Clin. exp. Immunol. (1975) 21, 185-201.

REVIEW

EXPERIMENTAL AUTOALLERGIC MYOSLTIS, POLYMYOSITIS AND MYASTHENIA GRAVIS AUTOIMMUNE MUSCLE DISEASE ASSOCIATED WITH IMMUNODEFICIENCY AND NEOPLASIA R. L. DAWKINS Royal Pcrth Hospital and Perth Medical Centre, Western Australia, Australia (Received 6 January, 1975) SUMMARY INTRODUCTION

I. EXPERIMENTAL AUTOALLERGIC MYOSITIS 1. 2. 3. 4.

Serological approach Histopathological approach Electrophysiological approach Features of experimental autoallergic myositis (a) Species and strain susceptibility (b) Pathology (c) Serology (d) Cell-mediated immunity (e) Passive transfer (f) Immunization schedules and disease course

(g) Nature of the antigen (h) Implications for human disease

II. POLYMYOSITIS 1. Definition and clinical features 2. Pathology 3. Serology 4. Cell-mediated immunity 5. Immune status 6. Significance of immunodeficiency III. MYASTHENIA GRAVIS 1. Definition and clinical features 2. Pathology 3. Serology 4. Cell-mediated immunity 5. Relationship between different features 6. Pathogenesis of neuromuscular block 7. Autoimmune muscle disease. Thymomata and immunodeficiency CONCLUSIONS

INTRODUCTION Human polymyositis and myasthenia gravis are of general immunological interest for at least two reasons. First, investigation of these diseases suggests that humoral immunodeficiency may be involved in the genesis of autoimmune muscle disease and may be a factor in the pathogenesis of myositis actually mediated by delayed hypersensitivity mechanisms. Secondly, in both diseases there is an association with neoplastic disease which appears to be related to the presence of humoral immunodeficiency. Since insights into these disorders may come from examining the effects of immunizing animals with skeletal muscle it is pertinent to review the current status of experimental autoallergic myositis (EAM) before analysing the human situation in detail. Correspondence: Dr R. L. Dawkins, Clinical Immunology Division, University Department of Pathology, Perth Medical Centre, Western Australia, 6008, Australia.

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I. EXPERIMENTAL AUTOALLERGIC MYOSITIS By analogy with the other experimental autoallergies (Waksman, 1962) this term is used to refer to the various consequences of immunization with skeletal muscle emulsified in Freund's complete adjuvant. Since some workers have restricted themselves to the serological, histological and electrophysiological effects it will be necessary to introduce these studies before attempting to characterize EAM as an entity. 1. Serological approach For many years myobiologists have been interested in the antibodies obtained by immunizing animals with skeletal muscle. At an early stage in its development immunofluorescence was applied to the localization of contractile proteins within the sarcomere (Holtzer, 1959, 1970; Ebashi and Nonomura, 1973). A continuing problem in this work has been the purification of antigens together with the fact that some of the contractile proteins appear to be more immunogenic than others. Not surprisingly, some aspects are controversial but irrespective of their precise biochemical nature, distinct antigens can be demonstrated at the M line, the A, I and Z bands, and at various junctional sites within the sarcomere. These antigens can be considered tissue-specific with the proviso that there is some cross-reactivity between skeletal and cardiac muscle. The immunological relationship between skeletal muscle actin and myosin and similar proteins found in smooth muscle and other non-muscle tissues remains to be determined (Trenchev, Sneyd & Holborow, 1974; Bray, 1974). Immunofluorescence using various antimuscle sera has been used to elucidate the changes which occur during myogenesis including the relationships between immunological and morphological maturation (Dawkins & Lamont, 1971; Dawkins, Aw & Simons, 1972; Papadimitriou & Dawkins, 1973). These studies are of relevance to human disease in that they indicate there is a multitude of muscle-specific antigens. However it is noteworthy that all of these are present in the cytoplasm and they have not been demonstrated on the muscle cell surface. 2. Histopathological approach Early attempts to determine the histological effects of immunization with skeletal muscle have been reviewed by Sobue (1968). The best known of these was the work of Pearson (1956) whereby immunization of rats with skeletal muscle and Freund's complete adjuvant led to the recognition of adjuvant arthritis. Since the first demonstration of a musclespecific lesion akin to those found in the other experimental autoallergies (Dawkins, 1963, 1965) numerous workers have examined the pathology of EAM (see below). In general, these studies have been designed to explore the possible relationship between the experimental disease and human polymyositis. Insufficient attention has been paid to similarities with other diseases.

3. Electrophysiological approach Following the suggestion of Simpson (1960) and the findings of Strauss et al. (1960) and Nastuk, Plescia & Otterman (1960) there were numerous attempts to reproduce the electrophysiological features of human myasthenia gravis by immunization with muscle. Early studies were uniformly negative (Strauss, 1963; Dawkins, 1963; Parkes, 1966; Vetters, Simpson & Folkarde, 1969) but in 1966 it was reported that immunization with muscle or

