG, and in a separate study, 16 normal rats were operated upon before x-irradiation.

X-Irradiation

Rats received a lethal dose (850 R ) of whole body irradiation from a cobalt-60 source approximately 8 weeks after thymectomy, and spleen cells (1-2 X los) were injected i.v. a few hours later. Two rats received in addition lo9 thymus cells. A single injection (40 mg) of tylosin (Eli Lilly and Co.) was given intramuscularly as prophylaxis against endemic chronic respiratory disease, and neomycin and polymyxin B were added to the drinking water for 2 weeks following irradiation.


Cell Preparations

Single cell suspensions of normal rat spleens and thymuses and immune rat lymph nodes were prepared in PBS with 10% heat-inactivated rat serum as described previously.lQ To kill T lymphocytes, spleen cells were treated with ATS plus fresh rat serum as a source of complement.?0 Clumped dead cells were removed by gravity sedimentation over undiluted normal rat serum. The efficacy of T-cell killing was assessed by testing spleen cells before and after treatment for their responsiveness to the T-cell mitogen phytohemagglutinin (PHA) as described previously.21

RESULTS

Cellular and Humoral lnzmune Responses of Normal Rats Challenged with AChR

Delayed Cutaneous Reactivity to AChR

Positive skin tests were typical of delayed-type hypersensitivity. Skin test sites appeared normal 4 hr after testing, showed maximum intensity of induration and erythema by 24 hr (ranging usually 1-2 cm in diameter) and were still positive, although waning in intensity, at 48 and 72 hr. Equivocal sites (<0.5 cm maximum induration) were scored as negative.

The onset of positive delayed cutaneous reactivity to eel AChR occurred 4 days after inoculation with AChR/adjuvant. Rats tested prior to day 3 postinoculation were all negative (TABLE 1). Those tested on day 3, unlike all other positive responders, showed no reaction until 48 hr after testing; their positive reactions lasted through 72 hr. The occurrence of a positive reaction in one of the 13 adjuvant controls cannot be explained.

TABLE 1

TIME COURSE OF DELAYED CUTANEOUS REACTIVITY

Incidence of Positive DTH* Reactions to

Immunogen/CFA Eel +Pertussis Day Tested Saline AChR PPD

Nil (no adjuvant) 0 o/ 3 o/ 3 o/ 2

Eel AChR (40 pmole) 1 2 o/ 2 o/ 2 o/ 2

4-13 0/18 18/18 16/17 18-21 o/ 5 2/ 5 4/ 5 51,57 n.t.S o/ 2 o/ 2

Saline 8-19 O/ 13 1/13 9/10

3 o/ 2 2/ 2 t 2/ 2 i

* DTH = delayed-type hypersensitivity. t All positive reactions were maximal at 24 hr (1-2 cm induration plus erythema)

$ n.t. =not tested. except these which were negative at 24 hr but became positive at 48 hr.


9 -

1 1 1 1 1 1 1 1 1 1 10 20 30 40 50

DAYS POST INOCULATION

+ $.

FIGURE 1 . Sequential antibody response of a rat inoculated with 350 pmole of eel AChR. This rat had two severe episodes of EAMG (+ + + ) : the first transient (days 8-12) and the second progressive (from day 30). Antibody to syngeneic muscle AChR (which appeared later) was directed at determinants of AChR shared by eel and rat since it could be totally removed by absorption with eel electric organ membranes." Titers are expressed as moles '"I-toxin binding sites precipitated per liter of serum.

Antibody Responses to AChR

The sequential antibody response of a rat inoculated with 350 pmole of eel AChR is shown in FIGURE 1. Antibody to eel AChR was detectable before the tenth day and rose to a plateau by day 27. By day 14 a low titer of antibody cross-reacting with syngeneic muscle AChR was demonstrable. The titer of antibody to muscle AChR rose sharply after day 25.

