Specificities of autoantibodies in autoimmune receptor diseases

Autoimmune Receptor Diseases

lmmunol Res 1988;7:218-231 �9 1988 S. Karger AG, Basel

0257-277X/88/0073-021852.75/0

Specificities of Autoantibodies in Autoimmune Receptor Diseases

M.H. De Baets

Department of Immunology, State University of Limburg, Maastricht, The Netherlands

Introduction

One of the most striking features of the immune system is the specific nature of the interaction of an antibody with a given antigen. Antibodies discriminate by means of their combining site between proteins or peptides differing by only one amino acid or between chemicals differing by one atom or three-dimensionally. The three-dimensional structure (conformation) of an antigen is probably more important than its chemical composition [ 1] for antibodies are probably directed against a particular electron cloud rather than specific chemical structures [2].

The specificity of antigen-antibody interaction is not absolute since antibodies also react with related antigens (so-called cross- reactive) sharing related but not identical determinants. The affinity of an antibody molecule for a cross-reacting antigen is lower but may be sufficient to cause a biological effect upon interaction, e.g. complement activation. Low-affinity antibodies clear antigens less effectively and may be responsible for the persistence of autoantigens [3].

The immune system is obviously involved in autoimmune disease. The basic abnormality is a loss of tolerance against the body's own constituents resulting in an im-

mune attack to a given organ or in non- organ-specific systemic lesions. It is beyond the scope of this paper to review all theories on the origin of autoimmunity. The inter- ested reader is referred to other reviews on this subject [3]. In this review we will con- centrate on the role of the characteristics of autoantibodies in autoimmune diseases.

Antigenic Anatomy of Autoantigens

Implicit in the hybridoma technology is the ability to produce monoclonal antibodies and this has allowed dissection of the antigenic anatomy of autoantigens. A given monoclonal antibody binds to a single antigenic determinant or epitope, i.e. to a specific area on the surface of a protein. The specificity of antibody responses is related to antigenicity and immunogenicity. Antigenic- ity implies the ability of antigens to be recognized by the immune system. Immunogenie- ity indicates the ability to elicite an immune response and is influenced by the available T cell repertoire, the activity of T helper/sup- pressor cells, the idiotype network and the major histocompatibility complex (MHC) [ 1]. The immunogenicity of autoantigens is low in view of the tolerant state of the T or B

Specificities of Autoantibodies in Autoimmune Receptor Diseases 219

cell repertoire [4]. The status of the idiotype network is ill-defined but evidence exists that the liter of auto-antiidiotypic antibodies is low at the beginning of autoimmune disease [5]. The MHC on antigen-presenting cells interacts with the antigen or with the antigenic fragments and thus enhances its immunogenicity [6]. Certain MHC sub- classes such as HLA-DR3 have an higher affinity for the antigen or form a more immunogenic antigen-MHC complex. This might explain why HL-DR3-positive patients with Graves' disease [7] or rheumatoid arthritis are more resistant to therapy [8]. MHC-linked genes have been found to con- trol the f'me specificity of antibody and T cell responses in mice and guinea pigs to a variety of protein antigens including myoglobin, albumin, lysozyme, cytochrome c and insulin [1].

Certain amino acid sequences on autoantigens are immunodominant i.e. the majority of the immune response is directed to a few epitopes. This restriction is typically seen for thyroglobutin in autoimmune thyroiditis [9] and acetylcholine receptors [ I 0] in myasthenia gravis and their respective autoimmune models, experimental autoimmune thyroiditis and experimental autoimmune myasthenia gravis. Several mechanisms can account for this immunodominance. Partic- ular three-dimensional structures on antigens may render certain determinants more immunogenic [ I ]. An alternative view is that immunodominance of a given region depends on regulatory mechanisms of the host, including specificity of T helper cells and idiotype networks [ 1 ].