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thymus could lead to an inflammatory lesion in the thymus designated 'experimental thymitis' and to electrophysiological changes resembling those found in myasthenia gravis (Goldstein & Whittingham, 1966, 1967; Goldstein & Hofman, 1968; Goldstein, Strauss & Pickeral, 1969). Only the diaphragm was examined pathologically, but it was stated that skeletal muscle was not affected. This work was confirmed by Kalden & Irvine (1969) and more recent studies have been consistent with Goldstein's hypothesis that a certain inflammatory lesion of the thymus leads to release of a neuromuscular blocking factor (Goldstein and Manganero, 1971; Kalden et al., 1969, 1970, 1973). Other groups, however, have been unable to reproduce either the inflammatory lesion in the thymus or the electrophysiological features (Kauffman, Rushworth & Wright, 1969; Vetters et al., 1969; Webb, 1969; Jones, Brennan & McLeod, 1971). The explanation for these divergent findings is not apparent, so that generalization is inappropriate at this time, but a number of aspects are of interest in relation to EAM. Attempts to produce an experimental model for myasthenia gravis first revealed that immunization of guinea-pigs with xenogeneic muscle could lead to the production of antibodies which react with thymic myoid cells as well as skeletal muscle (Goldstein & Whittingham, 1967; Kalden et al., 1969,1970). Although these myoid cells are extremely sparse in the adult thymus of higher vertebrates, this finding of antigenic cross-reactivity provided a simple explanation for the claim that anti-muscle immunity could induce inflammatory lesions within the thymus. However, it also posed some major issues which are still unresolved. Thus, if immunization with muscle leads to 'thymitis' by virtue of cross-reactivity between muscle and thymic myoid cells, then generalized myositis might be expected. Furthermore, by virtue of the same cross-reactivity, immunization with thymus could lead to myositis as well as 'thymitis'. These possibilities are especially pertinent since myositis per se could influence, if not induce, the observed electrophysiological changes (Dawkins, 1971) and appropriate controls were not included in the experiments reported. This aspect became even more relevant after Goldstein claimed that the thymus could release a second factor capable of inducing a toxic myositis (Goldstein & Manganero, 1971). In 1973, Kalden et al., attempting to clarify the situation, showed that immunization with muscle does lead to myositis as well as 'thymitis'. The electromyographic features were not described but it was concluded that neuromuscular block and 'thymitis' could occur independently of myositis. These findings are difficult to reconcile with any explanation of 'thymitis' in terms of crossreactivity between muscle and thymus, and they appear incompatible with Goldstein's suggestion that myositis is a consequence of 'thymitis'. Many other aspects remain to be elucidated; in the meantime it appears unwise to accept 'thymitis' or neuromuscular block as a specific consequence of immunization with muscle. On the other hand, as will be discussed below, the myositis which occurs may be as relevant to the inflammatory muscle disease of myasthenia gravis as it is to polymyositis (Dawkins, 1965; Currie, 1971; Kalden et al., 1973). 4. Features of experimental autoallergic myositis In this section the disease which follows immunization with muscle will be described, but no further reference will be made to reports concerned with only serological or electrophysical effects. (a) Species and strain susceptibility. Experimental autoallergic myositis (EAM) has been produced in guinea-pigs (Dawkins, 1965; Takayanagi, 1967; Dawkins, Eghtedari & Holborow, 1971; Currie, 1971; Kalden et al., 1973; Manghani, Partridge & Sloper, 1974a), rats

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(Dawkins, 1963; Kakulas, 1966; Parkes, 1966; Currie, 1971; Morgan, Peter & Newbould, 1971; Esiri & MacLennan, 1974, 1975) and possibly mice (Boehme, 1965; Tolnai, 1966) although in the last instance it is not clear whether the lesions were really similar to those seen in the other species. In spite of the use of multiple strains of guinea-pigs and rats there has been no systematic study of differing susceptibility. Preliminary experiments, however, suggest that there are substantial differences, Strain 13 guinea-pigs being more susceptible than some outbred strains (Dawkins, unpublished results). (b) Pathology. There is substantial agreement that the characteristic histological lesion is focal segmental myofibre necrosis with variable amounts of interstitial mononuclear infiltration (Dawkins, 1965; Kakulas, 1966; Currie, 1971; Esiri & MacLennan, 1974; Manghani et al., 1974a). Additional features include regeneration and perivascular inflammatory changes. Some workers have found more extensive necrotizing lesions especially in rats (Morgan et al., 1971; Kalden et al., 1973) but typically only one, or, at most a few, adjacent fibres are affected, producing relatively discrete foci. These features contrast with the more extensive granulomatous lesions spreading via fascial planes from the site of inoculation of Freund's adjuvant. The most likely source of confusion is focal injury of the type produced by intramuscular injection but infectious agents can probably lead to a similar appearance. The lesions of EAM are found in limb and paravertebral musculature. Relative sparing of the diaphragm may explain the failure to detect myositis in the early experimental 'thymitis' work (Kalden et al., 1973). The heart may be involved (Dawkins, 1963; Kalden et al., 1973) but other tissues including smooth muscle are spared. Most workers have not found significant abnormalities in the thymus even when myositis is present (Webb, 1970a; Dawkins et al., 1971). Features which correlate with the presence of myositis include elevations of serum creatine kinase (Morgan et al., 1971; Esiri & MacLennan, 1974; Manghani et al., 1974a) and a myopathic electromyogram (Dawkins, 1963). In this latter work fibrillation potentials and polyphasic motor units were found without evidence to suggest myasthenia. More work on this aspect is required. Curare sensitivity was demonstrated by a head-drop method but this appeared to be non-specific in that it occurred with atrophy as well as myositis. Gross clinical weakness has not been described. Notable defects in knowledge of the pathology of EAM exist in histochemistry to determine whether the focal distribution reflects selective fibre type involvement (Dawkins, Gibb & Holt, 1974), electron microscopy to elucidate the muscle-mononuclear cell interaction (Fulthorpe & Hudgson, 1974; Mastaglia, Papadimitriou & Dawkins, 1975) and direct immunofluorescence to detect whether immunoglobulin and complement are deposited in muscle. Further work is also required to examine the suggestion that the incidence of myositis is decreased by immunosuppressive drugs (Currie, 1971). (c) Serology. The possibility of anti-muscle antibody in EAM was considered by Takayanagi (1967) and Webb (1970b) but a distinction between histocompatibility and musclespecific antibodies of the type shown by Goldstein & Whittingham (1967) was not achieved until 1971 (Dawkins et al.). The precise sites of reaction of this antibody within the sarcomere have yet to be determined, but comparison with the patterns produced by antisera raised against various contractile proteins suggests that multiple antigens must be involved. Most sera appear to stain the I band and the M line (Dawkins, 1973b; Dawkins, Robinson & Wetherall, 1975) and therefore create patterns consistent with antibodies directed