Class of Antibody to AChR

Sera obtained from the rat illustrated in FIGURE 1 on days 10 and 35 were fractionated by sucrose-gradient ultracentrifugation (FIGURE 2) . At day 10 approximately one-third of the antibody activity sedimented with the 19-S marker and two-thirds with the 7-S marker. No cross-reactivity with muscle AChR was detectable in these fractions. At day 35 all the antibody activity to both eel and muscle AChR sedimented with the 7-S marker.

Analysis of the Contribution of Defined Lymphocyte Populations to the Pathogenesis of EAMG

Effect of Transferring Immune Lymph Node Cells to Normal Recipients

Two pools of cells were prepared from lymph nodes of donor rats inoculated 7 days previously with 100 pmole of eel AChR and/or adjuvants. Five normal rats received 6.6 x los AChR-sensitized lymph node cells and three received 4 X los adjuvant-sensitized lymph node cells. Over a 60-day period of observation, four of the five recipients of AChR-immune lymph node cells developed intermittent signs of EAMG ("+" severity), first appearing 6 to 20 days after cell transfer. Three of these four rats developed weakness characteristic of


20

16

12

8

4

0 4 8 12 16 20

SuCRog GRAMENT FRACTION Nos.

FIGURE 2. Sedimentation char- acteristics of the antibodies to eel and rat AChR shown in FIGURE 1. Serum applied to a 5%-30% sucrose gradient was centrifuged 6 hr at 4" in a Beckman 50.1 rotor at 3 x 105g. The position of tritiated mouse myeloma protein markers (19 S and 7 S ) on the gradient is shown by arrows. By day 10 most of the anti-AChR antibody sedimented as 7 S , suggesting an early switch from IgM to IgG production.

EAMG after being bled on the 19th day after cell transfer. No antibody to rat muscle AChR was detectable in the rat sera nor were EMG decrements induced by challenge with curare 63 days after cell transfer. In a subsequent experiment, five of six normal recipients of 6 X 10'; AChR-immune lymph node cells developed EMG decrements when challenged with curare 7 days after cell transfer.

Eflect of Thymectomy Early in the Chronic Phase of EAMG

Because of the occasionally beneficial effect of thymectomy on the course of MG in patients,5 11 rats with unequivocal signs of EAMG were selected from groups inoculated 35 days previously with varying doses of AChR to examine the effects of thymectomy and sham operation on their clinical course (TABLE 2) . Difficulty was encountered in anesthetizing the rats, since they appeared to be markedly resistant to ether, but recovery from surgery was uneventful. The clinical course of the thymectomized group did not differ significantly from those sham-operated.


EfJect of Treatment In Vivo with Antithyrnocyte Serum on the Response to AChR Challenge

The protocol of injecting ATS on days -1, f l , +3, and +5, with antigenic challenge on day 0, was chosen since it has been shown optimal for prolonging retention of skin homografts in mice and for suppressing induction of experimental autoimmune encephalomyelitis in rats.g2 Two groups of 12 rats were injected subcutaneously with 2 ml of either ATS or NRS. Six rats in each group were challenged at day 0 with 100 pmole of eel AChR with adjuvants; the remainder were challenged with adjuvants only. All the rats were bled on day 41. FIGURE 3 shows the subsequent weight patterns of these groups. The group that was treated with NRS and received only adjuvants failed to gain weight between days 12 and 20. This period coincided with peak severity of adjuvant-induced arthritis, which was of much less severity or did not occur at all in the groups treated with ATS. The progressive weight gain in the group treated with ATS and challenged with adjuvants reflects the absence of arthritis. The biphasic mean weight loss of the group treated with NRS and challenged with AChR illustrates that typical of EAMG in rats.’ All six of this group had transient acute EAMG (five “++” and one “+” in severity) commencing on day 8-9 and resolving by day 11-13. Five of these developed chronic EAMG by day 28, the sixth had not shown any further signs of EAMG by day 41 when accidental death occurred at bleeding. The weights of the group challenged with AChR and treated with ATS contrasted with the AChR group treated with NRS, rising progressively until day 20. Their weights then pla- teaued, diverging from the adjuvant group treated with ATS, and by day 28 commenced to fall. Only two of these six rats showed early signs of EAMG, both beginning on day 11, of “f” severity and resolving by days 12 and 13, respectively. Signs of chronic EAMG developed in all rats of this group commencing days 21-41. Antibody to muscle AChR was detectable on day 41 in the sera of both groups of AChR recipients, but in none of the adjuvant groups