The antigenic structure of several autoantigens has been studied in some detail. A large variety of membrane receptors are the target of an autoimmune attack (table I). The

complete amino acid sequence of the acetylcholine receptor has been determined [11- 15], including the main immunogenic region [16, 17], which allowed characterization of the immunogenic and antigen binding sites. Other membrane receptors are less well char- acterized. The thyrotropin receptor is a 200,000-dalton glycoprotein associated with gangliosides moieties. These glycolipids are important for the transmission of thyroid cell-stimulating signals of both TSH and thyroid-stimulating immunoglobulins [18-24]. One set of monoclonal antibodies derived from peripheral blood lymphocytes of patients with Graves' disease interacting with gangliosides have thyroid-stimulating activities. A second group of monoclonal antibodies interacting with the glycoprotein component of TSH receptor blocks TSH receptor function [21]. The insulin receptor is also a glycoprotein, composed of two distinct subunits with a molecular weight of 135,000 (el) and 95,000 (13) assembled with a stoichiome- try of ~213~ [25]. Autoantibodies against insulin receptor interfering with the binding and subsequent action of insulin to its receptor are responsible for some forms of severe insulin resistance seen in diabetics [26]. Persis- tent insulin-like action of anti-insulin receptor antibodies has also been found in patients with long-lasting hypoglycemia [27]. The opposite effect of these autoantibodies is most likely the consequence of a difference in their specificity. Monoclonal antibodies have been used to characterize the main immunogenic regions and their relation to receptor func- tions including the insulin binding site and the receptor kinase activity [28]. The specificities of other anti-receptor antibodies as mentioned in table I have not been determined yet because too little is known about the amino acid sequence of the receptor mol-

220 De Baets

Table I. Anti-receptor autoimmune disease

Discipline Receptor for Disease Reference

Neurology AChR myasthenia gravis 30

Endocrinology TSH Graves' disease (TSI) 31, 32 myxoedema (TGI block) 34 goiter (TGI) 33

insulin hyperglycemia 26 hypoglycemia 27

FSH amenorrhea 35

Nephrology PTH secondary hyperparathyroidism in patients with renal failure 36

Hematology transferrin anemia 37,38

Gastroenterology gastrin pernicious anemia achylia 39

Immunology lgA lgA deficiency 40 B2-adrenergic agonists asthma/allergic rhinitis 41, 42

TSI ~ Thyroid-stimulating immunoglobulins; TGI = thyroid growth-promoting immunoglobulins; TGI block = thyroid growth-blocking.

ecules. Further advance in the molecular anatomy of these molecules is needed to determine structure-function relationship and modulation of receptor function by autoantibodies. This also holds for the whole spec- trum of organ-specific and systemic autoimmune diseases [29].

thyroid disease. In contrast 40 or more determinants are recognized by antibodies to human thyroglobulin raised in rabbits [9, 43, 44]. Autoantibodies must meet certain requirements in order to induce autoimmune disease. The putative characteristic of autoantibodies are listed in table II.

Role of Antigenic Specificity of Autoantibodies in Relation to Disease

The molecular structure of most autoantigens is very complex and therefore a large number of antigenic determinants are expressed; the immune response to these autoantigens however is rather restricted. Typ- ically thyroglobulin, a 660,000-dalton glycoprotein, contains only 4-6 antigenic determinants relevant to human autoimmune

Autoantibodies Directed against Hidden or Secluded Antigens

These are not pathogenic unless the cell or organ containing these antigens is dam- aged. Lens a-cristallin released after an in- jury from the lens induces uveitis in experimental animals [45]. The secluded antigen theory however does not hold for the majority of autoimmune diseases. Most autoantigens circulate in the microenvironment in-


Table II. Characteristics of autoantibodies in relation to autoimmune disease

1 Pathogenic autoantibodies are directed against epitopes accessible to the immune system

2 Cross-linking of epitopes on membrane-associated autoantigens

3 Specificity in relation to biological effect: e.g. autoantibodies against the ligand-binding site may block or stimulate receptor molecules