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against tropomyosin, troponin, actinin or actin as well as M protein or perhaps heavy meromyosin. An alternative pattern involving the A and Z bands (Partridge, Manghani & Sloper, 1973) suggests that there may be antibodies to myosin, light meromyosin, or C protein. Further work will be required but it seems very likely that a number of antigens in the crude muscle homogenate are immunogenic. In spite of this apparent heterogeneity, however, sera from animals with EAM behave as though they contain a single musclespecific antibody which cross-reacts with cardiac muscle and thymic myoid cells but not smooth muscle or other tissues (Dawkins et al., 1971). Experiments using centrifugal fractions of skeletal muscle for immunization have indicated that other antimuscle but non-striation-binding antibodies may be produced (Namba & Muguruma, 1972; Kalden et al., 1973; Dawkins et al., 1974). These include antibodies which react with specific muscle fibre types and perhaps the sarcolemma. None of these muscle-specific antibodies has been shown to bind to surface antigens on viable muscle cells and none has been shown to be cytotoxic in vitro (Dawkins & Holborow, 1972; Dawkins &

Loewi, 1972). (d) Cell-mediated immunity. In the initial work it was shown that animals with EAM regularly possessed delayed cutaneous hypersensitivity to skeletal muscle (Dawkins, 1965; Currie, 1971). Lymphoid cells are cytotoxic to muscle cultures apparently by means of direct T-cell killing but further work is required to exclude K-cell and macrophage mechanisms (Kakulas, 1966; Currie, 1971; Dawkins, 1973b; Partridge & Smith, 1974). Lymphocyte transformation studies have suggested delayed hypersensitivity to a variety of syngeneic muscle antigens including at least one soluble component (Esiri & MacLennan, 1975). Spleen cells proved more sensitive than other lymphoid cells. Other workers have found evidence of inhibition of macrophage migration with myosin and tropomyosin (Manghani et al., 1974b). (e) Passive transfer. Passive transfer has been repeatedly demonstrated with lymphoid cells rather than serum (Takayanagi, 1967; Morgan et al., 1971; Currie, 1971; Esiri & MacLennan, 1974). In general, lesions were less frequent in recipients than in donors but the character was essentially the same and the latent period was reduced as expected. (f) Immunization schedules and disease course. Approximately 10 days after a single injection of xenogeneic muscle in Freund's complete adjuvant some animals will develop myositis (Dawkins, 1965; Kakulas, 1966; Morgan et al., 1971) but most workers agree that between two and four weekly injections are necessary for an incidence approaching 100% (Dawkins, 1965; Esiri & MacLennan, 1974; Manghani et al., 1974a). With further weekly injections the incidence appears to decrease, although some animals then show muscular atrophy rather than recovery (Dawkins, 1965; Currie, 1971; Esiri & MacLennan, 1974). This decline probably explains the claim that multiple injections can lead to a non-inflammatory dystrophy-like appearance (Tal & Liban, 1962). This uniphasic relatively transient course is paralleled by similar changes in delayed cutaneous hypersensitivity (Dawkins, 1965), potential of lymphoid cells for passive transfer and serum creatine kinase activity (Esiri & MacLennan, 1974). Thus some form of inhibition of cell-mediated immunity akin to 'desensitization' appears to occur. The antistriational antibody, however, follows a rather different course in that it may appear later and persist for many months (Dawkins et al., 1971; Manghani et al., 1974a; Dawkins, unpublished results). This dissociation of the humoral and cell-mediated responses is in accordance with the general view that T-cell mechanisms are involved in the pathogenesis of myositis but it

