The long-acting thyroid stimulator of Graves' disease

The Long-Acting Thyroid Stimulator

of Graves’ Disease*

GERALD BURKE, M.D., C.M.

Chicago, Illinois

A long-acting thyroid stimulator (LATS), distinct from pituitary thyrotropin (TSH), is found in the serum of some patients with Graves’ disease. Although serum LATS levels correlate with several parameters of thyroid activity in thyrotoxicosis, the role of this abnormal stimulator in the pathogenesis of Graves’ disease remains un- certain. High levels of LATS are frequently associated with pretibial myxedema bait the relation of LATS to the ophthalmopathy of Graves’ disease is less clear. Demon- stration that LATS is an immunoglobulin G (IgG) and evidence that the distribution of thyroid-stimulating activity in the polypeptide chains of the IgG parallels antigen-binding activity in known antibodies has led to speculation that LATS is an antibody to thyroid antigen(s). The evidence for and against this hypothesis is discussed in detail and it is concluded that present information is insufficient to confirm or disprove this postulate. Although both in viva and in vitro studies indicate that LATS and TSH act on the same thyroidal site(s), the precise mechanism of action of these thyroid stimulators has not yet been determined.

T HE long-acting thyroid stimulator (LATS) was first detected by Adams and Purves [I]

who observed that the intravenous injection of serum from a patient with thyrotoxicosis caused a prolonged discharge of la11 from the thyroid glands of suitably prepared guinea pigs. The response elicited by the thyrotoxic serum reached a maximum value sixteen to twenty-four hours after injection as cornpared with one and a half to three hours for pituitary thyrotropin (TSH). This observation was soon confirmed by Mc- Kenzie [Z] and Munro [.?I, using McKenzie’s modification of the assay method which entails the use of mice instead of guinea pigs. Subse- quent experiments demonstrated that thyrotoxic serum contained no substance which prolonged the thyroid-stimulating activity of added TSH [3,4]. The finding that LATS was active in hypophysectomized mice [3,5] indicated that its action was directly on the thyroid gland and did not involve stimulation of pituitary TSH secretion in the assay animal.

LATS has been shown to stimulate the thyroid of the guinea pig, mouse, rabbit [6], rat [7] and man [8]. Moreover, it has been clearly dernonstrated that serum containing LATS can

stimulate several other parameters of thyroid function in addition to discharge of radioiodine. Thus, LATS has been shown to increase thyroidal is11 uptake and protein-bound iodine [9], and to produce the histologic changes associated with increased thyroid activity [IO].

However, although LATS has long been rec- ognized as a true thyroid stimulator, its role in the pathogenesis of Graves’ disease is still disputed. Recent studies demonstrating that LATS is an immunoglobulin G (IgG) have led to speculation that LATS is an antibody to thyroidal antigen(s) [II], a point of view that affords an entirely new perspective to the problem.

This communication reviews the cumulative experience gained in the study of the long-acting thyroid stimulator during the past decade. Since many unsettled points of controversy remain, what follows is clearly but an interim report.

ASSAY METHOD

The method of McKenzie [o] is based on the in vivo labeling of mouse thyroid by intraperi- toneal injection of radioiodine, followed by the administration of exogenous thyroid hormone to suppress endogenous TSH. Stimulation of the

* From the Division of Metabolism and Endocrinology, Department of Medicine, Michael Reese Hospital and Medical Center, Chicago, Illinois 60616. This study was aided by a Grant [AM 11136-011 from the National Institute of Arthritis and Metabolic Diseases, U.S. Public Health Service. Manuscript received October 27,1967.

VOL. 45, SEPTEMBER 1968 435

436 Long-Acting Thyroid Stimulator of Graves’ Disease--Burke

mouse thyroid gland by the intravenous injection of TSH or LATS is assessed by serial measurement of blood radioactivity, the latter presum- ably representing thyroidal release of labeled hormone. Review of the numerous reports [ 77-791 from different centers employing the McKenzie bioassay reveals many minor differences in the strain of mouse used, the isotope of iodine selected and the dose administered, the route of administration of test serum, the method used for suppression of endogenous TSH secretion, the method of obtaining venous blood samples, and the time of blood sampling from the mice. Irrespective of the assay design employed, however, the delayed and/or prolonged release of thyroidal radioiodine effected by LATS remains the chief characteristic by which it is differentiated from TSH.

Initially, LATS values obtained by the Mc- Kenzie bioassay technic were expressed in terms of the radioiodine content of the blood at nine and at two hours as a percentage of the initial (zero hour) count [2]. A mean value greater at nine than at two hours was considered indicative of the presence of the stimulator, whereas TSH caused a greater increase at two than at nine hours. Subsequent experience in a number of laboratories [20,27] has shown that frequently a serum containing LATS produces a response in the assay at two hours almost equal to that at nine hours. Moreover, the maximum effect of LATS frequently occurs later than eight or nine hours, and the greater the response to the stimulator, the later is the maximum effect found. I n general, however, the greater the over-all response, the greater the likelihood of a significant difference between the two hour and nine hour values.

Basically three different methods have been used to record the magnitude of the assay response: (1) giving the absolute response obtained in the assay, which is our practice [ 791; (2) deducting 100 per cent from this figure [22], and, (3) deducting the mean effect of a control injection [20].

The statistical methods employed in the assessment of LATS bioassay data have been equally varied. There are those who rely entirely upon applying Student’s t test for assessing differences between the effects of test serum and the chosen control solution; others consider this criterion inadequate because control solutions (e.g., Dextran@) may occasionally effect a significant release of 1311 in the mouse bioassay [23]. McKenzie and Williamson 1201 recently re-

ported their experience with logarithmic trans- formation of the raw data, with subsequent adjustment by covariance analysis for the in- dependent variability of the zero hour count, and conclude that this approach is “statistically justifiable and of practical use” [20]. We have employed a repeated measurement, nested vari- able, l- or 2-factorial pooled S analysis of variance [24,25] for the LATS bioassay on the premise that since animals are randomly as- signed to different experimental groups, analysis of covariance is not a necessary step. How- ever, it would appear that these and other more elaborate statistical designs have not yet been in use long enough to permit definitive assessment of their utility in the LATS bioassay.