TABLE 2

COURSE OF EAMG IN RATS THYMECTOMIZED OR SHAM-OPERATED AT DAY 35

Dose of Preoperative AChR Severity of Maximum Day

(pmole) EAMG Surgery * Severity Killed

350 + Tx +++ 49 350 + STx +++ 49

11 1 i- STx +++ 43 111 i- Tx +++ 431 35 +-I- Tx +++ 79 35 i- STx +++ 49 11 + STx +++ 79 ll i- Tx + 97 11 + STx +++ 97

1 1 1 + Tx +++ 62 111 + STx +++ 62

* Txxthymectomy ( 5 ) ; STx=sham thymectomy ( 6 ) . t Found dead.

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Annals New York Academy of Sciences

- - - .- .- ~~ ~ *" u., 7" 7" "- W" -" -- DAYS AFTER CHALLENGE WITH AChR AND/OR ADJUVANTS

FIGURE 3. Weight changes in four groups of six rats treated with ATS or NRS on days indicated by arrows. Eel AChR (100 pmole) and/or adjuvants were injected on day 0. The adjuvant group treated with NRS (0) had severe adjuvant arthritis days 12-20. Arthritis was minimal in the groups treated with ATS. The AChR group treated with NRS (m) had two episodes of severe EAMG (+ + / + + +) (days 8-13 and progressive from day 28). Only two of the AChR group treated with ATS (m) showed early EAMG (+, days 11-13) but all developed chronic EAMG by days 21-41.

[mean titers ? standard error (SE) : ATS-treated = 45% 18.9 X lo-"' M; NRS- treated = 72.4 2 28.0 X lo-'" MI.

Eflect of Varying Degrees of T-Lymphocyte DepIetion on the Response to AChR Challenge

The experiments described so far suggest a central role for cellular immunity to AChR in the pathogenesis of EAMG, namely: the early onset of delayed- type hypersensitivity, the transfer of EAMG with lymph node cells and the suppression of the acute phase of EAMG by early treatment with ATS. T lymphocytes are responsible for delayed-type hypersensitivity reactions and are a major component of the rat lymph node cell population.22 T cells in the lymph nodes might be involved as helpers to B cells in the production of antibody to AChR. To ascertain the possible role of T cells in the pathogenesis of EAMG, rats were depleted of T cells by thymectomy and x-irradiation and were reconstituted with B cells with and without T cells before challenge with AChR/ adjuvant.

To assess hematologic recovery 4 weeks after irradiation and injection of

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B cells (with and without T cells as indicated in TABLE 3 ) , complete blood counts were performed on 13 thymectomized and 3 sham-operated rats before challenge with 100 pmole of AChR. Hematocrits of all 16 rats ranged from 42% to 50%. Peripheral blood lymphocyte counts were lower in the 7 thymectomized recipients of spleen cells treated in vifro with ATS (spleen B cells), but were lower than normal in all groups. The reduction in responsiveness to the T-cell mitogen PHA (the mean cpm above background fell from 1230 to 60) confirmed the lack of T lymphocytes in the spleen B cell population used for reconstituting the irradiated rats (TABLE 4) .

The majority of rats in all four groups (FIGURE 4) developed extremely severe and persistent adjuvant-type arthritis (+++), which commenced 6 days after challenge with AChR/adjuvant. Only the 3 sham-thymectomized rats (reconstituted postirradiation with spleen B cells) developed weakness and biphasic weight loss typical of EAMG. The weakness (++ and +++ severity) was later in onset than usual ( 12 to 18 days) and resolved by day 24, but recurred in two rats.