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also raises the possibility that antibody may be inhibitory and even protective. Other mechanisms may also protect against the development of EAM as is suggested by the finding that thoracic duct drainage can increase the severity of the myositis (Esiri & MacLennan, 1974). (g) Nature of the antigen. In general it has been found that xenogeneic muscle is more effective than allogeneic or syngeneic in producing EAM and this has been attributed to an adjuvant or carrier-like function of histocompatibility antigen (Dawkins et al., 1971). Irrespective of the source of muscle used for immunization there are immune responses which are directed against syngeneic muscle-specific antigens (Dawkins et al., 1971; Esiri & MacLennan, 1975). From the pathological and serological studies described above the antigen appears to be present in cardiac muscle as well as both major types of skeletal muscle. Although there has been no systematic study of the effectiveness of these different tissues for immunization it has been reported that administration of heart can induce myositis (Trisotto et al., 1970). Attempts to identify the responsible antigen by using centrifugal fractions for immunization have yielded conflicting results (Morgan et al., 1971; Kalden et al., 1973; Manghani et al., 1974a; Dawkins et al., 1974). More sophisticated methods are required to allow separation of the heterogeneous immune responses which must follow the use of relatively crude antigenic preparations. In vitro tests with more purified antigens (Manghani et al., 1974b; Esiri & MacLennan, 1975) have yet to contribute information as to the nature of the particular antigen which is involved in the pathogenesis of the myositis per se. (h) Implications for human disease. Although many aspects require further clarification, work on EAM strongly suggests that myositis may be produced by cell-mediated mechanisms directed against muscle antigens. Humoral responses may be irrelevant to pathogenesis or they could reflect some antibody-mediated protective function. Irrespective of their biological significance they are likely to provide a valuable means of detecting the presence of autoimmune reactions against skeletal muscle. There can be little doubt that attempts to promote EAM as a model of a particular human disease have been exaggerated. As pointed out by Currie (1971) the histological characteristics of human polymyositis, interstitial nodular polymyositis and myasthenia gravis overlap with those of EAM. Similarities and differences will be considered further below but it would appear more valuable to consider EAM as a model of a process (autoimmune myositis) rather than a syndrome complex. This seems especially so in relation to polymyositis and myasthenia gravis since these conditions are clinically heterogeneous but it may be even more so in connective tissue disease which can be associated with myositis. Thus myositis might be compared with synovitis or autoimmune haemolysis since these processes are found in association with many diseases characterized by immunological dysfunction. In this context it may be noted that lymphocyte-mediated cytotoxicity of the type seen in EAM has been shown to occur in some patients with connective tissue diseases and associated myositis (Currie et al., 1971; Kakulas, Shute & Leclere, 1972; Dawkins, 1973b) in addition to patients with polymyositis and myasthenia gravis. II. POLYMYOSITIS and 1. Definition clinicalfeatures Polymyositis refers to those chronic inflammatory disorders characterized by necrotizing

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myositis and symmetrical sustained weakness of proximal muscle groups. There are three criteria which must be met before the diagnosis can be sustained. (a) Objective evidence of symmetrical weakness of proximal muscle groups with relative sparing of distal and extraocular muscles and an excess of weakness over wasting. The weakness may wax and wane but it differs from myasthenia in that it does not disappear completely during the course of hours or days. (b) Systemic and/or local evidence of necrosis of skeletal muscle where systemic evidence includes elevated serum creatine kinase and local evidence consists of myofiber necrosis or regeneration implying previous necrosis. (c) Systemic and/or local evidence of inflammatory disease where systemic evidence includes fever and elevated erythrocyte sedimentation rate and local evidence consists of interstitial and/or perivascular mononuclear infiltration. These three criteria must show some temporal relationship. In addition it is necessary to exclude the possibility of infectious, neurogenic, genetic, metabolic, endocrine, toxic and physical disorders which may mimic polymyositis to varying degrees. This definition emphasizes the fact that the central and invariable feature of polymyositis is necrotizing myositis, but less constant features may be prominent. Foremost of these is the coexistence of specific skin lesions such as the heliotrope rash and Gottron's sign which allow the designation dermatomyositis. There is some evidence that skin involvement is a reflection of systemic vasculitis (Banker & Victor, 1966; Whitaker and Engel, 1972; Dawkins & Mastaglia, 1973b) so that there is a possibility that ischaemia contributes to the muscle pathology in these patients. However, skin and muscle involvement may follow separate courses and probably reflect quite different processes. Another characteristic of polymyositis is the frequent presence of features resembling those found in the connective tissue disorders. Thus synovitis may complicate polymyositis just as myositis may occur with rheumatoid arthritis. As mentioned above, experience with EAM suggests that myositis should be thought of as a process rather than a disease in itself. A third generally accepted feature of polymyositis is the co-existence of neoplasia especially in the elderly (Williams, 1959; Shy, 1962; Pearson & Currie, 1974) but not all agree (Bohan & Peter, 1975). The types of tumour found occur in approximately the same relative proportions as tumours in the population as a whole. However lymphoma may be rather more frequent than would be predicted by this rule. Further description of polymyositis and additional references are provided by Pearson (1966), Pearson & Currie (1974) and Bohan & Peter (1975). 2. Pathology The essential histopathological characteristic of polymyositis is myofiber necrosis with variable degrees of inflammatory infiltrate. In some instances the picture is described as myolysis without significant inflammatory infiltrate, but such cases should probably be excluded (Bohan & Peter, 1974; Dawkins & Mastaglia, in preparation). Although there is a resemblance to EAM the myositis is often more severe with a less focal character. The heart may be involved, but smooth muscle is spared and other pathological features can be explained in terms of: (i) the overlap with the connective tissue diseases; (ii) the local and metastatic effects of an associated tumour; and (iii) the systemic vasculitis which may occur especially in children with dermatomyositis. The finding of immunoglobulin in vessel walls (Whitaker & Engel, 1972) may belong in this last category but its significance is uncertain