The occurrence of nonspecific responses in the mouse bioassay thus remains an unsolved problem; clearly the point is of sufficient importance to warrant separate discussion.

SPECIFICITY OF THE LATS BIOASSAY

The McKenzie bioassay has been criticized in recent publications [ 78,221 for its relative lack of precision, but it remains the most widely used procedure for the detection of circulating LATS. Despite the many variations in assay methodol- ogy, there appears to be remarkably little dis- agreement among investigators as to the detection of high LATS levels with the mouse assay method. What remains in dispute, however, is the significance and/or specificity of smaller increases in blood radioactivity at the time of peak LATS effect. Thus the occasional demonstration of thyroid-stimulating activity in serum from euthyroid control subjects as well as in the albumin and globulin fractions thereof have been shown to be attributable to neither TSH nor LATS [22]. Concentration procedures of proved efficacy for LATS (vi& infra) may be ineffectual when applied to serum yielding an initial bioassay response index (per cent of zero hour blood radioactivity) below 300 [22].

These nonspecific responses to euthyroid serum are also the probable explanation of earlier reports [ 72,731 that LATS was present in the serum of euthyroid people, and was distrib- uted throughout all the serum protein fractions. Adams et al. [22] have therefore suggested that the bioassay demonstration of small amounts of TSH or LATS in serum is “unreliable” unless further evidence of specificity is found. The specificity of responses to TSH can readily be established by neutralization tests with TSH antiserum. Although the problem with low

AMERICAN JOURNAL OF MEDICINE

Long-Acting Thyroid Stimulator of Graves’ Ljisease --BZIT~CP 437

Ic\& t~f LATS is more difficult, demonstration of increased activity in an IgG concentrate as \\?!I as neutralization by antihuman IgG sntiserum provides some evidence of spccificit). [_?/,22].

DIFFERENCES BETWEEN LATS AND TSH

Earl\- studies clearly demonstrated that there were significant differences in the chemical and biologic properties of the two thyroid stimulators, quite apart from their distinctive time courses of action in the bioassay.

Antigenic Dzferences. Failure to neutralize serum LATS activity with antiserum to human or bovine TSH under conditions which resulted in complete inactivation of the homologous antigen established that these thyroid stimulators were antigenically distinct [26,27]. This premise has subsequently been confirmed by the studies of Dorrington and Munro [28] in which LATS- IgG antiserum as well as TSH antiserum were used. Each antiserum was effective only against the homologous antigen and no cross reaction between LATS and TSH was demonstrable.

Physicochemical D$erences. TSH can be readily separated from LATS by procedures such as the ethanol-salt percolation of Bates, Garrison and Howard [29] or Kennedy’s fractional precipitation with acetone [30]. These methods free TSH from serum IgG. No activity is recoverable by these procedures from serum containing only LATS.

TSH in serum has been shown to be more sus- ceptible than LATS to the action of the proteolytic enzymes [37] ; however, TSH appears to be more resistant to heat inactivation than is LATS [32].

Gel filtration of serum containing LATS or TSH has shown that the two stimulators have differing distribution coefficients in the gel and are recovered with different protein fractions. Thus, LATS is recovered in the 7s fraction, whereas TSH is found in the 4s fraction [37] (vide infra).

Biologic D~Jerences. When TSH is detected in serum, its level can be shown to decrease after the administration of l-thyroxine to the patient [4,33], whereas LATS levels are not affected by such treatment [4,34].

When injected into experimental animals, the biologic half-lives of the two stimulators differ markedly. Thus, LATS has a half-life of seven and a half hours after injection into the circulating blood of the rat compared with ten to twenty minutes for TSH [35,36].

VOL. 45. SEPTEMBER 1968

EVIDENCE FOR EXTRAPITUITARY ORIGIN OF LATS

An extrapituitary origin for LATS is suggested both by direct and indirect evidence. Pituitary glands, taken at necropsy from patients suffering from Gra\.es’ disease contain no detectable LATS [ 12,371. Furthermore, comparison of LATS levels in jugular venous blood and in mixed systemic venous blood obtained si- multaneously has not revealed any increase in LATS concentration in venous blood from the skull / 721.

LATS has been detected in the blood of hyperthyroid and euthyroid patients suffering from hypopituitarism after hypophysectomy or pituitary stalk section [27,38-401. In some instances, the LATS assays were performed without awareness of the existence of nonspecific responses (vide sujwa) but in at least four of the cases the values for the nine hour responses were high enough to make it probable that LATS was present. In such a case recently reported by Burke [27], LATS activity was restricted to the IgG fraction of the patient’s serum and was neutralized by antihuman IgG antiserum.

Indirect evidence for an extrapituitary origin of LATS is provided by reports of hyperthyroidism developing in hypopituitary patients after hypophysectomy [27,47,42], pituitary stalk section [38], postpartum pituitary necrosis [43] and g”Y pituitary implantation for acromegaly [44].

EVIDENCE FOR NORMAL PITUITARY TSH SECRETION

MECHANISM IN GRAVES’ DISEASE

Using a TSH concentration method and proving identity of TSH by neutralization tests with antiserum, Adams and Kennedy [30] were able to demonstrate normal functioning of the TSH secretion mechanism in a woman with Graves’ disease and a high blood LATS level. In the untreated hyperthyroid state, this woman had no demonstrable circulating TSH; after she had been inadvertently rendered myxedematous by 1311 therapy, her blood contained TSH as well as LATS, the former disappearing when she was given thyroxine in suppressive doses.