The group of seven thymectomized rats reconstituted with spleen B cells alone, failed to gain any weight over the course of 55 days of observation. This was most likely attributable to arthritis. None of the six thymectomized rats which received either untreated spleen cells (containing both B and peripheral T cells) or spleen B cells plus lo!' thymus cells developed signs of EAMG, but all showed loss of weight, commencing 12 days after AChR challenge, paralleling that of the sham-thyrnectomized group with EAMG. Unlike the sham-thymectornized group, however, these rats showed signs of recovery, evidenced by regain of weight, after day 33.

The rat sera were tested for antibody to eel AChR 45 days after challenge. All rats which had T cells (either from recovery of an intact thymus or from transfusion of thymus cells or splenic T cells) had anti-AChR antibody and there was no significant difference in their titers (TABLE 3). In contrast, anti- AChR antibody was detectable in only two of the seven thymectomized recipients of B cells alone and these titers were 30-fold lower than the mean titers of the rats possessing T cells. No rat showed an EMG decrement when tested 63 days after AChR challenge. Decrements were induced however by 8 pg of curare in all groups except the seven thymectomized recipients of spleen B cells alone (TABLE 3 ) . Sensitivity to this small dose of curare probably repre-

TABLE 4

EFFECT OF ANTITHYMOCYTE SERUM (ATS) ON RESPONSIVENESS OF SPLEEN CELLS TO A T-CELL MITOGEN, PHYTOHEMAGGLUTININ (PHA)

Spleen Cell Treatment

Incorporation of Mitogen 3H-Thymidine into DNA

in Culture (cpm +SD) * Nil Nil 5 0 2 21

PHA 1284k 150 ATS + complement Nil 2 4 2 10

PHA 83-1- 6

* Triplicate tubes of 5 x 10' viable leucocytes were cultured 48 hr at 37°C with 5% COz. 1 pCi of aH-labeled thymidine was added for the final 6 hr.


5 -40

Y t

TI* B SPLEEN

TX + WHOLE SPLEEN

T r B SPLEEN + T CELLS

SHAM T r + B SPLEEN

-501

t

5 I0 15 20 25 30 35 40 45 50 55 DAYS AFTER CHALLENGE WITH AChR

FIGURE 4. A total of 16 rats were thymectomized (Tx) and three were sham thymectomized; 8 weeks later all received 850 R and 1-2x 10* nonimmune spleen cells intravenously. Spleen populations treated in vitro with antithymocyte serum + complement are called B because they lack T cells (TABLE 4). All were challenged 4 weeks later with 100 pmole AChR and all developed severe and persistent adjuvant arthritis. Only the sham-Tx group showed signs of EAMG; however, the weight patterns of all groups possessing T cells (splenic or thymic) paralleled that of the sham-Tx group with EAMG between days 5 and 40 after inoculation.

sents a reduction of safety factor in rats possessing T cells since 8 pg of curare did not cause a decrement in any of 32 normal rats inoculated with adjuvants only.

DISCUSSION

Rats inoculated once with heterologous AChR protein with adjuvants de- velop autoimmunity to skeletal muscle AChR.21 l l ~ l4 This is evidenced clinically as two episodes of EAMG.',11 The first episode occurs 8 days after inoculation, is acute in onset and transient; the second occurs 25-30 days postinoculation and is chronic in its course. Transient infiltration of muscles with inflammatory cells, which are predominately mononuclear, occurs in the acute phase of EAMG.?a In this paper we have described both cell-mediated immunity and antibody to AChR occurring in rats in response to inoculation with AChR.

Cell-mediated immunity to AChR preceded the clinical and histologic onset of EAMG, being detectable by skin testing 4 to 5 days postinoculation. Skin test sites were consistently negative until the fifth day postinoculation when skin sites tested at both day 3 and 4 (48 and 24 hr earlier, respectively) became positive. Positive skin reactions thereafter peaked in intensity 24 hr after testing. Thus the cells responsible for delayed-type hypersensitivity reactions were first evident in the tissues on day 4. As EAMG progressed into its chronic phase, rats lost specific delayed cutaneous reactivity to eel AChR and later also became anergic to the mycobacterial antigen PPD (a component of the adjuvant).