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since inflammatory changes were not found and hypocomplementaemia is rarely, if ever, present (Dawkins & Mastaglia, 1973b; Bohen & Peter, 1975; Leddy et al. 1975). As in the case of EAM serum creatine kinase and electromyography probably reflect the degree of necrotizing myositis which would be seen in a muscle biopsy if there were no sampling problems. 3. Serology In spite of many exhaustive searches there is no definite evidence of antimuscle antibody in polymyositis. One study (Caspary, Gubbay & Sterne, 1964) has been interpreted as showing that anti-crude myosin and anti-crude muscle antibodies, which occurred in approximately 500% of patients with various forms of muscle disease, are a consequence of injury to muscle. However, a very similar incidence was shown in blood donors and we have never been able to demonstrate antimuscle antibodies in animals after various forms of muscle injury (Dawkins, unpublished results). It appears much more likely that the reported antibodies were due to non-specific reactions, but details were not given and assessment is difficult. Other workers have shown that non-specific reactions can occur and have failed to demonstrate true anti-muscle antibody (Strauss, 1968; Fessel & Raas, 1968; Aarli, 1972; Dawkins, 1973b). One recent study (Nishakai & Homma, 1972) reports the finding of antimyoglobin antibodies in polymyositis, but this has yet to be confirmed using adequate controls. Considering the evidence as a whole, it can be concluded that an obvious humoral anti-muscle response has yet to be demonstrated and that there is certainly no antibody akin to that found in EAM. This conclusion raises the possibility that there might be some restraint on humoral responsiveness in polymyositis. It was therefore of interest to determine the frequency of 'irrelevant' autoantibodies such as anti-nuclear factor (ANF). Many authors have claimed that non-muscle autoantibodies are very common in polymyositis but not all agree (Caspary et al., 1964; Currie et al., 1971) and a recent survey has suggested that the frequency may have been exaggerated due to the inclusion of patients with clear-cut systemic lupus erythematosus (SLE), rheumatoid arthritis or progressive systemic sclerosis complicated by myositis (Dawkins & Mastaglia, 1972; Dawkins, 1973a). 4. Cell-mediated immunity In contrast to the serological evidence there is little doubt that there is a cell-mediated reaction to skeletal muscle. Lymphocyte transformation with human muscle appears to be increased especially in the more active cases (Currie et al., 1971; Esiri, MacLennan & Hazelman, 1973). Lesser increases may be seen in other disorders, such as polymyalgia rheumatica, but this has been attributed to the use of crude muscle containing vascular antigens (Esiri et al., 1973). Lymphocyte-mediated cytotoxicity to muscle can be demonstrated by a variety of techniques (Currie et al., 1971; Kakulas et al., 1972; Johnson, Fink & Ziff, 1972; Dawkins & Mastaglia, 1973a). There are a number of problems with each of these systems and the actual mechanism of the myotoxicity is not clear, but evidence to date suggests that the process assayed reflects direct T-cell killing of muscle target cells. Antilymphocyte serum appears to be inhibitory in vitro (Currie et al., 1971) and treatment of the patient with immunosuppressive therapy decreases cytotoxicity pari passu with the suppression of delayed hypersensitivity to ubiquitous antigens (Dawkins & Mastaglia, 1973a).

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5. Immune status The evidence provided above suggests that the myositis of polymyositis is due to a T cellmediated cytotoxic reaction with a relative or absolute absence of the type of humoral response seen in EAM. As a consequence, tests of immunological status were undertaken. Cell-mediated immunity was shown to be normal unless patients were on immunosuppressive therapy. In contrast, in a group of sixteen patients it was found that 5000 had moderately depressed IgG concentrations, 25% low isohaemagglutinin titres and 5000 poor humoral responses to tetanus toxoid immunization (Dawkins, 1973a,b). These studies should be extended, but they suggest that polymyositis is associated with a humoral immunodeficiency state. It is relevant that polymyositis has been reported to occur in patients with gross hypogammaglobulinaemia (Gotoff, Smith & Sugar, 1972). This association assumes particular importance since the immune defect is genetically determined, and clearly antecedes the development of polymyositis. Tumours which may induce a depression of cell-mediated immunity have been reported to be associated (Currie et al., 1971) but this situation seems much less significant because the immune defect appears relatively late and has not been shown to precede or even coexist with active polymyositis. A hereditary complement deficiency has been described in a patient with dermatomyositis (Leddy et al., 1975). 6. Significance of immunodeficiency On the basis of the evidence cited above we have suggested that polymyositis may be a consequence of pre-existing immunodeficiency (Dawkins, 1973a,b; Dawkins & Zilko, 1975). This defect could be genetically determined and it is of interest that familial polymyositis has been reported (Howard & Thomas, 1960; Lambie & Duff, 1963; Lewkonia and Buxton, 1973). Alternative explanations may be possible but this speculation does pose some questions of general immunological interest. It must be emphasized that the postulated defect must be subtle and selective and could be expressed transiently. One consequence of such a defect could be the development of autoimmune disease due to the loss of a humoral mechanism which is involved in the maintenance of tolerance. Antibody may normally prevent the emergence or activity of self-reactive T cells. At the same time the defect could lead to the development of neoplasia due to a parallel failure of humoral immunosurveillance. In this context it is of interest that the types of tumours which coexist with polymyositis are found in similar proportions to those occurring in the general population. Thus, polymyositis may be a more significant example of immunodeficiency leading to neoplasia than severe forms of hypogammaglobulinaemia with which essentially only lymphoid tumours are associated. It is noteworthy that selective IgA deficiency appears to lead to an increased incidence of a large variety of tumours in addition to autoimmune diseases (Kersey, Spector & Good, 1974). Although this explanation for the coexistence of tumours with polymyositis requires further experimental support it appears inherently more likely than conventional dogma which views polymyositis as an aberrant anti-tumour reaction. It would be difficult to postulate that all the tumours associated with polymyositis possess muscle-specific antigens. Apparently only one patient has been adequately studied from this point of view and this work provided no clear evidence of cross-reactivity involving cell-mediated or humoral immunity (Dawkins, 1973b). Early work appears to be based on the possibility of only serological cross-reactivity (Grace and Dao, 1959; Curtis, Heckaman & Wheeler, 1961; Alexander & Foreman, 1968) and none of these studies provide any real basis for the cross-