ROLE OF LATS IN THE PATHOGENESIS OF

GRAVES’ DISEASE

If a pathogenetic role for LATS in the hyperthyroidism of Graves’ disease is to be accepted, it might be anticipated that this abnormal thyroid stimulator would be present in active disease and absent following “cure” of thyrotoxi-

438 Long-Acting Thyroid Stimulator of Graves’ Disease-Burke

cosis. A factor which has precluded definitive establishment of this tenet is the finding that LATS has been detected in only 40 to 60 per cent of whole serum from patients with Graves’ disease [ 72,79,45]. These observations might obviously reflect inadequate sensitivity of the bioassay, and reports of positive assays obtained after the use of concentrating procedures [466] permit the postulate that all patients with Graves’ disease have this stimulator in the blood. Parallel concentration studies on serum from euthyroid control subjects have consistently failed to demonstrate LATS activity [79,46].

Another situation giving rise to confusion re- garding the role of the stimulator is that in which a patient, following thyroid ablative therapy, has become euthyroid despite the continued presence of LATS in the blood. Re- cent studies in such patients by McKenzie [47] have to some extent clarified this anomaly. Five patients with high levels of circulating LATS were successfully treated for hyperthyroidism after repeated doses of radioiodine. Circulating LATS was still found in high concentration as long as two years thereafter. The per- sistence of Graves’ disease was indicated by the failure of thyroid function to be suppressed by exogenous thyroid hormone and by thyroidal unresponsiveness to exogenous TSH. It was concluded that although these patients had sufficient functioning thyroid tissue to remain euthyroid, they were not hyperthyroid because the remaining tissue was incapable of any greater response to stimulation [47].

Analogous observations have been made [&I in five euthyroid patients with infiltrative ophthalmopathy and positive serum assay for LATS who had never been hyperthyroid. It was concluded that in all five patients hyperthyroidism had failed to develop, even though LATS was present in their plasma, because some prior lesion had limited the capacity of the thyroid gland to respond to thyroid-stimulating hormones [48].

McKenzie [47] has examined serum from sixteen patients with autonomous hyperfunctioning thyroid adenomas, all but three having clinical hyperthyroidism; in no instance was a positive assay obtained when the serum was tested for both LATS and TSH.

NEONATAL GRAVES’ DISEASE

Neonatal thyrotoxicosis is a rare condition, and since White’s original description of the entity in 19.12 [49] only forty-two cases have

been reported in the world literature [50-521. In each instance the mother was hyperthyroid during pregnancy, or had a past history of thyrotoxicosis, usually associated with exophthalmos. The disease in infants is stormy but transitory, and except for six reported deaths, the neonates recovered completely within one to three months. This has led to the suggestion that some factor, transferred transplacentally from mother to fetus, was responsible for initiating symptoms in the newborn infant.

LATS has now been reported in the serum of six infants with neonatal thyrotoxicosis [50-521. The maternal serum was also positive for LATS in these cases and this has led to the postulate that neonatal thyrotoxicosis is caused by LATS crossing the placenta and reaching the fetal circulation. This hypothesis has been used to explain the self-limited nature of neonatal thyrotoxicosis which usually resolves sponta- neously within three to twelve weeks; this course correlates fairly well with the estimated six to seven day half-life of LATS in neonatal serum [57]. The transitory character of the disease in infants may therefore be a reflection of the rate of catabolism and ultimate disappearance of this exogenous protein.

LATS AND INFILTRATIVE OPHTHALMOPATHY

There has been considerable speculation re- garding a possible etiologic role for LATS in the development of infiltrative ophthalmopathy in patients with Graves’ disease. The cause of the eye signs of thyrotoxicosis has remained obscure despite detailed studies on an exophthalmos- producing substance (EPS) which may be detected in animal pituitary extracts [53,54]. Dobyns and co-workers [53] and McGill [54] have reported the detection of EPS in the serum of patients with severe exophthalmic ophthal- moplegia. There has been conjecture that LATS and EPS may be identical. To date, only one group of investigators has reported correlative assays [75] and there was apparent lack of identity.

Difficulties in studying the role of LATS in the pathogenesis of thyrotoxic ophthalmopathy arise from the inconstant relationship between the progress of the eye signs and excessive thyroid activity in thyrotoxicosis. It is well recog- nized that deterioration in eye signs is not necessarily associated with increasing thyroid activity, and this also suggests that the etiologic factors responsible for these two aspects of Graves’ disease differ. A gross correlation of high values for


I,ong-Acting Thyroid Stimulator of C;ra\;es’ Disease--~ -Burke 439

LA?‘,i in instances of severe ophthalmopathy has kJC!l’ll reported [72,75,36,45], but here too, cxalllples of strongly positive assays and no ophthalmopathy, or conversely, severe ophthal- illopathy with negati1.e assays were found. In several detailed studies, moreover, a number of investigators [ 12,391 have failed to find an! consistent relationship between the severity of associated eye involvement in thyrotoxicosis and serum LATS levels.

Lipman et al. [55] recently examined the association between the presence of LATS in the serum and the various features of Graves’ disease, singly and in combination, in a series of 439 subjects. The amount of LATS in the serum correlated more closely with the number of manifestations of Graves’ disease (hyperthyroidism, ophthalmopathy, dermopathy) than with the severity of any of these manifestations. They noted, however, that serial LATS determina- tions were of value in prognosticating the course, particularly of ophthalmopathy, which usually improved with glucocorticoid therapy.