4 Idiotypic characteristics

ducing tolerance at the T and/or B cell level [46]. Some epitopes on autoantigens however are not accessible to the immune system, including cytoplasmic determinants of membrane receptors. In patients with myasthenia gravis or animals with experimental autoimmune myasthenia gravis (EAMG) circulating antibodies to the acetylcholine receptor (ACh_R) are directly involved in the pathogenesis of the neuromuscular transmission defect [47]. These autoantibodies are directed against a few extracellular epitopes desig- nated as the main immunogenic region (MIR) of the AChR [10]. AChR devoid of the MIR (after reduction and carboxymethylation; RCM-AChR) are still immunogenic but do not induce EAMG in experimental animals. Analysis of the specificity of the immune response against this modified AChR revealed that most antibodies are directed against cytoplasmic determinants of the AChR [48]. Reduction and carboxymethylation according to the procedure of Bartfeld and Fuchs [49] do not dissociate AChR into its subunits [48]. Epitope analysis of the RCM-AChR with a panel of 29 monoclonal antibodies revealed a specific loss of the MIR [48]. Mild trypsinization leaves the MIR unaffected but destroys all other epitopes. Only 40% of the polyclonal autoanti-

bodies from rats with EAMG react with RCM-AChR whereas 75 % react with trypsin- ized AChR (t-AChR).

The combined results obtained with RCM-AChR and t-AChR are interesting in several aspects. First, reduction and carboxymethylation - a procedure that destroys conformational dependent determinants - has an opposite effect compared to trypsinization, a procedure that probably removes linear polypeptides in between conformational dependent determinants. Second, RCM-AChR lacks the MIR whereas t-AChR probably contains the MIR only. Finally, the results obtained with polyclonal antibodies suggest that 75 % of the polyclonal antibody response is directed against the MIR, which is in agreement with monoclonal antibody- blocking experiments reported by Tzartos et al. [101.

Rats immunized with RCM-AChR showed serum antibody concentrations against Torpedo and rat AChR of 2.7 +__ 4 ~ r and 353 ___ 69 pM, respectively. The serum antibody concentration of antibodies cross-reactive with rat AChR in animals immunized with native Torpedo AChR was 760 __. 41 pAL Thus, deletion of the MIR reduced the cross-reactive antibody elicited by (denaturated) Torpedo AChR by about 50%. None of the rats immunized with RCM-AChR showed AChR loss (table III). These animals contained greater quantities of antibodies complexed to the receptor at the neuromuscular j unction compared to animals immunized with native Torpedo AChR.

In order to relate the lack of pathogenicity of RCM-AChR to antibody specificity, we analyzed if anti-RCM antibody bound to extracellular parts of AChR incorporated in native membranes or to buried or cytoplas-

222 De Baets

Table III. RCM-AChR does not induce EAMG

I st injection Boost AChR concentration AChR AChR Antibody pmol/animal loss complexed complexed

% pmol/animal to AChR, %

RCM RCM 73+_6.2 0 31 +_8.9 42+_8 Native AChR native AChR 31 _ 7 42 _+_+ 11 12 _+ 2.8 41 +- 16 CFA only CFA only 59_+ 8.7 0 0 0

Female Lewis rats were immunized on day 1 and received a booster injection on day 30. All animals were sacrificed 42 days after the first injection. Both native AChR (15 ttg) and RCM-AChR (i 5 ~tg) were given in complete Freund's adjuvant (CFA).

Table IV. Specificities of antisera against RCM and native AChR

lmmunogen Antibody activity (%) that

binds to remains in membranes supernatant

RCM 3t • 71 +6 Native AChR 79_+4 21 +_4

Serum (20 ttl) from rats immunized with RCM- AChR (15 ttg) or native AChR was diluted (1/20) in PBS-BSA-NaN3 (10raM Na phosphate, 5mg/ml BSA, 10 rru~,I azide) and incubated with an excess of native Torpedo membranes (30 ttmol) in a total vol- ume of 100 ttl overnight at 4 *C. The antibody activity in the supernatant was determined by radioimmu- noassay. The amount of antibody remaining in the supernatant was compared to that of a serum sample, to which no membranes had been added.

mic determinants. It is clear from table IV

that most of the anti-RCM antibody activity (69 %) does not bind to native membranes

and is therefore directed to determinants buried in the membranes or to cytoplasmic surface of AChR. In contrast, the antibody

activity against native AChR was absorbed

by native membranes and thus directed to extracellular parts of AChR.