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reaction hypothesis. Immune complex deposition might be considered, but this would appear more relevant to dermatomyositis than polymyositis as a whole. Thus it seems most likely that the myositis and the neoplasia are independent manifestations of some underlying predisposition, especially since the tumour may not appear until after the diagnosis of polymyositis. One possible argument against the common predisposition hypothesis would be the much quoted improvement which occasionally occurs after removal of the associated tumour. However, these situations are extremely complex in view of the immunosuppressive effects of tumours and anti-tumour therapy including radiotherapy and surgery. Another possible implication of the postulated immunodeficiency state relates to the finding of virus-like particles in occasional patients with polymyositis. Some of these viruses may be involved in the production of a true viral myositis which can be confused with classical polymyositis but others, and especially those seen in chronic polymyositis, appear more likely to be incidental 'passengers', possibly reflecting an immunodeficiency state. A predisposition to viral infection could also be invoked to explain at least some of the associated tumours especially those involving lymphoid tissue. Finally the immunodeficiency hypothesis may have implications for therapy. Various forms of immunosuppressive therapy have been used (Benson & Aldo, 1973; Haas, 1973; Metzger et al., 1974; Bohan & Peter, 1974) but there is no consensus as to the most effective drug. The ideal agent may be a specific suppressor of T cell-mediated reactions without detrimental affects on any humoral protective mechanism. The report of polymyositis occurring after penicillamine therapy could be viewed in terms of the inhibitory effect of this drug on humoral rather than cell-mediated immunity (Schraeder, Peters & Dahl, 1972).

III. MYASTHENIA GRAVIS 1. Definition and clinicalfeatures The defining feature of myasthenia gravis is the presence of a specific form of neuromuscular block leading to excessive fatigability (Simpson, 1960; Strauss, 1968; Rowland et al., 1973; Engel et al., 1974). Since a consistent and specific pathological characteristic has never been demonstrated, the disease must be defined in clinical and electrophysiological terms. The thymus is histologically abnormal in a majority of patients but changes range from hyperplasia with a few germinal follicles to thymoma. Less commonly, examination of skeletal muscle shows inflammatory changes, but these vary from scattered collections of lymphocytes to frank myositis. Serum antibody reactive with skeletal muscle may be found but only in approximately one third of all cases. Hypocomplementaemia occurs in some patients, but substantial fluctuations around the normal mean may be more characteristic than significantly depressed levels. On the basis of these and other clinical features it has been suggested that myasthenia gravis is an autoimmune disease (Simpson, 1960; Strauss et al., 1960; Nastuk et al., 1960) but, in spite of numerous attempts, the variability in the pathological and immunological findings has never been explained and the abnormalities referred to have never been shown to be directly related to neuromuscular block. On this occasion the evidence will be reviewed in the light of EAM and it will be suggested that two quite different processes lead to neuromuscular block and autoimmune muscle disease. Thus, some of the immunopathological variation can be explained to the extent that these two processes vary independently of each other and that muscle disease only occurs in a minority of patients.

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2. Pathology

Examination of the thymus may reveal thymic hyperplasia or thymoma but occasionally there is neither or even both. The histological features of thymomata vary considerably and there is no clear relationship between particular appearances and associated clinical features (Korn et al., 1967). Skeletal muscle may be completely normal or may show relatively non-specific atrophic changes, but myositis is well recognized. Although the inflammatory changes are generally mild and restricted to lymphorrhages there appears to be a spectrum with rare patients exhibiting the clinical and pathological picture of polymyositis. In the last group a thymoma is a regular feature (Rowland et al., 1973; Namba, Brunner & Grob, 1974). Statistical analyses have shown that there is also a relationship between milder inflammatory changes and the presence of a thymoma (Oosterhuis, Bethlem & Feltkamp, 1968). Myocarditis has been reported in autopsy studies; this may also be related to the presence of myositis and a thymoma. Other pathological features of myasthenia gravis can be explained in terms of diseases such as autoimmune thyroiditis which commonly coexist. 3. Serology Approximately one third of all patients have been shown to have an IgG complement fixing anti-striational antibody which is very similar to that found in EAM. Almost all patients with an associated thymoma have this antibody but so do at least one quarter of patients with a thymoma without myasthenia. As in the case of EAM the precise nature of the antigen concerned is unknown but it is common to skeletal muscle, heart and thymic myoid cells. Although the predominant immunofluorescence pattern is striational, subsarcolemmal staining may be seen (Aarli, 1972; Dawkins, 1973b). Reaction with surface antigens on viable muscle cells has never been demonstrated. Initial claims that the antibody reacted with the A band of the sarcomere were revised after glycerinated fibres and better techniques were used and there is now general agreement that all, or almost all, sera bind to the I band (Strauss & Kemp, 1967; Strauss, 1968). In this respect the thymoma-myasthenic antibody appears to resemble that found in EAM but it differs in that there is staining of the A-I junction rather than the M line (Dawkins et al., 1975). One attempt to apply immunoperoxidase techniques led to the suggestion that this antigen is located on the sarcoplasmic reticulum rather than the thin filament (Mendell, Whitaker & Engel, 1973), but it will be necessary to reconcile this localization with previous work using immunofluorescence and especially the distinctly linear pattern which varies with changes in sarcomere length (Strauss & Kemp, 1967; Dawkins et al., (1975). Other methods have also been used to characterize the antigen(s), but these have yielded a surprising array of findings. Attempts to employ isolated contractile proteins generally reveal loss of antigen with successive purification. Irrelevant non-muscle autoantibodies are found in higher than the expected frequency irrespective of whether patients possess anti-striational antibody. Anti-nuclear factor and anti-thyroid antibodies are especially common but anti-parietal cell, anti-smooth muscle and anti-reticulin may also occur (Dawkins et al., in preparation). As mentioned above, hypocomplementaemia may be found, but its precise frequency and significance is unknown (Nastuk et al., 1960; Strauss, 1968; Dawkins et al., 1975). An ade-

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quate study of complement components has not been undertaken but, to date, there is as much evidence of hyposynthesis or inhibitors as utilization and there is no obvious relationship to the presence of autoantibodies including the anti-striational antibody.