Kriss et al. [56] related the appearance of LATS in the circulation and development of ophthalmopathy to 1311-induced thyroid injury, suggesting that LATS is an antibody whose formation is a consequence of physical injury to the thyroid with resultant release into the circulation of thyroid antigen. In direct contrast to these data, however, have been the findings of Burke [57] indicating dissociation between thyroid injury and formation of LATS in thyrotoxicosis. We have performed serial LATS assays on serum obtained from thirty thyrotoxic patients prior to and at various intervals following radioiodine treatment. Eighteen euthyroid patients treated with 1311 for intractable angina pectoris [58] were used as controls. No evidence of 1311-induced LATS formation was noted on bioassay of serum obtained from six weeks up to eighteen months after treatment in either the thyrotoxic or euthyroid patients. No correlation was noted between the development of infiltrative ophthalmopathy after treatment and the detection of circulating LATS nor was there any evident relationship between anti- thyroglobulin antibody levels and thyroid- stimulating properties of serum obtained before and after radioiodine treatment.

LATS AND PRETIBIAL MYXEDEMA

Although several attempts have been made to implicate LATS in the development of pretibial myxedema, including the formulation of an

VOL. 45, SEPTEMBER 1968

hypothesis which would identify it as an antibody involved in a delayed type of derlllal alIti- gen-antibody reaction I/7], there is as )ret no convincing evidence for a direct role of LATS in the pathogenesis of this enigmatic skin lesion.

Kriss and co-workers [ 171 ha\-? elllphasized that very high LATS levels are frequently found in the serum of patients in whom localized myxedema has developed as a complication of thyrotoxicosis. Pimstone et al. [ 151 detcrted high levels of LATS in serum from thyrotoxic patients with localized pretibial myxederna and reported extraction of low levels of LATS activit) from biopsy specimens of the abnormal skin. However, in all large series reported there are patients with extremely high LATS levels who have never had pretibial myxedema, and not all patients with this complication necessarily ha1.e high levels of LATS [.59]. Benoit and Greenspan [60] recently reported their experience with glucocorticoid therapy in patients with pretibial myxedema. Although all of the eight patients in whom serum LATS was measured were found to have elevated levels, changes in serum LATS did not seem to correlate well with improvement in the skin lesion. Kriss et al. [67], on the other hand, observed a general trend toward a reduction in serum LATS levels following local Auo- cinolone (Synalar@) treatment of pretibial myxedema lesions. In any event, the observation that localized myxedema is a good “marker” for a high serum LATS level has proved to be of great value to all workers in the field.

RELATIONSHIP OF LATS TO THYROID FUNCTION

IN THYROTOXICOSIS

By combining the assay of unconcentrated serum with the application of concentration procedures to inactive samples, Carneiro et al. [46] showed that LATS may be demonstrated in more than 85 per cent of the patients with thyrotoxicosis, whereas their control experiments in euthyroid subjects were uniformly negative. Thus, the substantial proportion of iuactive serum in thyrotoxicosis (vi& supru) is no longer a major obstacle to consideration of the potential importance of LATS in the pathogcnesis of the disease.

Other supporting evidence for the view that LATS causes the increased thyroid activity of thyrotoxicosis comes from the significant correlation observed by these workers [62] between circulating LATS levels and Is11 turnover rate, particularly when related to thyroid mass in a series of fifty-five patients with thyrotoxicosis.


These workers concluded from the data that LATS was the ultimate cause of the increased thyroid activity in these patients. The important question remains, however, whether this is also true of those thyrotoxic patients in whom concentration procedures are required to demonstrate LATS.

CHEMICAL CHARACTERIZATION OF LATS

Initial attempts to extract LATS from serum by means of starch block electrophoresis [36] or by ion-exchange chromatography on DEAE cellulose [3,36,63] were unrewarding, with considerable loss of activity. The observations of Adams and Kennedy [64] that LATS could be recovered in high yield with gamma globulin after cold ethanol precipitation stimulated more detailed studies of the association of LATS with this fraction. The isolation of LATS with im- munochemically pure IgG from active serum has since been described in reports from several laboratories [ 77,79,65-671. In most of these studies the initial concentration of IgG was performed either by salting out [ 77,65,67] or by ethanol precipitation [66] prior to ion- exchange chromatography on either DEAE cellulose [65-671 or DEAE Sephadex [77]. Such preparations showed a tenfold increase in LATS activity per unit weight of protein compared with that of the original serum proteins and sedimented in the ultracentrifuge as a single peak at a corrected rate (Szo,,) of 6.5/6.68. Im- munoelectrophoresis and gel diffusion against antiwhole human serum showed only the single precipitation arc characteristic of IgG, and injection of purified preparations into rabbits produced monospecific antiserum indicating the immunochemical homogeneity of the IgG. Miyai and Werner [68] have isolated LATS-IgG from an active serum by DEAE cellulose chromatography and then subfractionated the IgG on carboxymethyl cellulose. They demonstrated a heterogeneous distribution of LATS activity in the IgG subfraction and achieved a thirty to thirty-sevenfold concentration of LATS compared with that of the original serum.

The close association of LATS and IgG has been confirmed by neutralization studies with specific antiserum. Thus, LATS activity in both unfractionated serum and in the purified IgG obtained therefrom is abolished following incubation with antiserum to human IgG [ 7 7,65,69].

The abundant evidence that LATS is intimately associated with IgG has given rise to the

hypothesis that the activity is an integral part of the complex protein so designated [ 7 7,651. Using technics designed to disrupt the various types of noncovalent bonding between or within protein molecules, attempts have been made to dissociate the thyroid-stimulating activity of LATS from active IgG. Exposure of LATS-IgG to high concentrations of sodium chloride, urea [23,70], guanidine hydrochloride or formic or acetic acids [77] failed to dissociate any smaller active protein or peptide from the IgG and did not alter the time-course of response in the bioassay. Beall and Solomon [72] have shown that LATS activity cannot be transferred to new IgG molecules in a dialysis bath or bound to cellulose, thus supporting the idea that LATS is an integral part of the IgG molecule.