These results suggest that RCM-AChR is not pathogenic because its most immunogenic parts are located on the cytoplasmic surface o f native AChR. Consequently, the

majority of antibodies against RCM do not bind to AChR in vivo and can only bind to solubilized AChR which is freed from its surrounding membrane. The majority of

anti-native AChR antibodies bind to the MIR located on the extracellular part of AChR. Similar results were obtained by

Froehner [50] and Froehner et al. [51] with polyclonal antisera and monoclonal antibodies against sodium dodecyl sulfate-dena-

tured receptor or its subunits. Only 10-20%

of the determinants recognized by these antisera are accessible to antibodies when the

receptor is membrane-bound. Most of the monoclonal antibodies to denatured AChR

bound to membrane-incorporated AChR only when membranes were made permeable with saponin [51, 52]. These findings also

hold in vivo since anti-RCM-AChR mono-


Table V. Passive transfer of EAMG with monoclonal antibody against AChR

Monoclonal Specificity Injected anti-rat AChR antibodies muscle AChR fmoUg

antibodies, pmol muscle

157 RCM-AChR 100 0.46 _ 0.01 158 RCM-AChR 150 0.45 _.+. 0.04 164 RCM-AChR 100 0.48 _+ 0.06 35 native AChR (MIR) 40 0.27 -+ 0.02

saline 0.52 _+ 0.03

Monoclonal antibodies (concentrated tissue culture supernatant) were injected intraperitoneaUy into female Lewis rats. All animals were sacrificed 48 h later and the AChR content in muscle was measured by radioim- munoassay and expressed as femtomoles ~25I-ct-bungarotoxin binding sites per gram muscle.

clonal antibodies (No. 157, 158 and 164) do not cause AChR loss when injected into rats (table V).

Autoantibodies against Membrane-Associated Autoantigens Are Abte to Cross-Link These Antigens

This phenomenon, called antigenic modulation, results in increased internalization and degradation rate of autoantibody-antigen complexes, Unless the target organ reacts to this immunological assault with a compensatory increased synthesis, the number o f membrane-associated antigens is de- creased. A typical example of antigenic modulation is seen in myasthenia gravis and its experimental model (EAMG). Antibodies against AChR increase the degradation of AChR on cultured muscle cells in vitro [53- 56]. This effect of anti-AChR sera depends on the divalent structure of immunoglobulin G. Univalent Fab anti-AChR fragments are ineffective, but cross-linking of AChRs on muscle cells by intact anti-AChR antibodies

or Fab fragments and anti-IgG induces a 2- fold increase in the degradation o f AChR on muscle cells in tissue culture. Muscle cells in tissue culture display no compensatory increase of AChR synthesis, since AChR synthesis is already maximal. It is not known whether in patients with myasthenia gravis or animals with EAMG there is a compensatory increase in AChR synthesis. It is also not known if groups of muscle fibers with increased AChR synthesis are less suscepti- ble to the modulating effect in vivo. Proce- dures known to increase protein synthesis might ameliorate signs and symptoms of myasthenia. For this reason the effect of unilateral limb denervation and administration of anabolic steroids on AChR loss in EAMG was studied. Unilateral limb denervation, a procedure known to increase the AChR content of muscle, protects the denervated leg against antibody- mediated AChR loss in both acute and chronic EAMG [57]. Treat- ment with anabolic steroids, a more practi- cal therapeutic procedure than denervation, is also protective against chronic EAMG

[571.