4. Cell-mediated immunity Using various migration inhibition techniques a number of groups have reported hypersensitivity to muscle and/or thymus (Goust, Gastaigne & Moulias, 1972; Alpert et al., 1972; Armstrong, Novak & Falk, 1973; Vejjajiva et al., 1975). Differences in methodology make these studies difficult to interpret, but in one study it was claimed that there was a correlation between migration inhibition and the titre of anti-muscle antibody as determined by immunofluorescence (Alpert et al., 1972). With the assay used in EAM and polymyositis it has been found that lymphocyte-mediated cytotoxicity to skeletal muscle is restricted to those patients with anti-striational antibody and in this group there is a quantitative relationship between cytotoxicity and antibody titre (Dawkins, 1973b; Dawkins et al., 1975). 5. Relationship between different features From the aforegoing it is apparent that thymoma, myositis, anti-striational antibody and delayed hypersensitivity to muscle tend to occur together in a proportion of patients, but that these features bear no direct relationship to the presence of neuromuscular block, thymic hyperplasia, hypocomplementaemia or irrelevant autoantibodies. Thus patients can be divided into group A with myasthenia gravis plus thymomata and autoimmune muscle disease resembling EAM and group B with simple myasthenia gravis. Further support for this division comes from recent studies of HL-A antigen frequencies (Fritze et al., 1974; Feltkamp et al., 1974). If all patients are considered there is a significant association with HL-A8 but this is especially so for those in group B whereas in group A patients there appears to be an increased frequency of a first series antigen which may be HL-A2 (Feltkamp et al., 1974) or HL-A3 (Fritze et al., 1974). Furthermore group A patients are generally male with a relatively late onset whereas young adult female predominance in group B suggests a parallel with diseases such as SLE which may also be associated with HL-A8. Further epidemiological studies are required but at this time these data can be interpreted as indicating that all patients with myasthenia gravis are suffering from a process which leads to neuromuscular block and that in some of these there is also autoimmune muscle disease. 6. Pathogenesis of neuromuscular block It is apparent that many of the features which have been thought to support a role for autoimmunity in the pathogenesis of neuromuscular block are more relevant to autoimmune muscle disease. However, this does not apply to the presence of thymic hyperplasia, hypocomplementaemia, HL-A8, irrelevant autoantibodies and epidemiological characteristics similar to those found in SLE. Furthermore, the transmission of myasthenia from mother to neonate suggests the presence of a humoral factor which has some of the characteristics of IgG. For these reasons the recent claim that some patients have an IgG antibody which reacts with the acetylcholine receptor (Almon, Andrew & Appel, 1974) is of great potential interest. Conceivably, the presence of HL-A8, irrelevant autoantibodies and female predominance reflect a predisposition to autoimmune disease and to the development of this neuromuscular blocking antibody, but there is still no explanation for the hypocomplement-

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aemia and the relationship to thymic hyperplasia. Furthermore, the frequent benefit of thymectomy must be explained. Another feature of these patients which requires further study is the increase in the cytotoxicity of peripheral lymphocytes after mitogen stimulation Dawkins et al., 1975). 7. Autoimmune muscle disease, thymomata and immunodeficiency For the past decade the association of anti-muscle antibody and thymoma appears to have been accepted as just one of many confusing features of myasthenia gravis. Some have suggested the antibody might be more relevant to thymic myoid cells than skeletal muscle but these cells are not a feature of thymomata. With the characterization of EAM and the recognition that the antibody is associated with myositis, it appears much more likely that the true relationship is between thymomata and autoimmune muscle disease rather than 'antimyoid' antibody. Further support for this notion is provided by the description of a disease occurring in the mastomys and characterized by thymoma, polymyositis and antistriational antibody (Strauss et al., 1968). Furthermore, it is now accepted that frank polymyositis may be associated with thymomata (Rowland et al., 1973; Namba et al., 1974). It is therefore necessary to explore the possibility that there may be some underlying predisposition, such as immunodeficiency, which predisposes certain individuals to the development of both conditions. Some support for this possibility comes from the well known association between thymoma and immunodeficiency (Souadjian et al., 1974) and from early studies which showed that some patients with myasthenia gravis may have defective humoral responses (Kornfeld et al., 1965; Adner et al., 1966). Recent preliminary studies have shown that group A patients generally have this defect, whereas most patients without thymomata or autoimmune muscle disease are normal in this respect (Dawkins et al., 1975). This finding is of particular interest in view of the apparent relationship to the presence of a first series HL-A antigen since this could be readily explained in terms of inheritance of particular immune response genes. Much more work will be required to substantiate these findings, but a working hypothesis attributing a primary role to immunodeficiency appears justified at this time. Thus certain patients may be predisposed to both autoimmune muscle disease and thymomata. It remains to be shown whether this predisposition is similar to that postulated in polymyositis, but it might be predicted that there will be sufficient differences to explain the fact that patients with thymoma generally have anti-muscle antibody. Furthermore, it will be necessary to explain the predominance of tumours within the thymus, although there is some evidence that extra-thymic tumours are more frequent than in the general population (Papatestas, Genkins & Kark, 1974). Finally, although the evidence argues against a role for thymoma or myositis in the pathogenesis of neuromuscular block, it must be assumed that the underlying defect does increase the risk of various autoimmune diseases including myasthenia gravis, thyroiditis and systemic lupus erythematosus.