PROTEOLYSIS OF LATS-IgG

McKenzie [37] has studied the effects of various proteolytic enzymes on LATS activity in whole serum. LATS activity was not affected by trypsin, subtilopeptidase, pancreatic proteases or papain. The lack of effect of papain digestion is disputed by other workers [II], however. Dorrington et al. [67] as well as Meek and co- workers [65] have hydrolyzed purified preparations of LATS-IgG in a cysteine-EDTA acti- vated system, according to the method of Porter [73]. Three fragments of approximately equal size were obtained and the sedimentation rate was reduced from 6.6s to 3.5s [67]. The reduction in molecular size from 150,000 to 50,000 [74] was accompanied by a striking change in the time-course of thyroid stimulation in the mouse bioassay [65,69] ; the prolonged response of LATS was converted to a short-acting one resembling that of TSH and the magnitude of peak response was diminished. DEAE cellulose chromatography of the papain digest separated the Fat,-fragments from the F,-fragments; only the Fat,-fragments were found to have thyroid- stimulating activity [69].

The change in time-course of response following limited proteolysis of LATS-IgG led Meek et al. [65] to suggest that LATS was a thyrotropin-like substance bound to antibody, whereas Dorrington and co-workers [69] concluded that this change reflected a shorter biologic half-life of the fragments compared with that of intact IgG as a consequence of the reduction in molecular size. Using an in vitro system for the study of 1311-release from whole mouse thyroids [75], Dorrington and Munro [59]


Long-Acting Thyroid Stimulator of Graves’ Disease---Burkrz 441

found that F,t,-fragments cause a response equal to an equivalent concentration of LATS-IgG. These data suggest that when renal and other clearance rates are inoperative, proteolysis does not reduce thyroid-stimulating activity of LATS- IgG to the extent suggested by the diminished in civo response.

Despite the several differences between TSH and LATS, the evidence that a short-acting moiety is extracted from a long-acting molecule has frequently been interpreted to favor the view that LATS is but a conjugated form of the hypo- physeal hormone [65]. The repeated demonstration of only conventional TSH activity in pituitary extracts of patients suffering from Graves’ disease during life, the demonstration of a normal pituitary-thyroid axis in Graves’ disease, and the occurrence of Graves’ disease with demonstrable circulating LATS in hypopituitary patients, as discussed in detail earlier in this paper, provide ample clinical evidence that TSH is not part of LATS. Meek [76] has recently reported formation of LATS by human thyrotropin-antibody complexes, but the eight hour responses obtained therewith in the mouse bioassay differed not at all from those obtained with the uncomplexed TSH. Evidence has been obtained with specific antiserum indicating that the thyroid-stimulating activity of Fab-fragments remains antigenically distinct from TSH [69,77]. The short-acting activity of a LATS-IgG papain digest was annulled on incubation with antiserum to IgG and F,, (but not with antiserum to F,), whereas antihuman TSH was without effect. Conversely, human TSH responses were abolished only by homologous antiserum.

Similar findings have been reported with pepsin [67,69,77]. In the absence of a reducing agent, proteolysis of IgG with pepsin yields a single fragment with a sedimentation rate of approximately 5s (MW, lOO,OOO), equivalent to two F,,-fragments joined by a single disulfide bridge [77]. Peptic digests of LATS-IgG retain their LATS activity. However, the addition of a reducing agent such as cysteine lowers the sedimentation rate to 3.2s and, as after papain digestion, a short-acting assay response is obtained [67,69,77]. The results with pepsin digestion of LATS-IgG provide additional evidence that molecular size plays an important role in determining the time-course of thyroid stimulation.

Reduction studies on LATS-IgG by Meek


et al. [6.5] and by Dorrington and co-workers [66] have yielded conflicting results. The latter group found that treatment of LATS-IgG with 2-mercapto-ethanol and alkylation of the free sulfhydril groups with iodoacetamide did not significantly reduce LATS activity or change the time-course of response. Meek et al. suggested that there was a change to a short-acting response following reduction and that this resided in the heavy A chain only. The finding of thyroid-stimulating activity in the separated A chain has not been confirmed by other investigators [47,66], and indeed, Dorrington et al. [66] have shown that in their hands treatment with acetic or propionic acid (as carried out in separating A and B chains) caused loss of nearly all thyroid-stimulating activity.

INTERACTION OF LATS WITH THYROID

TISSUE IN VITRO

The accumulated evidence that LATS activity is an inherent property of specific IgG molecules has led to several studies evaluating the possibility that LATS may be an antibody (vi& in@). The primary thrust of the investigations has been directed toward study of the interaction between LATS and thyroid tissue in oitro.

Kriss and co-workers [77] incubated LATS serum with homogenates of dog thyroid as well as of other tissues. Some reduction in LATS potency was observed after incubation with all but splenic homogenates, but the loss was tenfold higher with thyroid tissue. These workers were also able to demonstrate progressive reduction in thyroid-stimulating activity following the incubation of LATS serum with human thyroid slices.

In direct contrast to these data, however, have been the observations of Pinchera et al. [ 761 that biologic activity of serum containing LATS was neither inactivated nor altered by incubation with slices or cell membranes of toxic thyroid glands.

Conflicting data as to the organ specificity of LATS inhibition have been reported by Dorring- ton et al. [78] on the one hand, and El Kabir and co-workers [79] on the other. Although the former group showed no inhibition of LATS by a homogenate of human myometrium under conditions in which thyroid preparations showed maximum inhibitory activity, El Kabir et al. noted that incubation of LATS-positive serum with homogenates of kidney and muscle as well as of thyroid resulted in inhibition of LATS


activity. These workers were unable to ac- count for the partial LATS inhibition by tissues other than thyroid or for the failure of this inhibitory activity to increase with increased tissue concentration. Both groups have apparently made the tacit assumption that inhibition of LATS activity is equivalent to its absorption by organ homogenates or subcellular thyroid fractions without any direct evidence validating this contention. That this does occur, and that such an interaction represents an antigen-antibody reaction has been inferred from the data of Beall and Solomon [72,80]. These investigators have demonstrated (organ-specific) neutralization of LATS by thyroid microsomes and have reported concentration of LATS activity by adsorption to and acidic elution from these thyroid microsomal fractions. The eluted material contained a much larger quantity of LATS in proportion to its IgG content than the original serum. This is said to represent the in vitro

counterpart of the technic of antibody absorption and has been cited by these workers as evidence for an antigen-antibody relationship between thyroid fractions and LATS [72,80].