224 De Baets

The role of antigenic modulation in other autoimmune receptor disease is less well- defined. Chronic exposure of insulin receptors to autoantibodies results in receptor desensitization, i.e. a loss of insulin-mediated receptor function [58]. The mechanism of desensitization involves a decrease in receptor affinity rather than antibody-mediated receptor loss. The acute effect of anti-insulin receptor antibodies is similar to those of anti-thyrotropin receptor antibodies [58]. Both anti-bodies mimic hormone action in the receptor molecule. The biological activity of anti-insulin receptor antibodies re- quires antibody bivalency whereas the receptor blockade can be induced by monovalent antibodies [59]. Similarly monovalent anti- thyrotropin receptor antibodies induce a short-acting response similar to thyrotropin. Long-acting stimulating responses require bivalent antibodies to induce receptor cross- linking leading to thyroid follicular cell stimulation [60-63].

Fine Specificity of Autoantibodies May Be Directly Related to Their Biological Effect

Antigenic modulation and receptor cross- linking play a central role in this stimulating process. The f'me specificity of anti-receptor antibodies may determine if they are able to cross-link receptor molecules. Antibodies against AChR can act as internal cross-linkers or intermolecular cross-linkers, thereby inducing antigenic modulation whereas internal cross-linkers are not able to aggregate receptor molecules [64].

Antibodies against membrane receptors can exert an agonist-like effect. Antibodies to the thyrotropin receptor from patients

with Graves' disease stimulate thyroid cell function, i.e. thyroid hormone secretion. An- tibodies to the insulin receptor found in some patients with hypoglycemia have an insulin-like effect [27]. Insulin binds to the extracellular part of its receptor. This signal activates a tyrosine kinase associated with the ~-subunit of the insulin receptor [65]. Receptor cross-linking by intact anti-insulin receptor antibodies from patients with severe insulin resistance but not by their monovalent Fab fragments activates the insulin receptor kinase [65]. Anti-insulin receptor antibodies also block the binding of insulin to its receptor and may therefore be directed to the ~t-subunit. Possibly anti-insulin receptor antibodies from patients with prolonged hypoglycemia may directly bind to the 13-subunit and subsequently activate the tyrosine kinase. However, these patients have also a population of antibodies that inhibit the binding of insulin to its receptor. Similarly to patients with Graves' disease [66] both agonistic and antagonistic antibodies may be present in the same patient. The balance between agonist and antagonist antibodies may determine whether hypoglycemia or insulin resistance develops.

Antibodies against the thyrotropin receptor are involved in various thyroid diseases including hyperthyroidism of Graves' disease, hypothyroidism and euthyroid nonen- demic goiter. The thyrotropin receptor is an integral membrane protein with a molecular weight of about 200,000 daltons expressed on thyroid epithelial ceils and fat cells [24]. Complex gangliosides are also present on thyroid membranes [ 18, 22]. These glycolipids are important for the transmission of thyroid-stimulating signals of both TSH and thyroid-stimulating immunoglobulin [20- 23]. A large panel of antibodies reacting with


Table VI. Reactivity of autoantibodies to thyrotropin receptors

Antibody activity Disease Reference

Long-acting thyroid stimulator Long-acting-thyroid-stimulator protector Thyrotropin-binding-inhibiting immunoglobulins Thyroid-stimulating immunoglobulinsdantibodies Immunoglobulins blocking thyrotropin-induced thyroid

hormone synthesis Thyroid growth-stimulating immunoglobulins Immunoglobulins that block thyrotropin-induced thyroid growth

Graves' disease 31 Graves' disease 69 Graves' disease 32 Graves' disease 66 Graves" disease 66 primary myxoedema 70 goiter 33 primary myxoedema 34