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FIGURE 5. Proposed immunopathogenesis of EAMG. This sequence of events was synthesized from the immunologic, ultrastructural," and electrophysiologic 14.31 events occurring in rats inoculated on day 0 with AChR and adjuvants. Antigen entering regional lymph nodes (LN) initiates an immune response by interacting with macrophages and T and B lymphocytes. Approximately 80% of lymphocytes entering the circulation are T. By day 4 postinoculation T cells mediating delayed-type hypersensitivity to AChR are detectable in remote tissues (by skin testing). By day 8 the motor end-plate region is densely infiltrated with inflammatory cells (and presumably antibody molecules also) and the underlying muscle is the target of destruction." By day 12 inflammatory cells are not detectable but postsynaptic ultrastructural damage


Because of the complexity of factors that could be influencing cellular immune responses in vivo at this stage, further analysis will require investigation in vitro.

Delayed-type cutaneous reactivity is a classic property of thymus-derived (T) lymphocytes and is mediated by pharmacologically active “lymphokines,” which are released by the immune T cell on contact with the antigen to which it has been sensitized.25 An early event in the lesion accompanying delayed hypersensitivity reactions is an increase in vascular permeability.z6 At 24-48 hr after contact between an immune T cell and its antigen there occurs massive infiltration of inflammatory cells, predominately mononuclear in character. Presumably this is the mechanism responsible for the accumulation of inflammatory cells at the neuromuscular junction 8 days after inoculation with AChR.23 Both the pleomorphic nature of the cells infiltrating the motor end-plate region 23

and the early time of onset of acute phase EAMG2. l 1 are most likely due to the use of B. pertussis Zi as additional adjuvant.

Antibody to eel AChR was detectable readily in serum at the time of onset of the acute phase of EAMG and by day 10 postinoculation more than half of the antibody sedimented as 7 S and the rest as 19 S, suggesting that a switch from IgM to IgG production had occurred very early. By day 35 all detectable anti-AChR antibody was 7 S (presumably IgG). The 7-S antibody, which appeared later, cross-reacted with rat muscle AChR and presumably represented a population of antibody with a high affinity to determinants shared by both eel and rat muscle AChR.ll As EAMG progressed into its chronic phase, the titer of cross-reacting antibody continued to climb.

Transfer of EAMG to normal rats by immune lymph node cells further suggested that T cells might play an important role in the pathogenesis of EAMG. Thymectomy after the onset of the chronic phase of EAMG did not alter the course of the disease. EAMG induction failed however in rats in which T cells had been virtually eliminated by a combination of thymectomy, x-irradiation. and reconstitution with a population of spleen cells depleted of T lymphocytes. Moreover, no detectable anti-AChR antibody was produced in the absence of T cells. The presence of an intact thymus, or transfusion of thymocytes or splenic T cells, appeared to be necessary for both production of antibody to AChR and for immunopharmacologic blockade of motor end-plate function.28 This indicates that T cells are involved in EAMG not only as cytotoxic effectors (which give rise to delayed-type hypersensitivity reactions) but also as helpers for the B cells in their production of antibody to AChR.

Considerable recent evidence attests to the existence of heterogeneity in the peripheral T-lymphocyte :In The response we observed after early treatment in vivo with antithymocyte serum might be explained on the basis of functional heterogeneity of the rat peripheral T-cell pool. Thus it could be envisaged that inhibition of the early episode of EAMG reflected a differential suppression of development of delayed-type hypersensitivity responses by ATS. The subsequent onset of chronic EAMG and the presence, 41 days postinoculation, of significant titers of antibody to AChR indicate that helper T-cell function was relatively unimpaired.