Parallel studies on the interaction of thyroid microsomes and TSH have yielded conflicting results. Dorrington et al. [78] and Burke [87] were unable to demonstrate consistent inhibition of TSH by thyroid cell fractions under conditions when LATS inhibition was maximal. Beall and Solomon [SO], on the other hand, reported consistent inhibition of bovine TSH by incubation with thyroid microsomes.

Because of the singular lack of physicochemical [I, 7 7,64,65] and immunologic [ 7 7,27,28] identity of LATS and TSH, the nature of the interaction between LATS-IgG and the thyroid has been investigated further by determining whether the prior in vitro exposure of thyroid microsomes to TSH modified the subsequent interaction of LATS and these subcellular thyroid fractions. The significant modification of microsomal inhibition of LATS under these conditions, as reported by Burke [87], suggests that the LATS and TSH molecules have similar, nonantigenic thyroid-stimulating groupings, and that there may be other than an immunospecific basis for the inhibition and/or absorption of LATS by thyroid microsomes. Obviously, these data do not exclude the alternate possibility that even though these stimulators may act on the same thyroidal site, TSH does so by physio- logic bonding and LATS through an antigen-

antibody reaction. Support for the former postulate is derived from the apparent reversibility of the interaction between thyroid microsomes and LATS-IgG as evidenced by “elution” of the absorbed IgG following serial incubation of the microsomal pellet with the LATS immunoglobulin [87]. Moreover, in unpublished studies, and in direct contrast to the observations of Beall and Solomon, we have eluted IgG with LATS activity from microsomal pellets previously incubated with LATS serum by incubation with phosphate-buffered saline solution, conditions inappropriate for dissociation of antigen-antibody complexes.

Adams and Kennedy 1821 recently reported the occurrence in Graves’ disease of an IgG which protects LATS from neutralization by an extract of thyroid gland. This LATS “protector” was found in a greater proportion of thyrotoxic serum than was LATS. Since the LATS “protector” appears not to compete with LATS for the thyroid cell sites on which LATS exerts its thyroid-stimulating action [82], but does compete with LATS in the reaction with thyroid extract, it has been suggested by these investigators that LATS possesses two different groupings which react with thyroid cell components. One may be the thyroid-stimulating grouping, not possessed by the LATS “protector,” possibly similar to TSH. The other grouping may be responsible for the neutralization reaction with thyroid extract and may be shared by the LATS “protector.”

MODE OF ACTION OF LATS

Hormonal action through a primary effect on RNA or protein synthesis is frequently suggested. Inhibition by antibiotics of TSH and LATS has been offered as evidence that these thyroid stimulators also act by this means by Kriss and co-workers [ 7 71. The influence of TSH and LATS on protein and RNA synthesis in the mouse thyroid in vivo has been studied by Mc- Kenzie [83] in conjunction with measurement of their potency in releasing thyroid iodine. It was found that TSH and LATS increased amino acid incorporation into thyroid protein, as did administration of antithyroid drugs; actinomycin D or puromycin prevented the stimulation in both situations. Release of thyroid 1261 after intravenous injection of TSH or LATS was not inhibited unless the antibiotics were given eight to fifteen hours earlier. No clear qualitative differences in the effects on the two stimulators

AMERICAN JoUXCNAL OF MEDICINE

Long-i\cting Thyroid Stimulator of Graves’ Disease---Burke -143

were: yccn, implying an action at the same site(s) in the thyroid. Fresh protein or RNA synthesis tllus does not appear necessary for acute effects of TSH or LATS, but the short time interval ~~~andatory for the inhibiting effects of antibiotics sltggests that stimulation of thyroid iodine rcleasc is mediated by a protein with a rapid tLnnover rate 1831.

Shishiba and co-workers [84] compared the early actions of LATS and TSH upon two thyroidal responses related to hormonal secretion, namely, intracellular appearance of colloid droplets and release into the blood of preformed colloici-stored radioiodine. They concluded that the early phase of stimulation of the thyroid by LATS i$ qllalitatively similar to that due to TSH, but has a longer latency, and that this might reflect a difference in the initial process of stimulation by the two materials.

Scott et al. [85] studied the comparative effects of LATS and TSH on the intermediary metabolism of thyroid tissue in vitro. It was foullcl that LATS increased the uptake and oxidation of glucose and enhanced the formation of lactate by sheep thyroid slices in a manner entirely analogous to the action of TSH. LATS also increased phospholipogenesis (as indicated by 32P incorporation), although the specificity of individual phospholipid response differed slightly from that noted with TSH. Thus LATS stimulated incorporation of 32P orthophosphate into lecithin and monophosphatidylinositol to about the same degree whereas TSH action is directed mainly toward the latter phospholipid. In contrast to TSH, LATS consistently increased the labeling of neutral lipids from UJ4C glucose, although their effects on decreasing the ratio of radioactive triglyceride to diglyceride were similar. These investigators concluded from these studies that LATS and TSH may be characterized by having common “receptor” sites on the cell membrane (thus producing similar metabolic responses), but may also have other sites which differ in chemical nature, thus resulting in differential effects on thyroidal intermediary metabolism.

McKenzie [86] has recently presented evidence that the effects of both TSH and LATS on thyroid iodine discharge and uptake may be mediated by cyclic AMP. This nucleotide is formed by the action of adenyl cyclase on adeno- sine 5 ‘-triphosphate and the enzyme appears to reside in the cell membrane [87]. Thus a vital moiety in the proposed pathway of thyroid

vol.. 45, SEPTEMBER 1968

stimulation may bc readilv acccssiblc. w both TSH and LATS.