Table VII. Anti-receptor antibodies exerting agonist-like effects

Receptor Cellular response Reference

TSH thyroid hormone secretion 32 cell multiplication

Insulin glucose uptake 25, 27 Prolactin casein synthesis 68

cell multiplication Epidermal growth factor cell multiplication 71 Catecholamine adenylate cyclase activity 72 Muscarine ACbR muscle cell contraction 73 Nicotine muscle cell contraction 74, 75

thyrotropin receptors or their surrounding structures associated with several autoimmune thyroid diseases (table VI) have been described. It is tempting to speculate that differences in specificities of these thyrotropin receptor antibodies result in a different biological effect. Indeed, monoclonal antibodies which stimulate thyroid hormone synthesis in vitro (TSI activity) all react with gangliosides [21]. Monoclonal antibodies against the glycoprotein compartment of the TSH receptor block receptor function (thyrotropin-binding inhibiting immunoglobulins or thyroid synthesis-blocking immunoglobulins). Patients with Graves' disease have a

mixture of circulating antibodies with thyroid blocking and stimulating capacities. The balance between agonist and antagonist immunoglobulins determine if hyper- or hypothyroidism develops [66].

Several other anti-receptor antibodies with agonist-like effects have been generated (table VII). Antibodies to the prolactin receptor have only been induced experimen- tally. The prolactin receptor consists of a single chain of glycoprotein [67]. Prolactin, similarly to antibodies against the prolactin binding site, stimulates casein biosynthesis and mammary cell multiplication. Mono- clonal antibodies may have antagonist or ag-

226 De Baets

onist activities. Both agonist and antagonist monoclonal antibodies compete with prolactin and prevent its biological effects on mammary cells. This suggests that the binding domain could be relatively complex, containing a small portion essential for trigger- ing the receptor. Antibodies against the trig- gering site mimic hormone action, whereas antibodies with other specificities block hormone binding. Another possibility to explain the differences between agonist and antagonist antibodies involves cross-linking of membrane receptors. A monoclonal antibody against the prolactin receptor lost its prolactin-like activity when reduced to Fab fragments [68].

Fab fragments however differ from their proband immunoglobulin molecule not only by their monovalency but also by their lower affinity. It is more likely that the differences in specificity and thus primary and tertiary structure of the antibody binding sites account for their agonist or antagonist activities. Similarly discrete molecular manipula- tions of neurotransmitters can result in a change from agonistic to competitive antagonistic properties. Hydrophobic aromatic rings bound to acetylcholine bind to acces- sory hydrophobic binding areas on the receptor molecule and thus stabilize the hydrophobic relation between the receptor and its lipid surrounding [76]. Highly polar agonists promote the polar characteristics of the receptor protein, thus destabilizing its relationship to the membrane lipid and increas- ing the microaggregation of receptors resulting in cell activation. Similarly anti-receptor antibodies containing a few hydrophobic side chains in the vicinity of the antigen combining site (paratope) are probably an- tagonists whereas those containing hydro- philic residences may be agonists.

Specificity of Autoantibodies Is Closely Linked to Their Idiotype

The idiotype of autoantibodies can be probed with a set of antiidiotypic antibodies that closely mimic the structure of autoantigens as they fit in the antibody combining site. In other words, the combining site is complementary in shape (i.e. the internal image) to a part of the three-dimensional profile of an antigen molecule [77] or antiid- iotype (anti-Id). These anti-Ids, called internal images, are expected to cross-react with all autoantibodies reacting with a given epitope on an autoantigen. In view of the restricted specificity of autoantibodies a limited number of anti-Id-producing clones are expected to be found. These cross-reactive anti-Id have a potential immunoregulatory role, so that administration of minute amounts of anti-Id can profoundly alter the Id-anti-Id network [78]. Perturbations of this Id-anti-Id network could be involved in the induction of autoimmune diseases.

Antiidiotypic antibodies directed at anti- hormone antibodies can mimic hormone action by acting as internal images of the site reactive with the receptor for that hormone [79]. Anti-hormone antibodies (abl) elicited spontaneously or by active immunization of experimental animals induce antiidiotypic antibodies (ab2), which in turn induce anti- antiidiotypic antibodies (ab3). The antibody-combining site of ab2 is the internal image of the hormone and may therefore mimic the hormone action on the receptor. Antibody 3 resembles antibody 1 and is able to bind to the hormone. This "internal image' hypothesis has been tested in various experimental systems. Anti-Id against insulin antibodies interact with insulin receptors and mimic insulin action [80, 81]. Similarly an-