CONCLUSIONS The evidence presented suggests that immunization with crude skeletal muscle leads to a variety of immunological responses. One of these results in myositis apparently via cellmediated mechanisms. A similar reaction appears to be responsible for the myositis found in human polymyositis and in association with thymomata and various autoimmune disorders. Humoral immunodeficiency may predispose to the development of this form of myositis and

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possibly to the tumours and other autoimmune diseases which are commonly associated. The neuromuscular block of myasthenia gravis is not directly related to autoimmunization against muscle but may be due to an antibody reacting at the neuromuscular junction. Further study of these conditions may provide valuable insights into the relationship between immunodeficiency, autoimmune disease and neoplasia. REFERENCES AARLI, J.A. (1972) Localisation and properties of an acid-soluble muscle antigen reacting with antibodies in myasthenia gravis sera. Acta. path. microbiol. scand. 80, 453. ADNER, M.M., ISE, C., SCHWAB, R., SHERMAN, J.D. & DAMESHEK, W. (1966) Immunologic studies of thymectomised and non thymectomised patients with myasthenia gravis. Ann N. Y. Acad. Sci. 135, 536. ALEXANDER, S. & FOREMAN, L. (1968) Dermatomyositis and carcinoma. A case report and immunological investigations. Brit. J. Derm. 80, 86. ALMON, R.R., ANDREW, C.G. & APPEL, S.H. (1974) Serum globulin in myasthenia gravis. Science, 186, 55. ALPERT, L.I., RULE, A., NORIO, M., Korr, E., KORNFELD, P. & OSSERMAN, K. (1972) Studies in myasthenia gravis: cellular hypersensitivity to skeletal muscle. Amer. J. clin. Path. 58, 647. ARMSTRONG, R.M., NOVAK, R.M. & FALK, R.E. (1973) Thymic lymphocyte function in myasthenia gravis. Neurology, 23, 1078. BANKER, B.Q. & VICTOR, M. (1966) Dermatomyositis (systemic angiopathy) of childhood. Medicine, 45, 261. BENSON, M.D. & ALDO, M.A. (1973) Azathioprine therapy in polymyositis. Arch. intern. Med. 132, 547. BOEHME, D. (1965) The influence of experimental allergic muscular dystrophy on the reticuloendothelial system. J. reticuloendothel. Soc. 2, 47. BOHAN, A. & PETER, J.B. (1975) Polymyositis and dermatomyositis: a critical review. New Engl. J. Med., 292, 344 (I) and 403 (II). BRAY, D. (1974) Antiactin. Nature (Lond.), 251, 187. CASPARY, E.A., GUBBAY, S.S. & STERNE, G.M. (1964) Circulating antibodies in polymyositis and other muscle wasting disorders. Lancet, ii, 941. CURRIE, S. (1971) Experimental myositis: the in vivo and in vitro activity of lymph node cells. J. Path. 105, 169. CURRIE, S., SAUNDERS, M., KNOWLES, M. & BROWN, A.E. (1971) Immunological aspects of polymyositis. Quart. J. Med. 40, 63. CURTIS, A.C., HECKAMAN, J.H. & WHEELER, A.H. (1961) Study of the autoimmune reaction in dermatomyositis. J. Amer. med. Ass. 178, 571. DAWKINS, R.L. (1963) An investigation into the possibility of immunologically-produced disease of skeletal muscle. B. med. Sci. thesis, University of Western Australia, Perth. DAWKINS, R.L. (1965) Experimental myositis associated with hypersensitivity to muscle. J. Path. Bact. 90, 619. DAWKINS, R.L. (1971) Pathogenesis of myasthenia gravis. Brit. med. J. iv, 235. DAWKINS, R.L. (1973a) The pathogenesis of polymyositis. Excerpta Med. 299, 161. DAWKINS, R.L. (1973b) Cytotoxicity, muscle-specific antigens and their relevance to diseases of muscle. M.D. thesis, University of Western Australia, Perth. DAWKINS, R.L., AW, E.J. & SIMONS, P.J. (1972) The persistence of tissue-specific antigen in muscle cultures growing in vitro. Immunology, 23, 961. DAWKINS, R.L., EGHTEDARI, A. & HOLBOROW, E.J. (1971) Antibodies to skeletal muscle demonstrated by immunofluorescence in experimental autoallergic myositis. Clin. exp. Immunol. 9, 329. DAWKINS, R.L., GIBB, D.G.A. & HOLT, P.G. (1974) Demonstration of immunological differences between different muscle fibre types. Basic Research in Myology (ed. by B. A. Kakulas), p. 605. Excerpta Medica, Amsterdam. DAWKINS, R.L. & HOLBOROW, E.J. (1972) Surface antigens of differentiated muscle cells in monolayer demonstrated by immunofluorescence and cytotoxicity. J. immunol. Methods, 2, 1. DAWKINS, R.L. & LAMONT, M. (1971) Myogenesis in vitro as demonstrated by immunofluorescent staining with antimuscle serum. Exp. Cell Res. 67, 1.

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