The postulate that LATS and TSH act on the same site(s) in the thyroid is supported by the recent demonstration by Burke j&q’] elf the competitive interaction of these thyroid stinllllators ir viva. These studies reveal that TSH given OJK

to two hours prior to or concomitant with the administration of LATS results in significant decrease in mouse thyroidal twenty-folu~ horn response to the latter stimulator. 111 like manner, LATS-IgG given two hours before 0.8 I~II- TSH almost completely prevented response (1311

release) to the latter although so~ne control mice exhibited a greater total response to larger doses of either agent. McKenzie [83] pre\.iollsly reported similar data. Although these studies suggest a similar site of action of these thyroid stimulators, more information will undoubtedly be required concerning details of thyroid gland response to TSH stimulation before any specific mechanism(s) for LATS action can be formu- lated.

IS LATS .4N ANTIBODY?

The accumulated evidence intimately linking LATS with IgG was reviewed earlier. These studies indicate that the thyroid-stimulating activity of LATS is an inherent property of specific IgG molecules and have led to the postulate that LATS is an antibody to thyroidal antigen(s) .

Chemical [89] and enzymatic [73,77] degra- dation of IgG with specific antibody activity has clearly indicated that the binding sites are located within the Fab-fragments. As noted earlier, similar localization of thyroid-stimulating activity has now been established for LATS-IgG.

The association of LATS with IgG has prompted the study of LATS production by lymphoid tissue, which is known to be the site of IgG synthesis. McKenzie and Gordon [37] have cultured white blood cells from the blood of a thyrotoxic patient with circulating LATS. In the presence of phytohemagglutinin, which is known to stimulate IgG synthesis by lympho- cytes [90], low levels of LATS activity were detected in the supporting medium. Incorpora- tion of 14C-labeled amino acids into IgG isolated from the cultures confirmed that synthesis of IgG occurred in this system.

Miyai et al. [91] recently confirmed and amplified these findings. They found that most of


the resultant LATS activity was present in the IgG fraction of the lymphocyte culture medium and was partially neutralized by anti-IgG antiserum. On the premise that lymphoid tissue obtained from a thyrotoxic patient with high LATS levels might prove a fruitful source of LATS-producing cells, Pinchera et al. [92] performed LATS assays on extracts of lymphoid tissue (including Delphian lymph node and spleen) col- lected at autopsy from such a patient. None of the samples tested elicited LATS response however.

Suppression of LATS production by corticosteroids has been demonstrated by several workers [ 77,75,60,93] and has been cited as a demonstration of the well known immunosup- pressive action of corticosteroids [59]. The significance of these findings is clouded, however, by the not infrequent dissociation under these cir- cumstances between progression of those thyrotoxic manifestations (e.g., ophthalmopathy) pre- sumed due to LATS, and circulating LATS levels.

The frequent demonstration of autoantibodies to components of thyroid tissue in thyrotoxicosis [94] and the occurrence of disorders of immunologic tolerance [95] to which there may be genetic predisposition in patients with Graves’ disease does not provide evidence that LATS is an antibody. Thus, Major and Munro [ 721 were unable to obtain any correlation between the presence of autoantibodies to thyroid components and serum LATS activity in a large series of patients with thyrotoxicosis.

If LATS is an antibody, a complementary antigen must exist and its most likely site would be the thyroid. Although the demonstration of LATS-IgG absorption by thyroid microsomes has been cited by some [59] as illustrating the hypothetical antibody-antigen reaction which results in stimulation of the intact thyroid cell, the “protective” action of TSH in these circum- stances [87] suggests, as indicated earlier, that there may be other than an immunospecific basis for this reaction.

Of greater significance, however, is the recent demonstration in three laboratories [96-981 that antiserum with LATS activity has been obtained in rabbits immunized with homogenates [98] and/or microsomal fractions [96,97] of human thyroid tissue. Although McKenzie [98] has suggested that prolonged maintenance of antibody production by “booster” immunization can lead to increased thyroid function, experi-

mental Graves’ disease in these animals has not been produced. Solomon and Beall [97] indicate that their rabbit LATS did not produce thyroid hyperfunction in the animals. The data obtained by both groups strongly suggest an induced thyroiditis in these animals [97,98], which in itself may result in significant eleva- tion of various parameters of thyroid function.

Although we have confirmed that rabbit antiserum to human thyroid microsomes may possess LATS activity, such activity decreased rapidly after cessation of immunization. More- over, there was no correlation between the thyroid-stimulating propertiesof theantiserum so obtained and the titer of precipitating antibodies to human microsomes, the antigen employed. To date we have been unable to demonstrate immunologic cross reaction between rabbit antiserum with LATS activity and mouse thyroid tissue in vitro, nor was there any demonstrable laboratory evidence of thyroid hyperfunction in the rabbits during the eight weeks of immunization or thereafter (Burke, G. Un- published data).

In discussing the putative antibody role of LATS, it is important to note that conventional immunologic studies directed toward evaluation of LATS as an antibody to thyroidal antigen(s) have, to date, been largely negative. Thus, Dorrington et al. [59], using a “sandwich” technic with fluorescein-labeled rabbit antihuman IgG, were unable to demonstrate any localization of LATS-IgG in frozen thyroid slices. Blum and co-workers [99] recently described an immunofluorescent technic for the simultaneous detection TSH and LATS in human serum. In these studies TSH was found to be localized in the nucleus of the thyroid cell, whereas cytoplasmic fluorescence was observed with twenty-seven of twenty-eight serum specimens obtained from patients with Graves’ disease. However, it is as yet unclear whether the latter finding is due to the presence in the serum of LATS, LATS-“protector” [82], or anticyto- plasmic antibodies [ 7001 without LATS activity. To date, we have been unable to confirm these findings with serum from patients with Graves’ disease, using both conventional indirect immunofluorescence [707] or the more sensitive technic of mixed antiglobulin immunofluorescence [702]. We have, however, confirmed that nuclear fluorescence is readily demonstrable with TSH using the former technic [Burke, G. Unpublished data].