tiidiotypic antibodies against 13-adrenergic ligands, thyrotropin, retinol-binding proteins, and formyl peptide chemoattractant interact with receptors for their respective ligands [82]. This system is also relevant for autoimmune diseases. Antiidiotypic antibodies pre- pared against antibodies to the acetylcholine anologue Bis Q interact with AChR. In vivo, these anti-Id block the binding of acetytcho- line to its receptors and cause myasthenia gravis [83]. Many of these systems have been criticized because of their artificial nature. Anti-ligand antibodies however occur spontaneously in patients with autoimmune diseases including anti-thyroid hormone antibodies in patiens with autoimmune thyroiditis [84].

Autoantibodies and their respective idiotypes form an autoimmune network [78] distinct from the aUoreactive network. Both networks however may interact with each other [85]. The immune system's primary activity is self-recognition, like a 3-month- old baby first observes the movements of his own hands before signals from the outside world are catching its attention. This self- centered immune system could be deleteri- ous unless the immune system also produces a complementary set of anti-idiotypes, i.e. anti-antiself molecules [86]. This high inter- connectivity of the neonatal B cell repertoire decreases during adult life, probably by interaction with the external environment, i.e. viruses and bacteria. This pertubation of the neonatal autoimmune network may be phys- iological but may also result in autoimmunity. For example, immunization of experimental animals (C57B1/J/mice) with the rabies virus results in autoimmunity to AChR by expansion of autoreactive clones through autoimmune and alloreactive network interaction. Indeed idiotypes expressed on au-

toantibodies to the AChR are also expressed on rabies viral envelope [Verschuuren et al., unpubl observations]. Not all idiotypes may be equally important to the economy of the immune system [87]. Only a limited set of idiotopes within the entire idiotype repertoire are immunogenic and play a primary role in autoimmune regulation. This particular set of regulatory idiotopes (Ri) elicits ab2 (anti-Ri), which in turn induce the produc- tion of a small amount of ab3 and high titers of Ri.

These regulatory idiotypes have been amply demonstrated in several experimental systems. Murine monoclonal antibodies against phylogenetically conserved epitopes (Id62) on thyroglobulin express cross-reactive idiotopes at the paratope [88]. This idiotype is also expressed neonatally in normal mice. Injection of antiidiotypic antibodies induces anti-thyroglobulin antibodies ex- pressing Id'62 [89]. Mice hyperimmunized with Id62 are suppressed in their immune response against thyroglobulin; this suppres- sion occurs at the T cell level [90].

Antibodies against the AChR in patients with myasthenia gravis and its experimental model are of restricted specificity but don- ally heterogeneous [91]. In EAMG the full spectrotype of antibodies against AChR is expressed early after immunization (day 10) in the period preceding clinical disease and remains constant during the chronic phase of the disease. This result suggests that the spectrotype of anti-AChR does not affect the expression of the disease [92]. The spectrotype of antibodies bound to muscle is similar to the population of circulating antibodies. This refutes the existence of a particular antibody clone with greater affinity or specificity for muscle AChR and therefore a higher pathogenic potential in EAMG. The analysis

228 De Baets

of the idiotype of these antibodies bound to the target organ might provide further in- sight into the pathogenesis of autoimmune diseases. If tissue-bound autoantibodies are idiotypically restricted, this could have important therapeutic implications for specific immunosuppression.

Acknowledgments

We would like to thank Prof. Dr. P.J.C. van Breda Vriesman for his critical review of this manuscript. The secreterial expertise of Mrs. F. Teng-Vangroot- loon is greatly appreciated. This study was supported by grants from the Beatrix foundation and MEDI- GON.

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88 Zanetti M, De Baets M, Rogers J: High degree of idiotypic cross-reactivity among murine monoclonal antibodies to thyroglobulin. J Immunol 1983;131:2452-2457.

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Dr. M.H. De Baets Department of Immunology State University of Limburg POB 616 NL-6200 MD Maastricht (The Netherlands)

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Specificities of autoantibodies in autoimmune receptor diseases