The preliminary observations we have presented concerning the time course of cellular and humoral immune responses to AChR should be considered in relation to the evolution of clinical,?. * histologic,23 and electrophysiologic events l’, 31 subsequent to these responses. In FIGURE 5 we have schematically

(loss of junctional folds and widened synaptic space) persists in the presence of high titers of antibodies to muscle AChR. A patient with MG would be represented by the final picture in frame 7.


proposed a sequence of events to explain the immunopathogenesis of EAMG. The elicitation of positive delayed cutaneous reactivity to AChR by skin testing 4 days postinoculation indicates the presence of immune T cells in the tissues at this time. The massive infiltration of the motor end-plate region with mononuclear inflammatory cells on day 8 , z 3 is most likely triggered 24-48 hr earlier by the release of lymphokines by an immune T cell interacting with readily accessible antigenic determinants of AChR at the neuromuscular junction. AChR in the postsynaptic membrane of an intact neuromuscular junction is probably not readily accessible for immune interactions with cells, but antibody molecules appear to have free access to the end plate.3z Lytic enzymes released in the inflammatory response consequent to immunologic interaction in the motor end-plate region would lead to exposure of the postsynaptic membrane structures which appear to be the primary target of attack by mononuclear cells.23 Cellular attack on the postsynaptic membrane is probably mediated not only by immune T cells, but also by nonimmune B cells and macrophages rendered cytotoxic in the presence of anti-AChR antibodies."! 3 L In this acute phase of EAMG, interruption of neuromuscular transmission could be in part due to inflammatory cells causing physical separation of nerve endings from the postsynaptic membrane of muscle. Increased sensitivity to curare l4 and small miniature end-plate potentials 31 persist after the disappearance of inflammatory cells (after approximately day 12). This later impairment of neuromuscular transmission is no doubt due to the continued presence of anti-AChR antibodies, which from day 25 comprise a high titer of 7-S antibody with affinity for syngeneic muscle AChR. It is conceivable that persisting antibody interferes with regeneration of the damaged end plate, which remains simplified in ultrastructure with widening of the postsynaptic space.23

The patient presenting with clinical MG is represented by the final frame in FIGURE 5 : impaired neuromuscular transmission,35 simplification of the ultrastructure of the postsynaptic membrane,23 and high titers of 7-S anti-human- muscle-AChR antib0dies.l' The events preceding this picture in human MG are not known. It has been reported recently that the occurrence of MG in man is associated with certain histocompatibility antigen^.^^-^^ From our knowledge of the linkage of genes controlling histocompatibility antigens in inbred animals 3'3 to genes controlling specific immune responses, it is likely that susceptibility to organ-specific autoimmunity in man is genetically deter- mined. The etiologic factor in MG, represented by the syringe injecting AChR and adjuvant in the animal model, EAMG, could be either an environmental immunogen (? viral) cross-reacting with AChR, or an altered autoantigen. This remains the greatest mystery of MG today.

SUMMARY

Rats inoculated intradermally with eel acetylcholine receptor protein ( AChR) with adjuvants developed autoimmunity to skeletal muscle AChR. This is evidenced clinically as two episodes of experimental autoimmune myasthenia gravis (EAMG), an acute phase that occurs early ( 8 days) and is transient, and a chronic phase ( 3 0 days) that is usually progressive. Positive delayed cutaneous reactivity appeared at day 4 and serum antibody to eel AChR was detectable by day 7 postinoculation. After day 25 the titer of antibody to syngeneic muscle AChR rose abruptly. Antibody to muscle AChR sedimented


as 7 S. Lymph node cells from rats sensitized to AChR were capable of transferring E A M G to normal recipients. Thymectomy after the onset of E A M G had no effect. Early treatment in vivo with antithymocyte serum suppressed acute but not chronic phase EAMG. Experiments combining thymectomy, x-irradiation and reconstitution with distinct populations of lymphocytes indicated that thymus-derived lymphocytes are required for induction of E A M G and antibody to AChR. These data suggest that both cellular and humoral responses to AChR, either sequentially or in combination, contribute to the pathogenesis of EAMG.

ACKNOWLEDGMENTS

We thank Dr. R. Hyman for reviewing this manuscript and Eli Lilly and Co. for gifts of B. pertussis and Tylosin. We gratefully acknowledge the ex- cellent technical assistance of Janet Clark, Joy Nieder, Ruth Rubin, and Millie Thompson.

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