Long-Acting Thyroid Stimulator of Graves’ Disease--Burke 445

Although antimicrosomal complement fixing antibodies were present in their rabbit antiserum with LATS activity [97], Real1 and Solomon [72] had previously reported that mixtures of hun~an LATS serum and thyroid microsomes do not fix complement, stating that “. , . it might. . .be concluded from this result that the reaction is not of an immunologic nature.” Kohler et al. [703] recently reported positive LATS response in the complement-deficient mouse. These studies indicate that the presence of a complete complement system was not necessary for LATS activity and the response was not increased by passive transfer of a complete complement source. Employing a variety of technics including passive cutaneous anaphylaxis, radio- isotope precipitation (double antibody) test and passive hemagglutination. Burke [79] recently reported failure of immunologic reaction between LATS (whole serum or purified IgG) and subcellular thyroid fractions. The uniformly negative data obtained under these circum- stances do not support (but do not negate) the hypothesis that LATS is an antibody to thyroidal antigen(s). Such an hypothesis therefore cannot be considered confirmed at the present time.

ADDENDUM

The following resume cites developments in LATS research published since compilation of this review.

Assay Method. Mason et al. [704] modified the bioassay method for TSH described by Good and Stenhouse [705] for the determination of LATS. With this design, which elimi- nates the factors of animal and day variation, and the residual effects of the initial test doses by use of the mice on two successive days, the reproducibility and precision obtained appear to be better than with previous modifications of the McKenzie method.

LATS and Zrgfltratiue Opthalmofiathy. Bauer and Catz [706] originally reported dramatic success in treating the progressive opthalmopathy of Graves’ disease by surgical and/or radioiodine ablation of the thyroid. In contrast, Werner et al. [707] found total thyroidectomy to be ineffective in altering the opthalmopathy of Graves’ disease; the presence and/or titers of serum LATS did not correlate with the severity of eye changes and were variably altered following ablation of the thyroid gland. Pequegnat et al. [708] subsequently reported similar data.


Hamilton and associates [IO!)] found no significant difference in the incidence of post- treatment opthalmopathy of Graves’ disease in a group of patients treated by subtotal thyroidectorny and that in a group treated with radioiodine.

Interaction of LATS with Thyroid Tissue in Vitro. Only a small portion (about 10 per cent) of the LATS originally present in serum can be liberated from thyroid microsomes by acid washing following incubation of the par- ticulate material with the stimulator [80]. This apparent disappearance of LATS has prompted consideration that LATS activity has been destroyed. The known presence of proteases and peptidases in thyroid extracts might then explain the apparent specificity of thyroid tissue in removing LATS activity. However, Beall and Solomon [770] recently presented data indicating that inhibition of thyroidal proteolytic activity does not inter- fere with the inhibition of LATS by thyroid microsomes. These studies do not support the hypothesis that LATS is destroyed by enzymes in thyroid microsomes and fortify the notion that the LATS-thyroid microsome interaction is specific.

In contrast to the findings of Beall and Solo- mon [80] and those of Dorrington and associates [78], Berumen et al. [777] reported that cell sap contains the greatest amount of LATS inhibitory activity to be found in the thyroid gland.

Wong and Litman [772] labeled with 1261 the protein eluate obtained by acid washing of thyroid microsomes incubated with LATS serum and suggested that an in vitro assay method for LATS analogous to the established immunoassays for polypeptide hormones is feasible.

Mode of Action of LATS. Burke 17731 recently confirmed the findings of Scott et al. [85] that LATS augments glucose oxidation and phospholipogenesis in sheep thyroid slices. Qualitative differences in the mode of action of TSH and LATS in vitro were suggested by the divergent response of these stimulators to inorganic iodide as well as to iodotyrosines [ 7 741.

Burke [775] compared the early effects of TSH and LATS on thyroidal phospholipogenesis. The data obtained demonstrate no latency in the early effects on LATS when compared to TSH and suggest that the initial interaction of both stimulators is rapid, firm


binding to a superficial site. Cashman and Meek [ 1761 reported similar findings in glucose oxidation studies. Shishiba et al. [777] demonstrated that, in dog thyroid slices, LATS causes dose- related stimulation of 14C02 production from glucose-lJ4C with a latency usually shorter than for TSH.

Is LATS an Antibody? Two current, different, hypotheses of the nature of LATS state that LATS is (1) an antibody of the IgG type, and (2) an IgG globulin but not an antibody. The B chains of each IgG antibody molecule may be characterized as either kappa (K) or lambda (X) in type but specific antibodies formed by a given individual possess both types of molecules. Kriss [778] recently demonstrated significant inactivation of LATS serum by both anti-K and anti-X antiserum. These data add further evidence of the immunoglobulin nature of LATS and indicate that LATS is not the product resulting from a monoclonal selection process.

Although additional data on the experimental production of LATS in vivo have been published [779-7271, these studies have not materially clarified the putative antibody nature of LATS. The stimulator produced by Beall and Solomon [779] and by McKenzie [720] effected release of murine thyroid radioiodine which was intermediate in time-course between TSH and LATS, whereas that obtained by Burke [727] was indeed long-acting.

Although we recently confirmed the finding of polar-solvent resistant cytoplasmic fluorescence using potent LATS serum and frozen human thyroid slices, there is no correlation between fluorescent titer of this serum and the magnitude of LATS activity therein [ 7221.

El Kabir et al. [723] reported the localization of ferritin-labeled LATS-IgG in the follicu- lar cell of the thyroid of the mouse. However, their studies suggest that the sites at which ferritin was visualized were not specifically the binding sites for LATS, but that LATS acts on the thyroid cell membrane to enhance endocytosis.

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The long-acting thyroid stimulator of Graves' disease